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Unravelling the interactions of the β7
integrin cytoplasmic domain
Yih-Chih Chan
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of
Philosophy, The University of Auckland, 2009.

Abstract
Integrins are dynamic cell adhesion molecules expressed on the cell-surface which transmit
signals through the plasma membrane, and play vital physiological roles in multicellular
organisms. The β7 integrins α4β7 and αEβ7 are leukocyte-specific, and evolved to form and
maintain the mucosal immune system of the intestine, which is an important immune barrier.
They participate in antigen presentation, leukocyte migration and homing, and other
leukocyte functions. α4β7 binds the mucosal vascular addressin MAdCAM-1, which is
expressed on specialised high endothelial venules at sites of chronic inflammation. The β7
integrins contribute to the initiation and maintenance of inflammation, in particular chronic
inflammatory disease, playing a key role in the migration of leukocytes into inflamed tissue.
It is important to understand integrin signalling mechanisms in order to be able to devise
therapeutic agents that can inhibit integrin function, and thereby attenuate inflammation. The
host laboratory identified a novel cell adhesion regulatory domain (CARD) within the β7
subunit cytoplasmic domain, which plays a key role in regulating β7-mediated cell adhesion.
The CARD comprises the small pseudosymmetrical motif YDRREY, which forms the basis
of the present study.
This study aimed to identify key signalling pathways involved in regulating the cell adhesion
function of the β7 integrins, to identify intracellular ligands that interact with the β7 CARD,
and to characterize their interaction. The adhesion of the thymic lymphoma TK-1 (α4β7 +
α4β1 - ) to MAdCAM-1 was used to investigate signalling via α4β7. A panel of chemical
inhibitors of cell signalling molecules was employed in TK-1 cell adhesion assays, revealing
that JNK, the src family of kinases, and myosin light chain kinases all potentially participate
in controlling α4β7-mediated cell adhesion.
Cell-permeable synthetic peptide technology was employed to investigate structure-function
relationships of the YDRREY CARD motif. Substitution of the flanking tyrosine residues of
the YDRREY motif with phenylalanines revealed that the tyrosines were critical for the
function of the CARD. However, the state of phosphorylation of the flanking tyrosines was
inconsequential. The core DRRE region was also important for CARD function. Mutagenesis
studies involving substitution of the three tyrosine residues in the β7 subunit cytoplasmic
ii

domain to phenylalanines disrupted α4β7-mediated adhesion of TK-1 cells to MAdCAM-1,
reinforcing the role of tyrosine residues in β7 activation.
The tyrosine kinases src and FAK were shown to bind directly to the YDRREY peptide. FAK
and autophosphorylated src was shown to bind to both phosphorylated and non-
phosphorylated forms of YDRREY, while non-phosphorylated src bound only the non-
phosphorylated form. The cytoskeletal proteins α-actinin, and paxillin formed complexes
with FAK and bound to the YDRREY peptide, whereas filamin disrupted src binding. FAK
and src were both present in immunoprecipitates of α4β7. Immunofluorescence studies
combined with confocal microscopy suggested both kinases co-localise with the β7 integrin
within the trailing edge uropod and at sites of pseudo supra-molecular complex (SMAC)
formation.
Heat shock protein (hsp) 70 was also identified as a potential intracellular ligand that
associates indirectly with the β7 subunit. Thus, a synthetic peptide encompassing the fulllength
β7 cytoplasmic domain bound to recombinant hsp70, but only in the presence of a TK-
1 cell lysate. Heat shock using fever-range temperatures activated α4β7-mediated cell
adhesion. A chemical inhibitor of hsp70 prevented Mn 2+ -induced adhesion of TK-1 cells to
MAdCAM-1, suggesting that hsp70 plays a key role in regulating signalling by α4β7. The
latter results suggested that other stressors might activate α4β7-mediated cell adhesion, and
in accord serum starvation of cultured cells also induced α4β7-mediated cell adhesion to
MAdCAM-1.
In summary, this study has provided novel insights into signalling pathways that regulate
signalling by β7 integrins. Structure-function relationships of the β7 CARD have been
investigated, and kinases that interact with the CARD have been identified. Activation of β7
integrin signalling by stressors including fever-range temperatures and serum starvation, and
identification of hsp70 as a molecule which associates indirectly with α4β7 are novel
discoveries. The information gained provides a platform of results which provide the basis for
generating agents that might have therapeutic potential for treating inflammatory diseases.
iii

Acknowledgements
Firstly, I would like to express my greatest appreciation to my chief supervisor, Associate
Professor Geoffrey W. Krissansen, for providing me the opportunity to conduct my doctoral
research project within his research group, his ever important advice and guidance throughout
these years, and his help in putting this thesis together.
I also extend my appreciation to my past and present co-supervisors Drs J-Zhong Bai,
Euphemia Leung, and Katherine Woods for their advice on the direction of my project.
Additionally I would like to acknowledge Dr Euphemia Leung for her help and her
tremendous knowledge of molecular biology techniques and experimental trouble shooting.
Further I would like to extend my appreciation to our laboratory manager Yi Yang for her
help in the preparation of recombinant proteins, molecular biology and tissue culture
techniques and management of the laboratory.
I would also like to acknowledge past and present research fellows in the laboratory, Drs
Kevin Sun, Jagat Kanwar, and Rupinder Kanwar for their input into this thesis.
To fellow students Antonio Cheung and Jiwon Hong, I would like to acknowledge their
invaluable support and friendship throughout these years.
To all my friends, I would also like to thank them for their encouragement and friendship
throughout this project.
I would also like to thank the Marsden Fund for their support of the project, and the Maurice
and Phyllis Paykel Trust, the Sir John Logan Campbell Trust, Department of Molecular
Medicine and Pathology, and the Faculty of Medical and Health Sciences Postgraduate
Student Association for supporting my attendance at the Queenstown Signal Transduction
Meeting in Queenstown, New Zealand and the Signaling by Adhesion Receptors Conference
in Massachusetts, United States.
Finally I would like to thank my parents Paul and Judy, my brothers Kai and Woei, my sisterin-law
Ying-Ying, and my wife Enid for their wonderful love, support and encouragement
throughout my life. I dedicate this thesis to them.
iv

Table of Contents
Abstract......................................................................................................................................ii
Acknowledgements...................................................................................................................iv
List of Figures...........................................................................................................................xi
List of Tables ..........................................................................................................................xiv
Abbreviations...........................................................................................................................xv
Chapter 1. Introduction ..............................................................................................................1
1.1. Integrins ....................................................................................................................................... 1
1.1.1 Integrin structures .................................................................................................................. 3
1.1.2 Integrins and their extracellular ligands............................................................................... 11
1.2. β7 integrins ................................................................................................................................ 13
1.2.1 The β7 subunit cytoplasmic domain .................................................................................... 13
1.2.2 Integrin α4β7........................................................................................................................ 14
1.2.3 Integrin αEβ7 ....................................................................................................................... 16
1.3. Integrin signalling ...................................................................................................................... 17
1.3.1 Models of integrin activation ............................................................................................... 19
1.3.2 Integrin regulation by inside-out signalling ......................................................................... 22
1.3.3 Intracellular ligands of the α-subunit ................................................................................... 27
1.3.4 Integrin outside-in signalling ............................................................................................... 28
1.3.5 Force sensing and focal adhesions....................................................................................... 30
1.3.6 Integrin crosstalk.................................................................................................................. 30
1.4. Integrins and immunological synapses, unique signalling structures ........................................ 31
1.4.1 Synapses formed between communicating immune cells.................................................... 31
1.4.2 Synapses termed kinapses are formed on migrating T cells ................................................ 33
1.5. Tyrosine kinases and cell signalling .......................................................................................... 33
1.5.1 The focal adhesion kinase (FAK) family............................................................................. 33
1.5.2 Src kinase family (SFK)....................................................................................................... 36
1.5.3 Integrin signalling, FAK and src.......................................................................................... 38
1.6. Heat shock proteins.................................................................................................................... 39
1.6.1 Hsp70 family........................................................................................................................ 40
v

1.7. Integrins and disease .................................................................................................................. 44
1.7.1 Hereditary disease................................................................................................................ 45
1.7.2 Inflammatory disease ........................................................................................................... 46
1.7.3 Other diseases ...................................................................................................................... 46
1.7.4 β7 integrins and disease ....................................................................................................... 47
1.8. Aims of this thesis...................................................................................................................... 49
Chapter 2. Materials and Methods...........................................................................................50
Section 1 – Materials ...............................................................................................................50
2.1. Chemicals, reagents, buffers and media..................................................................................... 50
2.1.1 Suppliers .............................................................................................................................. 50
2.1.2 Chemicals/Molecular biology reagents................................................................................ 52
2.1.3 Buffers/Solutions ................................................................................................................. 53
2.1.4 Tissue culture reagents......................................................................................................... 56
2.2. Molecular biology reagents........................................................................................................ 57
2.2.1 Mammalian cell lines........................................................................................................... 57
2.2.2 Insect cell line ...................................................................................................................... 57
2.2.3 Bacterial cell line ................................................................................................................. 57
2.2.4 DNA cloning vectors ........................................................................................................... 58
2.2.5 Enzymes/isotopes................................................................................................................. 58
2.2.6 Antibodies............................................................................................................................ 59
2.2.7 PCR oligonucleotide primers............................................................................................... 60
2.2.8 cDNA for cloning/protein expression .................................................................................. 60
2.2.9 Recombinant proteins .......................................................................................................... 61
2.2.10 Inhibitors of intracellular signalling pathways................................................................... 61
2.2.11 Peptides.............................................................................................................................. 62
Section 2 – Methods.................................................................................................................64
2.3. Molecular biology techniques.................................................................................................... 64
2.3.1 Preparation of competent cells............................................................................................. 64
2.3.2 Small scale preparation of plasmid DNA (miniprep) .......................................................... 64
2.3.3 Large scale preparation of plasmid DNA............................................................................. 65
vi

2.3.4 Linearization and dephosphorylation of plasmid vectors .................................................... 66
2.3.5 Isolation of plasmid inserts .................................................................................................. 66
2.3.6 Agarose gel electrophoresis ................................................................................................. 66
2.3.7 Ligation of DNA inserts into vectors................................................................................... 67
2.3.8 Transformation..................................................................................................................... 67
2.3.9 RNA isolation ...................................................................................................................... 67
2.3.10 Synthesis of cDNA by reverse transcriptase...................................................................... 68
2.3.11 Polymerase chain reaction (PCR) ...................................................................................... 68
2.4. Protein chemistry ....................................................................................................................... 69
2.4.1 Production and purification of GST fusion proteins............................................................ 69
2.4.2 Sodium dodecylsulphate polyacrylamide gel electrophoresis ............................................. 69
2.4.3 Staining of SDS gels ............................................................................................................ 71
2.4.4 Western blot analysis ........................................................................................................... 71
2.4.5 Stripping of nitrocellulose and PVDF membranes .............................................................. 72
2.4.6 Kinase assays ....................................................................................................................... 72
2.4.7 Coupling of proteins and antibodies to Sepharose............................................................... 74
2.4.8 Immunoprecipitation / pull-down assay............................................................................... 74
2.5. Cell biology................................................................................................................................ 75
2.5.1 Cell culture........................................................................................................................... 75
2.5.2 Production of recombinant proteins..................................................................................... 75
2.5.3 Spleen cell isolation ............................................................................................................. 76
2.5.4 Cell adhesion assay .............................................................................................................. 76
2.5.5 Enzyme-linked soluble E-cadherin-Fc-mediated adhesion assay. ....................................... 78
2.5.6 Cell transfection ................................................................................................................... 78
2.5.7 Immunofluorescence staining .............................................................................................. 78
2.6. Phylogenetic analysis................................................................................................................. 79
2.7. Statistical analysis...................................................................................................................... 79
Chapter 3. Results ....................................................................................................................80
3.1. Expression and cell adhesion properties of β7-integrins............................................................ 80
3.1.1 Confirmation of the expression of α4β7 and αEβ7 on TK-1 and MTC-1 cells ................... 80
vii

3.1.2 Production of recombinant forms of E-cadherin and MAdCAM-1 ..................................... 84
3.1.3 Comparison of activators of β7 integrin-mediated cell adhesion......................................... 85
3.2. The effect of pathway inhibitors on β7 integrin mediated cell adhesion ................................... 89
3.2.1 Genistein, a PTK inhibitor, inhibits the binding of TK-1 cells to MAdCAM-1 .................. 89
3.2.2 Involvement the MAP kinase kinase (MEK) pathway in TK-1 cell binding to
MAdCAM-1 ......................................................................................................................... 90
3.2.3 Jun N-terminal kinase (JNK) pathway................................................................................. 93
3.2.4 Epidermal growth factor receptor (EGFR) tyrosine kinase ................................................. 96
3.2.5 Src family of tyrosine kinases (SFK)................................................................................... 97
3.2.6 Myosin light chain kinase (MLCK) ................................................................................... 100
3.3. Properties of a cell adhesion regulatory domain in the cytoplasmic tail of the integrin β7
subunit .................................................................................................................................... 102
3.3.1 A cell-permeable YDRREY peptide inhibits β7 integrin-mediated adhesion to
MAdCAM-1 and E-cadherin .............................................................................................. 102
3.3.2 Defining the features of the YDRREY peptide required to inhibit β7-mediated
adhesion .............................................................................................................................. 104
3.3.3 Effect of NPLY on TK-1 cell adhesion ............................................................................. 111
3.4. Tyrosine kinases interact with and phosphorylate the YDRREY motif .................................. 113
3.4.1 Production of GST-β integrin cytoplasmic domain fusion proteins .................................. 113
3.4.2 Phosphorylation of GST-β7 cytoplasmic domain fusion proteins ..................................... 114
3.4.3 Tyrosine phosphorylation of the YDRREY peptide .......................................................... 115
3.4.4 Kinases in multiple cell types can phosphorylate the YDRREY motif ............................. 116
3.4.5 Phosphorylation of the YDRREY peptide by FAK, src and lck........................................ 117
3.4.6 Direct binding of FAK and src to the YDRREY motif...................................................... 119
3.4.7 Binding of YDRREY peptide variants by FAK and src .................................................... 120
3.4.8 Does src remain active after binding to the YDRREY peptide?........................................ 121
3.4.9 FAK and YDRREY interactions........................................................................................ 124
3.4.10 FAK and src bind synergistically to the YDRREY peptide............................................. 125
3.5. The impact of cytoskeletal proteins on the interaction of the YDRREY peptide with kinases 126
3.5.1 Filamin disrupts the binding of src to the YDRREY peptide ............................................ 126
3.5.2 Interaction of paxillin with the β7 subunit cytoplasmic domain........................................ 127
viii

3.5.3 FAK, src, and α-actinin bind the YDRREY peptide ......................................................... 128
3.6. In vivo interactions of β7 integrins with kinases ..................................................................... 129
3.6.1 Co-localisation of a fluoresceinated YDRREY peptide in living cells.............................. 129
3.6.2 Phosphorylation of the integrin β7 subunit in vivo............................................................ 131
3.6.3 Confirmation of FAK and src involvement in β7 integrin signalling ................................ 134
3.6.4 Detection of src in β7 integrin immunoprecipitates........................................................... 136
3.6.5 Detection of FAK in β7 integrin immunoprecipitates........................................................ 137
3.6.6 Localisation of interacting kinases in living cells.............................................................. 138
3.7. Localization of α4β7 with kinases in supra-molecular activation complexes (SMAC)........... 140
3.7.1 TK-1 cells bind to MAdCAM-1-coated magnetic beads ................................................... 141
3.7.2 α4β7 colocalizes with src in SMACs................................................................................. 142
3.7.3 α4β7 colocalizes with FAK in SMACs.............................................................................. 143
3.7.4 α4β7 colocalizes with lck in SMACs................................................................................. 144
3.8. Mutation of the β7 cytoplasmic domain and effects on cell adhesion ..................................... 146
3.8.1 Cloning of mutated variants of the integrin β7 subunit into mammalian expression
vectors................................................................................................................................. 146
3.8.2 Cloning of a wild-type integrin α4 construct ..................................................................... 148
3.8.3 Transfection of HEK-293T cells to express the α4 and β7 subunit plasmids .................... 149
3.8.4 Testing the ability of transfectants to adhere to integrin ligands ....................................... 153
3.9. Identification of binding partners of the β7 cytoplasmic domain ............................................ 155
3.9.1 Pull-down assay ................................................................................................................. 155
3.9.2 Mass spectrometry identifies heat shock proteins as ligands of the β7 subunit................. 156
3.9.3 A pull-down assay estabilishes that hsp70 interacts indirectly with the β7 subunit .......... 158
3.9.4 Coimmunoprecipitation of α4β7 and hsp70....................................................................... 159
3.9.5 Localization of hsp70 and hsp90 in TK-1 cells ................................................................. 161
3.9.6 Hsp70 colocalizes with intracellular β7 ligands following ligand-induced clustering of
α4β7 .................................................................................................................................... 163
3.9.7 Effect of heat shock on expression of hsp70 and the β7 subunit ....................................... 164
3.9.8 Effect of heat shock on activation of α4β7 is time dependent ........................................... 165
3.9.9 Fever-range temperatures can activate α4β7...................................................................... 166
ix

3.9.10 Heat shock leads to prolonged activation of α4β7 even after Mn 2+ -treatment ................ 167
3.9.11 An inhibitor of hsp70 blocks the binding of TK-1 cells to MAdCAM-1 ........................ 168
3.9.12 KNK-437 has no detectable effect on hsp70 and β7 expression...................................... 169
3.9.13 Serum deprivation activates α4β7.................................................................................... 170
Chapter 4. Discussion ............................................................................................................173
4.1. Preliminary characterization of α4β7 and αEβ7 expression and adhesion............................... 173
4.2. Analysis of β7 integrin signalling pathways............................................................................ 174
4.3. The YDRREY motif ................................................................................................................ 180
4.4. Phylogenetic study of the sequence of the YDRREY motif in the β7 subunit ........................ 181
4.5. The NPLY motif ...................................................................................................................... 183
4.6. Interaction of the YDRREY motif with tyrosine kinases ........................................................ 185
4.7. Probing the function of β7 subunit cytoplasmic domain tyrosine phosphorylation sites by
mutational analysis ................................................................................................................. 187
4.8. Interaction of the YDRREY motif with cytoskeletal proteins ................................................. 188
4.9. In vivo interactions of the YDRREY peptide with cellular kinases......................................... 191
4.10. α4β7 clusters within SMACs ................................................................................................. 193
4.11. The affect of stress on the function of β7 integrins................................................................ 193
4.12. Summary................................................................................................................................ 195
4.13. Future directions .................................................................................................................... 196
References..............................................................................................................................200
x

List of Figures
Figure 1.1 Integrin subunits and pairings................................................................................................3
Figure 1.2 Structure of the integrin heterodimer.....................................................................................4
Figure 1.3 Domains of the integrin α-subunit.........................................................................................5
Figure 1.4 Domains of the integrin β-subunit.........................................................................................7
Figure 1.5 Structure of the αβ integrin heterodimer................................................................................9
Figure 1.6 The amino acid sequence of the cytoplasmic domain of the human β7 subunit. .................13
Figure 1.7 Multistep model of T cell transmigration ............................................................................15
Figure 1.8 Switch blade model of integrin activation...........................................................................20
Figure 1.9 Deadbolt model of integrin activation .................................................................................21
Figure 1.10 Sequence alignments of the human integrin β subunit (a), and α subunit (b)
cytoplasmic domains. ........................................................................................................23
Figure 1.11 Models of the immunological synapse and kinapse ..........................................................32
Figure 1.12 Structural features and binding partners of FAK...............................................................34
Figure 1.13 Structural features and activation of src ............................................................................37
Figure 1.14 Domain structure of human hsp70 family members..........................................................42
Figure 3.1 Immunoblot analysis of an MTC-1 cell lysate with antibodies against the αE and β7
subunits..............................................................................................................................81
Figure 3.2 MTC-1 cells immunostained with an anti-β7 antibody. ......................................................82
Figure 3.3 TK-1 cells stained with the DATK32 antibody against the α4β7 complex.........................83
Figure 3.4 Analysis of purified recombinant E-cadherin-Fc and MAdCAM-1-Fc by SDS-
PAGE. ...............................................................................................................................85
Figure 3.5 Activation of TK-1 cell adhesion to MAdCAM-1 ..............................................................86
Figure 3.6 Activation of MTC-1 cell adhesion to E-cadherin ..............................................................87
Figure 3.7 Binding of E-cadherin to MTC-1 cells is specifically mediated by αEβ7...........................88
Figure 3.8 Effect of genistein on TK-1 cell adhesion to MAdCAM-1 .................................................90
Figure 3.9 Effect of PD98059 on TK-1 cell adhesion to MAdCAM-1.................................................91
Figure 3.10 Effect of SB203580 on TK-1 cell adhesion to MAdCAM-1.............................................92
Figure 3.11 Effect of JNK-I-1 on TK-1 cell adhesion to MAdCAM-1 ................................................94
Figure 3.12 Effect of JNK-I-2 on TK-1 cell adhesion to MAdCAM-1 ................................................95
Figure 3.13 Effect of AG99 on TK-1 cell adhesion to MAdCAM-1....................................................96
Figure 3.14 Effect of PP2 on TK-1 cell adhesion to MAdCAM-1 .......................................................98
Figure 3.15 Effect of damnacanthal on TK-1 cell adhesion to MAdCAM-1 .......................................99
Figure 3.16 Effect of radicicol on TK-1 cell adhesion to MAdCAM-1..............................................100
Figure 3.17 Effect of ML-7 on TK-1 cell adhesion to MAdCAM-1 ..................................................101
Figure 3.18 The amino acid sequence of the human β7 integrin cytoplasmic domain .......................102
Figure 3.19 Effect of the YDRREY peptide on TK-1 cell adhesion to MAdCAM-1.........................103
Figure 3.20 Effect of YDRREY peptide on MTC-1 cell adhesion to E-cadherin ..............................104
Figure 3.21 Effect of the phosphorylated xDRREx peptide on TK-1 cell adhesion to
MAdCAM-1 ....................................................................................................................105
Figure 3.22 Effect of the YDRGGGGREY peptide on TK-1 cell adhesion to MAdCAM-1 .............106
Figure 3.23 Effect of the YDGGEY peptide on TK-1 cell adhesion to MAdCAM-1 ........................107
Figure 3.24 Effect of the YEEEEY peptide on TK-1 cell adhesion to MAdCAM-1 .........................109
Figure 3.25 Effect of the pYDRREY peptide on TK-1 cell adhesion to MAdCAM-1.......................110
Figure 3.26 Effect of the pFDRREF peptide on TK-1 cell adhesion to MAdCAM-1........................111
xi

Figure 3.27 Effect of the NPLY peptide on TK-1 cell adhesion to MAdCAM-1...............................112
Figure 3.28 Production of GST-β subunit cytoplasmic domain fusion proteins, and analysis by
SDS-PAGE......................................................................................................................114
Figure 3.29 Phosphorylation of GST-β subunit cytoplasmic domain fusion proteins........................115
Figure 3.30 The YDRREY peptide is phosphorylated by a tyrosine kinase(s) in a TK-1 cell
lysate................................................................................................................................116
Figure 3.31 Kinases in multiple cell types can phosphorylate the YDRREY motif...........................117
Figure 3.32 Phosphorylation of the GGYDRREY peptide by recombinant FAK, src, and lck..........118
Figure 3.33 Sequence recognition of the YDRREY peptide by FAK, src, and lck ............................119
Figure 3.34 Analysis of the binding and phosphorylation of the YDRREY peptide by FAK, src,
and lck .............................................................................................................................120
Figure 3.35 FAK and src binding and phosphorylation of variants of the YDRREY peptide............121
Figure 3.36 Src bound to the YDRREY motif remains active............................................................122
Figure 3.37 Src binds to YDRREY but not to xDRREx.....................................................................123
Figure 3.38 Tyrosine phosphorylation of the YDRREY peptide affects the degree of FAK
binding.............................................................................................................................124
Figure 3.39 Do FAK and src bind synergistically to the YDRREY peptide?.....................................125
Figure 3.40 Filamin disrupts the binding of src to the YDRREY peptide..........................................127
Figure 3.41 Erk-phosphorylated paxillin binds to FAK and forms a complex with the YDRREY
peptide. ............................................................................................................................128
Figure 3.42 Binding of src, FAK, and α-actinin to the YDRREY peptide. ........................................129
Figure 3.43 The YDRREY peptide co-localises with FAK and src at focal adhesions in vivo ..........130
Figure 3.44 Tyrosine phosphorylation of the β7 subunit in vivo is dependent on the activation
status of cells ...................................................................................................................133
Figure 3.45 The Fib504 mAb specifically immunoprecipitates the β7 subunit from TK-1 cells .......135
Figure 3.46 Src is coimmunoprecipitated with the β7 integrin from TK1 cell lysates .......................136
Figure 3.47 FAK is coimmunoprecipitated with the β7 integrin from TK1 lysates ...........................137
Figure 3.48 FAK and α4β7 do not strongly colocalize on TK-1 cells ................................................138
Figure 3.49 Src appears to colocalize with α4β7 in the uropod..........................................................139
Figure 3.50 Lck and α4β7 do not appear to colocalize on TK-1 cells ................................................140
Figure 3.51 TK-1 cells bind to MAdCAM-1-coated magnetic beads.................................................141
Figure 3.52 α4β7 clusters with src in pseudo-SMAC .........................................................................143
Figure 3.53 α4β7 clusters with FAK in pseudo-SMAC......................................................................144
Figure 3.54 Clusters of integrin β7 and lck.........................................................................................145
Figure 3.55 Cloning strategy for constructing plasmids encoding the full-length β7 subunit with
mutated cytoplasmic tyrosines ........................................................................................147
Figure 3.56 Schematic diagram of the strategy used to generate a pcDNA6-V5his vector
encoding the full-length α4 subunit ................................................................................148
Figure 3.57 Transfection of HEK-293T cells with plasmids encoding the integrin α4 and β7
subunits............................................................................................................................149
Figure 3.58 Western blot analysis of HEK-293T cells transfected with plasmids encoding α4
and β7 varients.................................................................................................................150
Figure 3.59 α4β7 is expressed at the surface of HEK-293T cells transfected with α4 and β7
plasmids...........................................................................................................................152
Figure 3.60 Testing the ability of HEK 293T transfectants to adhere to MAdCAM-1 ......................154
Figure 3.61 SDS-PAGE analysis of proteins precipitated with a synthetic peptide comprising
the complete cytoplasmic domain of the β7 subunit .......................................................156
xii

Figure 3.62 Mass spectrometry identifies four heat shock proteins as potential β7 ligands...............157
Figure 3.63 A synthetic β7 cytoplasmic domain peptide precipitates recombinant hsp70 in a
pull down assay ...............................................................................................................158
Figure 3.64 Hsp70 is coimmunoprecipitated from a TK-1 cell lysate with the β7 subunit ................159
Figure 3.65 The β7 subunit is coimmunoprecipitated from a TK-1 cell lysate with hsp70................160
Figure 3.66 Localisation of hsp70 and α4β7 on TK-1 cells attached and spread on MAdCAM-1....161
Figure 3.67 Localisation of hsp90 and α4β7 on TK-1 cells attached and spread on MAdCAM-1....162
Figure 3.68 Hsp70 colocalizes with intracellular β7 ligands within SMAC formed with
MAdCAM-1-coated microspheres ..................................................................................163
Figure 3.69 Effect of heat shock on the expression of hsp70 and the β7 subunit by TK-1 cells........164
Figure 3.70 Effect of heat shock treatment on the binding of TK-1 cells to MAdCAM-1.................166
Figure 3.71 Fever-range temperatures induce TK-1 cell binding to MAdCAM-1 .............................167
Figure 3.72 The prolonged activation of TK-1 cells by heat shocking is not affected by Mn 2+
treatment..........................................................................................................................168
Figure 3.73 KNK-437 blocks TK-1 cell adhesion to MAdCAM-1 ....................................................169
Figure 3.74 Effect of KNK-437 on the expression of hsp70 and the β7 subunit................................170
Figure 3.75 Effect of serum deprivation on TK-1 cell adhesion to MAdCAM-1...............................171
Figure 3.76 Expression levels of the β7 subunit and hsp70 remain unchanged after serum
depletion ..........................................................................................................................172
Figure 4.1 Pathways which lead to integrin activation. ......................................................................176
Figure 4.2 Proteins which may interact with the β7 cytoplasmic domain ..........................................189
Figure 4.3 The location of zones on migrating TK-1 cells. ................................................................192
xiii

List of Tables
Table 1.1 Gene locations and structures of human integrin subunits......................................................2
Table 1.2 A list of integrins and their known extracellular ligands ......................................................12
Table 1.3 A list of integrins and their known intracellular ligands.......................................................18
Table 1.4 Overview of the proteins/pathways that are involved in FAK-mediated signalling.............36
Table 1.5 Essential features of members of the human Hsp70 family..................................................41
Table 1.6 Phenotypes of Hsp70 knockout mice....................................................................................43
Table 1.7 Ablation of integrin genes in mice and their resulting phenotypes.......................................45
Table 4.1 Summary of the effects of chemical inhibitors on β7-mediated cell adhesion ...................177
Table 4.2 Alignment of the first 20 amino acid residues of the β7 subunit cytoplasmic domain
of various animal species.................................................................................................182
Table 4.3 Alignment of the protein sequences from the distal region of the β7 subunit
cytoplasmic of various animal species ............................................................................184
xiv

Abbreviations
× g g-force
© copyright
°C degree Celsius
µ (prefix) micro-
A ampere
Å Ångströms
aa amino acid
Ab antibody
ABTS 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)
ADMIDAS adjacent to the MIDAS
AF Alexa Fluor®
APC antigen presenting cell
Arg arginine
ATCC American Type Culture Collection
ATP adenosine triphosphate
bp base pair
BSA bovine serum albumin
CARD cell adhesion regulatory domain
CCL CC chemokine ligand
CD cluster of differentiation
cDNA complementary DNA
CO collagen
Corp corporation
CXCL CXC chemokine ligand
DAG diacylglycerol
DEPC diethylpyrocarbonate
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
DTT dithiothreitol
ECM extracellular matrix
EDTA ethylenediaminetetra-acetic acid
eed embryonic ectoderm development
EGF(R) epidermal growth factor (receptor)
EM electron microscopy
ERK extracellular signal-regulated kinases
FA focal adhesion
FAK focal adhesion kinase
FITC fluorescein isothiocyanate
FLN filamin
fMLP formyl-methionyl-leucyl-phenylalanine
FN fibronectin
FRET Förster resonance energy transfer
g gram
GALT gut associated lymphoid tissue
GAPDH glyceraldehyde-3-phosphate dehydrogenase
HIMEC human intestinal microvascular endothelial cells
HIV human immunodeficiency virus
hr(s) hour(s)
Hsp heat shock protein
IBD inflammatory bowel disease
xv

ICAM intercellular cell adhesion molecule
IL interleukin
ILK integrin-linked kinase
IPTG isopropyl-β-D-thiogalactopyranoside
IS Immunological synapse
JNK c-Jun NH2-terminal kinase
kb kilobase
kDa kilodalton
L liter
LB Luria-Bertani
LFA-1 lymphocyte function-associated antigen 1
LIMBS ligand-induced metal ion-binding site
LN laminin
LPAM lymphocyte Peyer’s patch adhesion molecule
LPL lamina propria lymphocytes
m meters
M molar
m (prefix) milli-
mAb monoclonal antibody
MAdCAM mucosal addressin cell adhesion molecule
MAPK mitogen-activated protein kinase
MIDAS metal ion-dependent adhesive site
min minute
MLCK myosin light chain kinase
mRNA messenger RNA
n (prefix) nano-
NK natural killer
NMR nuclear magnetic resonance
OD optical density
p p-value
PBM perivascular basement membrane
PCR polymerase chain reaction
PFA paraformaldehyde
PIP2 phosphatidylinositol 4,5-biphosphate
PKA protein kinase A
PKC protein kinase C
PMA phorbol 12-myristate 13-acetate
PP Peyer’s patches
PSI plexin-semaphorin integrin
PTB phosphotyrosine binding
R9 polymer of 9 arginines
RGD arginine-glycine-aspartic acid
RIAM Rap1-GTP interacting adapter molecule
rpm revolutions per minute
RTK receptor tyrosine kinase
s second
SDS sodium dodecyl sulphate
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
SFK src family of kinases
SH src homology
SIEL small intestine intraepithelial lymphocytes
siRNA small interference RNA
SL spleen lymphocytes
SMAC supramolecular activation complex
xvi

TCR T-cell receptor
Tfb transformation buffer
TGF transforming growth factor
TIED ten β-integrin EGF-like repeat domains
TNF tumor necrosis factor
V volts
VCAM vascular cell adhesion molecule
vWFA von Willebrand Factor type A
wt wild-type (native form)
www world wide web
β-TD β-terminal domain
xvii

Chapter 1. Introduction
1.1. Integrins
Integrins are a family of cell adhesion molecules that mediate cell-cell, cell-extracellular
matrix (ECM) and cell-pathogen interactions (reviewed in Hynes 2002; Krissansen et al.
2007). They are widely distributed with diverse functions, and play a central role in cell
adhesion and migration. Integrins play vital physiological roles during the lifespan of an
organism, from embryogenesis and organogenesis to maintaining haemostasis by mediating
cell proliferation, survival, the immune response, and wound healing (reviewed in Hynes
2002; Krissansen et al. 2007).
Integrins have been found to exist in metazoan organisms, from the simplest metazoan
sponges to the most complex eukaryotes (reviewed in Hynes 2002). No homologues have
been detected in prokaryotes, plants or fungi (Whittaker et al. 2002). Integrins are formed
from two non-covalently associated type I transmembrane glycoprotein subunits, named the
α- and β-subunits. The α- and β- subunits are totally distinct with no detectable homology
between them, and are encoded by two separate supergene families located on various
chromosomes (Table 1.1; reviewed in Lam and Loftus 2005, Krissansen and Danen 2007,
Takada et al. 2007).
In addition to their adhesive functions, integrins are dynamic cell-surface receptors for cell
signalling that can propagate signals across the plasma membrane in both directions, termed
“inside-out” and “outside-in” signalling (reviewed in Hynes 2002). Inside-out signalling
refers to the transmission of signals from within a cell to the outside extracellular
environment, altering the affinity of integrins for their ligands. Conversely, outside-in
signalling is the transmission of signals from outside of the cell by integrin-ligand interactions
leading to changes inside the cell. Signals delivered from inside and outside of the cell
impinge on the integrin cytoplasmic regions, which serve as important regulators of integrin
signalling and function (reviewed in Hynes 2002; Krissansen et al. 2007).
1

Table 1.1 Gene locations and structures of human integrin subunits
Integrin subunit Synonym Gene location
α1 CD49a 5q11.2
α2 CD49b 5q23–q31
α3 CD49c 17q21.33
α4 CD49d 2q31.3
α5 CD49e 12q11–q13
α6 CD49f 2q31.1
α7 - 12q13
α8 - 10p.13
α9 - 3p21.3
α10 - 1q21
α11 - 15q23
αIIb CD41b, GPIIb 17q21.32
αv CD51 2q31–q32
αE CD103 17q13
αL CD11a 16p11.2
αM CD11b, Mac-1 16p11.2
αX CD11c 16p11.2
αD - 16p11.2
β1 CD29 10p11.2
β2 CD18 21q22.3
β3 CD61 17q21–q23
β4 CD104 17q25
β5 - 3q21.2
β6 - 2q24–q31
β7 - 12q13.13
β8 - 7p15.3
(Adapted from Lam et al. 2005; Krissansen et al. 2007; Takada et al. 2007)
Integrin-ligand binding is regulated by changes in integrin affinity and avidity, which allows
cells to change shape and migrate (reviewed in Hynes 2002). It involves integrin diffusion
and clustering within the plasma membrane, which ultimately affects cytoskeletal connections
with the extracellular milieu. Extracellular interactions involving integrins include cell-matrix
adhesion where integrins bind to ECM proteins (Geiger et al. 2001), cell-cell adhesion which
leads to “immunological synapse” formation in the case of immune cells (Sims et al. 2002),
transendothelial migration of leukocytes which is important for leukocyte infiltration of
inflamed tissues (Barreiro et al. 2007), and cell-pathogen adhesions (Forsyth et al. 1998).
Disruption of integrin function and signalling contributes to a wide variety of diseases
including, cancer, inflammatory disease, thrombosis and infection (reviewed in Hynes 2002;
Krissansen et al. 2007). In order to understand integrin involvement and contribution to
2

various diseases, it is necessary to understand integrin signalling pathways to elucidate the
control of integrin function. Of particular interest are the integrin cytoplasmic domains, and
their binding/interacting partners which regulate integrin function.
1.1.1 Integrin structures
There are 18 different integrin α chains and 8 different β chains (Table 1.1) which associate
non-randomly in a divalent cation-dependent fashion forming at least 24 different αβ hetero-
dimeric integrin molecules, each with discrete receptor binding functions (Figure 1.1;
reviewed in Hynes 2002). Alternative splicing of certain integrin subunits gives rise to even
greater numbers of different integrin structures and parings, with the different alternatively
spliced forms having divergent functional attributes (reviewed in Hynes 2002). Integrins can
be broadly grouped by ligand specificity into laminin (LN)-binding, collagen (CO)-binding,
and Arginine-Glycine-Aspartic acid (RGD)-binding integrins, and leukocyte-specific
receptors (Figure 1.1; reviewed in Hynes 2002). They can be further defined on the basis that
some integrin α-subunits have an inserted domain called the I-domain (reviewed in
Krissansen et al. 2007).
Figure 1.1 Integrin subunits and pairings.
A diagram depicting the pairing of different integrin α and β subunits. A heterodimer is formed by the
association of particular α and β subunits as denoted by the lines connecting the α and β subunits. Integrins are
broadly grouped into those that bind laminin (red), collagen (purple), and the RGD motif (blue), or by restriction
to leukocytes (yellow). (Adapted from Hynes 2002; Krissansen and Danen 2007).
3

Integrins, being type-1 transmembrane proteins, contain three domains; namely an N-terminal
extracellular domain, a single transmembrane domain, and a C-terminal cytoplasmic domain
(Figure 1.2).
Figure 1.2 Structure of the integrin heterodimer
A cartoon image of an integrin heterodimer, depicting the different domains present in the integrin α and β
subunits. (Adapted from Luo et al. 2007)
Extracellular domain: The extracellular domain comprises two separate regions that form a
large globular head and a long stalk region (reviewed in Luo et al. 2007). The globular head
of the α and β subunits contains multiple domains and mediates ligand binding. The stalk is
also comprised of multiple domains and is important for integrin activation. Details of these
domains will be discussed below.
Transmembrane domain: The single transmembrane segment connects the extracellular and
cytoplasmic domains. It is thought to mediate the transmission of signals between the
cytoplasmic and extracellular domains arising from conformational changes in either domain
(reviewed in Luo et al. 2007).
Cytoplasmic domain: Integrin cytoplasmic domains are typically short (13-70 amino acids,
aa), with the exception of the cytoplasmic domain of the β4 subunit (1072 aa; (Hogervorst et
al. 1990). They mediate integrin-cytoskeletal interactions and signal transmission, and play a
central role in integrin activation (reviewed in Krissansen and Danen 2007). Overexpression
of particular proteins that bind to the cytoplasmic domains can result in integrin activation
(Vinogradova et al. 2002).
4

Integrin α-subunits
The 18 different α-subunits share 18 to 63% aa identity and are up to 1104 aa residues in
length, with a mature size of 120-210 kDa (reviewed in Krissansen et al. 2007). As mentioned
above, each integrin subunit contains an extracellular domain, a transmembrane domain, and
a cytoplasmic domain. The extracellular domain is comprised of five distinctive domains
based on their structure, refered to as the β-propeller, I-domain, thigh domain, Genu domain,
and the calf domains (Figure 1.3; reviewed in Luo et al. 2007)
Figure 1.3 Domains of the integrin α-subunit
A cartoon depiction of the integrin α-subunit, showing the different domains in a linear form. Indicated are the
β-propeller domain, I domain, thigh domain, genu region, calf domains, transmembrane domain, and the
cytoplasmic domain. (Adapted from Luo et al. 2007).
The β-propeller domain: The N-terminal half of the α subunit contains a globular head
termed the β-propeller domain, which is made up of seven β-sheet repeats (~60 aa each)
formed by four anti-parallel β-strands, folded around a pseudo-symmetric axis similar to the
structure of the heterotrimeric G-protein β-subunit (Springer 1997; Xiong et al. 2001; Xiao et
al. 2004). In addition, there are four Ca 2+ binding motifs in the propeller β-sheet repeats 4-7.
In integrins which lack the I-domain (described below), the β-propeller domain appears to
directly participate in ligand binding (Humphries 2000). In contrast, the β-propeller domain
may cooperate in ligand binding (Yalamanchili et al. 2000) or play no role in integrins which
possess the I-domain (Shimaoka et al. 2001).
5

The I-domain: Almost half of integrin α-subunits contain an I (insertion or interaction)
domain [also referred to as an αA, αI or von Willebrand Factor type A (vWFA) domain]
(Humphries 2000). The subunits which possess an I-domain include α1, α2, α10, α11, αM,
αL, αD, αX, and αE. The I-domain is an inserted sequence of ~190 aa residues comprising
three EF-like motifs at the top of the β-propeller, between the second and third β-sheet
repeats. The I-domains fold independently of the β-propeller; and adopt the dinucleotide-
binding or Rossmann fold, with seven α-helices surrounding six hydrophobic β-strands
arranged as a central β-sheet (Lee et al. 1995). At the top of the β-sheet is a metal-ion-
dependent adhesive site (MIDAS) motif. Within the I-domain are two conserved serines, two
aspartic acid residues, and a threonine, which coordinate a Mg 2+ cation to an acidic residue in
the ligand. Divalent cations are known to be universally required for ligand-binding by
integrins, and the MIDAS motif is important for ligand binding. The I-domain exists in either
an open (high affinity) or a closed (low affinity) conformation (Humphries 2000). For non-I-
domain integrin α-subunits (α3, α4, α5, α6, α7, α8, α9, αV, and αIIb) the third β-turn of the β-
propeller is directly involved in ligand binding (Humphries 2000).
Stalk region: The stalk region is formed from three β-sandwich domains, and links the
globular head and transmembrane domain (Xiong et al. 2001). The upper part of the stalk
region is designated the thigh domain and the lower part the calf-1 and calf-2 domains (Xiong
et al. 2001). A small Ca 2+ -binding loop is located between the thigh and calf-1 domains,
named the genu domain (Xiong et al. 2001). The genu domain is the key pivot point for the
switchblade extension of the α subunit (described below).
Transmembrane domain: The α-subunit transmembrane domain, formed from single-pass α-
helices, anchors the subunit to the plasma membrane. Its association with the β-subunit
transmembrane domain is important in integrin activation (Lau et al. 2008a; Lau et al. 2008b).
Cytoplasmic domain: Integrin α-subunit cytoplasmic domains are short sequences of 15-58 aa
residues. They are highly divergent, except for a membrane proximal GFF(R/K)R motif,
which appears essential for β-subunit association and stability (described below; reviewed in
Krissansen et al. 2007).
6

Integrin β-subunits
The 8 different integrin β-subunits share 28 to 55% aa identity and range in size up to 778 aa
residues in length, with a mature size of 90-130 kDa (reviewed in Krissansen et al. 2007). The
extracellular region contains a globular head and a stalk region. The globular head contains
three highly conserved domains, the I-like domain, the PSI domain, and the hybrid domain
(Luo et al. 2007). The stalk region consists of cysteine-rich integrin epidermal growth factor
(EGF)-like domains (I-EGF), and a C-terminal β-tail domain (Figure 1.4).
Figure 1.4 Domains of the integrin β-subunit
A cartoon picture depicting the integrin β-subunit and the different domains in a linear form. Indicated are the
PSI domain, hybrid domain, I-like-domain (β I-domain), I-EGF domains 1-4, β-tail, transmembrane domain, and
the cytoplasmic domain. (Adapted from Luo et al. 2007).
The PSI domain: The extreme N-terminal region of integrin β-subunits termed the plexinsemaphorin-integrin
(PSI) domain is rich in cysteine residues (Zang et al. 2001). It shares
sequence homology with the cysteine-rich membrane proteins plexins and semaphorins
(Figure 1.4; Zang et al. 2001). The PSI region contains seven cysteines which cooperate to
restrain the integrin in an inactive conformation (Zang et al. 2001).
I-like domain: The N-terminal region of integrin β-subunits contains a highly conserved
domain located between aa residues 100-340, termed the I-like-domain (β I-domain; Figure
1.4; Krissansen et al. 2007). The I-like-domain contains a conserved metal-binding DXSXS
sequence (where X denotes any amino acid other than cysteine) which resembles the MIDAS
motif of integrin α-subunits (Lee et al. 1995). Distinguishing features of I-like-domain
regions are the presence of additional cation binding sites, termed adjacent to the MIDAS
7

domain (ADMIDAS), and the ligand-induced metal ion-binding site(s) (LIMBS; Xiong et al.
2001). The I-like-domain of the integrin β-subunit is physically associated with the α-subunit
β-propeller domain. The β-subunit I-like-domain of integrins which lack the α subunit I-
domain appears to bind ligands directly. It indirectly regulates ligand-binding in integrins that
contain an α-subunit I-domain (Lu et al. 2001b). Several non-conserved residues within this
region determine ligand specificity.
Mutational studies have shown that the LIMBS functions as a positive regulatory site, and the
ADMIDAS as a negative regulatory site (Chen et al. 2003; Mould et al. 2003a; Chen et al.
2006a). The addition of Mn 2+ or removal of Ca 2+ can increase ligand-binding affinity and
adhesiveness of almost all integrins (Shimaoka et al. 2002). In most integrins, Ca 2+ can have
both positive and negative regulatory effects, in that high concentrations of Ca 2+ inhibit
adhesion, whereas low concentrations of Ca 2+ synergize with suboptimal Mg 2+ concentrations
to support adhesion (Shimaoka et al. 2002). The LIMBS mediates the synergistic effects of
low Ca 2+ concentrations, whereas the ADMIDAS mediates the negative regulatory effects of
higher Ca 2+ concentrations, which are competed by Mn 2+ (Shimaoka et al. 2002).
The hybrid domain: An Ig-like hybrid domain is composed of sequences from either side of
the I-like-domain (Figure 1.4; Luo et al. 2007). It forms a β-sandwich structure looped out
between the I-like domain and the PSI domain. The hybrid domain plays a central role in
conformational activation of integrins, as described in more detail below (Luo et al. 2007).
The stalk region: The stalk region contains four internally disulfide-bonded repeats of a
cysteine-rich motif located between aa residues 340-700, termed I-EGF1-4 (Figure 1.4; Luo
et al. 2007). The cysteine-rich EGF-like repeats are reminiscent of the EGF repeats of the
protein Ten β integrin EGF-like repeat domains (TIED) protein (Berg et al. 1999a). No
specific functions have been attributed to the stalk region apart from the fact that it may
facilitate ligand binding by ensuring the globular head extends beyond the glycocalyx of a
cell (Kim et al. 2003a; Calderwood 2004).
Transmembrane domain: Like the integrin α-subunit mentioned above, the integrin β-subunit
transmembrane domain contains single-pass transmembrane α-helices which anchor the
integrin β-subunit to the plasma membrane. Its interaction with the α-subunit is thought to be
important in integrin activation (described below; Lau et al. 2008a; Lau et al. 2008b).
8

Cytoplasmic domain: Integrin β-subunit cytoplasmic domains are typically short sequences of
47-66 aa residues, with the exception of the β4 integrin subunit (1018 aa residues). The β4
integrin cytoplasmic domain contains two pairs of C-terminal fibronectin (FN) type III
repeats for binding to the keratin cytoskeleton (de Pereda et al. 1999). Most integrin β subunit
cytoplasmic domains contain one or two NPxY motifs (where x is any amino acid), which
upon tyrosine phosphorylation are phosphotyrosine-binding (PTB) domains that interact with
a wide variety of signalling and cytoskeletal proteins. The integrin β subunit cytoplasmic
domain recruits several signalling and cytoskeletal proteins, which are discussed in detail
below.
The integrin αβ heterodimer:
Integrins appear to assume multiple different conformations (Ginsberg et al. 2005), in
particular bent and extended conformations as evidenced by crystallography, nuclear
magnetic resonance (NMR), electromicroscopy (EM) and Förster resonance energy transfer
(FRET) studies (Figure 1.5).
Figure 1.5 Structure of the αβ integrin heterodimer
A cartoon representation of the integrin αβ heterodimer in an inactive state on the left and the active
confirmation on the right. The inactive bent conformation is a schematic representation of the resolved crystal
structure of αVβ3 (Xiong et al. 2001). The active extended conformation has been drawn to depict the different
integrin subdomains. (Figure adapted from Wegener et al. 2007).
9

Crystallography uncovered the structures of the large extracellular portions of the integrin
heterodimers αvβ3 (Xiong et al. 2001; Xiong et al. 2002) and αIIbβ3 (Xiao et al. 2004). αVβ3
was found to exist in a bent formation, in which the N-terminus was folded back towards the
cell membrane (Xiong et al. 2001). A sharp bend was located between the thigh and calf-1
domains within the genu domain of the αV subunit, and approximately between EGF domains
1 and 2 of the β3 subunit. It was proposed that this bent conformation represented a low-
affinity state, while the extended conformation was a high-affinity state (Jin et al. 2004;
Nishida et al. 2006). However, the bent form had also been found to bind ligands with high
affinity, as EM images show bent αVβ3 in complex with a fragment of fibronectin (Adair et
al. 2005) and crystalised αVβ3 integrin in the bent conformation can bind to cyclic RGDsequence-containing
peptide (Xiong et al. 2002). In addition, some integrins are thought to
exist in an intermediate-affinity state (Chigaev et al. 2001).
The membrane proximal regions of the α and β integrin subunit cytoplasmic domains in the
low-affinity state are believed to be stabilised by a salt bridge between a conserved arginine
in the α-subunit cytoplasmic domain, and an aspartic acid in the β-subunit cytoplasmic
domain, and by hydrophobic contacts (Arnaout et al. 2005). Mutational analysis and
computational modelling suggest the transmembrane domain stabilizes the inactive state, such
that disruption of the transmembrane domain leads to integrin activation (Luo et al. 2005;
Partridge et al. 2005). Several models of integrin activation propose orientational changes in
the stalk region, however recent studies support a model which involves separation of the
transmembrane stalk region (Kim et al. 2003a; Calderwood 2004), as seen in Figure 1.5.
Interactions of the membrane-proximal regions of the α and β subunit cytoplasmic domains
are believed to regulate integrin activation by stabilising integrins in low affinity states,
whereas separation of the cytoplasmic domains and their stabilisation with cytoskeletal
proteins is thought to cause integrin activation, as discussed further below (Luo et al. 2007).
The three-dimensional structure of the integrin αIIb and β3 transmembrane segments of
activated αIIbβ3 was recently resolved. The αβ extracellular domains were shown to connect
to the transmembrane domains at different crossing angles (Lau et al. 2008a; Lau et al.
2008b).
10

1.1.2 Integrins and their extracellular ligands
Integrins and their extracellular ligands are listed in Table 1.2. Of note, several integrins bind
to many ligands via the tripeptide RGD motif, which is common to many ECM proteins
(Elices et al. 1991). Hence, the complexity of integrin-ligand recognition can be partially
attributed to the ability of certain integrins to interact with multiple ligands. Other integrins
such as αEβ7 have only one identified ligand so far, namely E-cadherin in the case of αEβ7
(reviewed in Krissansen et al. 2007). Several integrins bind in common to the same ligand.
For example, the matrix proteins FN, LN, and CO; and the immunoglobulin superfamily cell
adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell
adhesion molecule-1 (ICAM-1) are each recognized by more than one integrin (reviewed in
Krissansen et al. 2007). Integrins are either widely distributed or restricted to certain cell
types, hence the environment in which they reside and need to function may account for the
various ligand specificities.
11

Table 1.2 A list of integrins and their known extracellular ligands
Integrin Ligands
Laminin receptors α3β1 LN; TSP; CO; FN; epiligrin; α3 80kDa fragment; fibrillin-2; MMP-1;
ADAMs
α6β1 LN; CO; ADAMs; epiligrin; midkine
α7β1 LN, ADAMs
α6β4 LN
Collagen receptors α1β1 CO; LN
α2β1 CO; LN; MMP-1; chondroadherin
α10β1 CO
α11β1 CO
RGD receptors α5β1 FN; FG; ADAMs
α8β1 FN; NN; TN; LAP; OP; VN; POEM; MAEG; QBRICK
αvβ1 FN; VN; FG; OP; LAP; fibrillin-2, agrin; L1-CAM
αvβ3 FN; VN; FG; VWF; OP; TN; BSP; TSP; LAP; CD31; ADAMs; MMP-2;
uPA; uPAR; ICAM-4; CD31; fibrillin-1; osteoadherin; L1-CAM;
developmental endothelial locus-1; agrin; lactadherin; metargidin; MMP-2;
fibrin; Cyr61; Fisp12; microfibril-associated glycoprotein2; tropoelastin
αIIbβ3 FN; VN; FG; OP; CO; TSP; VWF; CD40L; prothrombin; ICAM-4; VWF;
L1-CAM; factor H
αvβ5 VN; BSP; OP; LAP; FN; CO; lactadherin; sialoprotein; PAS-6/7, betaig-h3;
ADAMs; MFG-E8, P2Y2 receptors
αvβ6 FN; VN; TN; LAP
αvβ8 FN; VN; CO; LN; LAP; fibrin
α4/α9 receptors α4β1 FN; VCAM-1; MAdCAM-1; OP; FG; TSP; uPA; VWF; JAM-2; annexin;
coagulation factor XIII, transglutaminase; ADAM28; ADAMs
α9β1 TN; FN; OP; LN; CO; VWF; VCAM-1; ADAMs; uPA; transglutaminase,
coagulation factor XIII; VEGF-C and VEGF-D
Leukocyte-specific α4β7
integrins
FN; MAdCAM-1; VCAM-1; ADAMs
αEβ7 E-cadherin
αDβ2 ICAM-3; VCAM-1; FN; FG; VN; plasminogen; Cyr61
αLβ2 ICAM1-5, CO; JAM-1
αMβ2 ICAM-1; VCAM-1; FG; FN; VN; LN; CO; iC3b; factor X, galectin-3,
complement factor H, CD23, ICAM-4; neutrophil inhibitory factor; Cyr61;
GP1bα; serum factor x; JAM-3; Thy-1; RAGE; lipoprotein a; DC-SIGN;
oligodeoxynucleotides; heparin; elastase; plasminogen; MMPs; β-glucans;
high molecular weight kininogen; myeloperoxidase; azurocidin; haptoglobin;
denatured proteins; lipopolysaccharides; pertussis toxin
αXβ2 FG; ICAM-1; CO; iC3b; CD23, lipopolysaccharides, RAGE; Thy-1
Notes: ADAM, a disintegrin and metalloprotease; BSP, bone sialic protein; CO, collagen(s); FG, fibrinogen; FN,
fibronectin; iC3b, inactivated complement component 3b; ICAM, intercellular adhesion molecule; LAP, TGFβ latency
associated protein; LN, laminin(s); MAdCAM-1, mucosal addressin cell adhesion molecule-1; MMP, matrix
metalloprotease; NN, nephronectin; OP, osteopontin; RAGE, receptor for advanced glycation end-products; TG, tiggrin;
TN, tenascin; TSP, thrombospondin; VCAM-1, vascular cell adhesion molecule-1; VN, vitronectin; VWF, von Willebrand
factor. (Adapted from Krissansen et al. 2007)
12

1.2. β7 integrins
The integrin β7 (βp) subunit is made up of 798 aa with a predicted mass of 87 kDa (Yuan et
al. 1990). When resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) it has a reduced size of 105-110 kDa (Parker et al. 1992). The gene is located at
12q13.13, and is 10 kb in length with 14 exons (Table 1.1; Baker et al. 1992).
Complementary DNA (cDNA) encoding the human β7 subunit was first isolated from T
lymphocytes (Yuan et al. 1992). The β7 subunit forms two integrins by heterodimerisation
with the integrin α4 and αE subunits, forming α4β7 and αEβ7 respectively (reviewed by
Krissansen et al. 2007). The integrin α4β7 is expressed by mucosal lymphocytes, natural
killer (NK) cells, and eosinophils (Holzmann et al. 1989), while αEβ7 is highly expressed by
intraepithelial lymphocytes, and only a small minority of peripheral blood lymphocytes (Cerf-
Bensussan et al. 1987).
1.2.1 The β7 subunit cytoplasmic domain
The integrin β7 subunit cytoplasmic domain is 52 aa in length (Figure 1.6; Yuan et al. 1992).
It contains a conserved 2-LLv-iHDR-9 motif and an NPxY motif at positions 29-32.
1 11 21 31 41 51
| | | | | |
β7 KLSVEIYDRR EYSRFEKEQQ QLNWKQDSNP LYKSAITTTI NPRFQEADSP TL
Figure 1.6 The amino acid sequence of the cytoplasmic domain of the human β7 subunit.
The amino acid sequence of the integrin β7 subunit cytoplasmic domain with key residues indicated. In bold
letters are potentially phosphorylatable tyrosine/serine/threonine aa residues, and underlined is the conserved
NPxY and NPRF motifs. The conserved LLv-iHDR motif is also present from residues 2-9. Highlighted in blue
is the β7 cell adhesion regulatory domain.
It also contains several potentially phosphorylatable serine, threonine and tyrosine residues as
indicated in Figure 1.6. The β7 subunit cytoplasmic domain binds filamin more tightly than
does the β1 cytoplasmic domain (Pfaff et al. 1998). Increased filamin (FLN) binding was
found to restrict integrin-dependent cell migration by inhibiting transient membrane
protrusion and cell polarization (Calderwood et al. 2001). The filamin binding site in the β7
cytoplasmic domain was mapped to residues 35-40, which are necessary for high affinity
13

inding of FLNa (one of the two most widely expressed members of the filamin family). The
amino acid isoleucine at positions 36 and 40 was found to be critical for filamin binding
(Calderwood et al. 2001). Filamin binding supported low levels of cell migration of β7
integrins, in contrast, filamin binding to β1 integrins was weaker with higher levels of cell
migration (Calderwood et al. 2001). Splice variants of filamin lacking a 41 aa segment
interacted more strongly with the β7 tail than did full-length filamin, suggesting alternative
splicing of filamin may negatively regulate cell migration (Travis et al. 2004).
The β7 cytoplasmic domain contains the NPxY motif that is important for integrin activation
and signalling by talin and filamin (Pfaff et al. 1998). An additional motif “YDRREY”
uniquely present in the β7 cytoplasmic domain (see Figure 1.6) was identified as a cell
adhesion regulatory domain (CARD) of the β7 subunit (Krissansen et al. 2006b). A peptide
containing the YDRREY motif was found to specifically block the binding of α4β7 to
MAdCAM-1.
Various PTB domain-containing proteins including Dok1, Numb, Dab1, Dab2, and tensin
were shown to associate with the β7 cytoplasmic domain (Calderwood et al. 2003). The
human WD repeat protein WAIT-1 which is homologous to embryonic ectoderm
development (eed; a member of the superfamily of polycomb group proteins), was found to
specifically bind to the cytoplasmic domain of the β7 subunit but not to the β1 or β2
cytoplasmic domains (Rietzler et al. 1998). However, its functional significance is not fully
known. In studies of the human immunodeficiency virus (HIV) nef protein it was found that
nef recruited eed (a repressor of transcription) from the nucleus to the plasma membrane,
enabling transcriptional derepression (Witte et al. 2004). Similarly, activated integrin
receptors recruited eed from the nucleus, suggesting a link between membrane-associated
activation and transcriptional derepression (Witte et al. 2004).
1.2.2 Integrin α4β7
α4β7 (lymphocyte Peyer’s patch adhesion molecule-1, or LPAM-1) preferentially binds to the
mucosal addressin cell adhesion molecule-1 (MAdCAM-1; Holzmann et al. 1989). It
mediates the binding of lymphocytes to high endothelial venules of Peyer’s patches (PPs) in
the small intestine, but not to peripheral lymph nodes (Holzmann et al. 1989). It is important
in the homing of lymphocytes specifically to intestinal sites where MAdCAM-1 is expressed
on the endothelial cells of PPs, mesenteric lymph nodes, and capillaries of the lamina propria
14

(Berlin et al. 1993; Briskin et al. 1993). Additionally, α4β7 mediates the adhesion of T-
lymphocytes within the lamina propria to microvessels at the tips of the intestinal villi, but
not to the postcapillary venules of the PPs (Berlin et al. 1993; Briskin et al. 1993). α4β7 binds
to the first Ig-domain of MAdCAM-1 with assistance from the second Ig domain. It also
binds to RGD-containing peptides, VCAM-1, and the CS-1 fragment of fibronectin (Chan et
al. 1992; Ruegg et al. 1992).
α4β7 plays an essential role in lymphocyte extravasation across the endothelium (Hogg et al.
2003). After selectin-mediated tethering and fast rolling of lymphocytes on the vessel wall,
the integrin α4β7 mediates slow rolling and firm attachment of lymphocytes in conjunction
with the β2 integrins prior to transendothelial migration (refer to Figure 1.7). α4β7 is also
critical for the transmigration of mast cell progenitors into the small intestine, but not the
lungs (Gurish et al. 2001); haematopoietic progenitor cell homing to bone marrow (Masellis-
Smith et al. 1997); and the migration of eosinophils to the intestine (Brandt et al. 2006).
Figure 1.7 Multistep model of T cell transmigration
A cartoon representation of the multistep model of T cell migration across the endothelium (above), and a table
showing the adhesion molecules involved in each step of the transmigration process (below). (Figure adapted
from Hogg et al. 2003).
15

Chemokines can activate α4β7 to adhere and migrate on MAdCAM-1. The immobilised CC
chemokine ligands (CCL), 21, 25, 28, and CXC-chemokine ligand (CXCL) 12 all converted
the rolling adhesion of human lymphocytes on MAdCAM-1 to static arrest (Hogg et al.
2003). However, only CCL21 and CXCL12 also triggered a motile phenotype characterized
by lamellipodia and uropod formation. α4β1-VCAM-1 and α4β7-MAdCAM-1 interactions
were reported to operate independently to support lymphocyte adhesion under flow (Hogg et
al. 2003). Different chemokines may act in concert in triggering integrin-mediated arrest and
promoting motility and transendothelial migration (Miles et al. 2008). In addition to
activation by chemokines, α4β7 is also regulated by transforming growth factor (TGF)-β1
(Lim et al. 1998; Bartolome et al. 2003), as well as the chemoattractants interleukin (IL)-8
and formyl-methionyl-leucyl-phenylalanine (fMLP; Sadhu et al. 1998).
α4β7 is critical for the homing of lymphocytes to the gut and mucosal sites which are
normally chronically inflamed (Butcher et al. 1999). The formation of the gut-associated
lymphoid tissue (GALT) is severely impaired in β7 gene knockout mice due to the failure of
the β7 -/- lymphocytes to adhere to blood vessel walls at the site of transmigration into the
GALT (Wagner et al. 1996). β7-deficient mice also show aberrant distribution of mast cells in
the small intestine, and are more susceptible to Cryptosporidium parvum at the early stages of
infection (Mancassola et al. 2004).
1.2.3 Integrin αEβ7
The second α subunit that associates with the β7 subunit is the αE subunit (CD103), which
was discovered in the late 1980’s (Cerf-Bensussan et al. 1987). It forms the αEβ7 integrin
(HML-1, αIEL or M290 in mice) whose expression is restricted almost exclusively to T cells
and dendritic antigen presenting cells (APC) in the mucosal immune system (Kilshaw 1999).
It is particularly prominent on intraepithelial lymphocytes in the gut (Kilshaw 1999). The
distribution of αEβ7 is largely attributed to the restricted expression of the αE subunit,
because the β7 subunit in association with α4 is widely distributed on T and B cells, mast
cells, activated macrophages, and eosinophils (Kilshaw 1999). In inflammatory diseases, αE
is detectable on T cells in the rheumatoid joint (Baumgart et al. 1996), CD8 T cells
infiltrating the kidney tubular epithelium during acute rejection of renal allografts (Yuan et al.
2005), and in lachrymal glands in Sjogren’s syndrome (Fujihara et al. 1999).
16

αEβ7 null mice show a reduction in the numbers of mucosal T cells and display T cell-
dependent dermatitis of unknown etiology (Lefrancois et al. 1999; Schon et al. 1999).
Interestingly αEβ7 null mice transplanted with pancreatic islet cell allografts did not reject the
implants, suggesting impaired systemic immunity (Feng et al. 2002).
The αE subunit uniquely contains an X-domain N-terminal to the I-domain (Shaw et al.
1994), which is important for ligand binding. The only ligand for αEβ7 discovered so far is E-
cadherin, which is an epithelial homophilic adhesion molecule (Kilshaw 1999). However,
αEβ7-transfected K562 cells were found to bind to human intestinal microvascular
endothelial cells (HIMEC) independently of E-cadherin, suggesting a role for αEβ7 in
lymphocyte homing or interaction with the intestinal endothelium, mediated by a ligand that
is yet to be identified (Strauch et al. 2001).
Expression of αEβ7 is induced by TGF-β, which is prominent in all tissue microenvironments
in which αE-expressing cells are found (Kilshaw et al. 1990). TGF-β caused a rapid increase
in the expression of αE messenger RNA (mRNA) in mouse T lymphoma cells (Robinson et
al. 2001). TNFβ was found to differentially regulate αEβ7 in mouse intestinal lymphocytes
(Ni et al. 1995). TNFβ was found to have no effect on αEβ7 expression in small intestine
intraepithelial lymphocytes (SIEL), partially downregulated αEβ7 expression in lamina
propria lymphocytes (LPL) and strongly upregulated in spleen lymphocytes (SL). Thus,
TNFβ regulated expression of αEβ7 depends on the microenvironments in which cells reside,
where the lymphocytes may play different roles (Ni et al. 1995).
1.3. Integrin signalling
Extracellular ligand-binding generates intracellular signals (outside-in signalling), and
conversely ligand-binding is regulated by signals from within the cell (inside-out signaling;
reviewed in Krissansen et al. 2007). Hence, integrins serve as a transmembrane bridge
between the extracellular milieu and actin microfilaments of the cytoskeleton. The short
integrin cytoplasmic tails lack intrinsic enzymatic activity, therefore there is the necessity for
the recruitment of adaptor molecules and signalling proteins for effector function and integrin
regulation (reviewed in Krissansen et al. 2007). The integrin cytoplasmic domain therefore
plays a crucial role in signalling, as it serves as the interface through which many signalling
and cytoskeletal molecules exert their actions. A list of the many intracellular ligands
identified for integrins is given in Table 1.3.
17

Table 1.3 A list of integrins and their known intracellular ligands
Integrin tail
α Calreticulin, caveolin, GTP-bound G(h)/TG2, ancient ubiquitous protein 1 (Aup1)
α1 Paxillin, talin, α-actinin, FAK, actin
α2 F-actin
α3 DRAL/FHL2, nucleotide exchange factor Mss4, putative tumour suppressor protein BIN1
α4 Paxillin
α5 Tap-20, TIP-2/GIPC, nischarin
α6 TIP-2/GIPC
α7 DRAL/FHL2
α8 ?
α9 Paxillin, spermidine/spermine N (1)-acetyltransferase (SSAT)
α10, α11 ?
αv ?
αL Rap1?
αM, αX, αD ?
αIIb Talin, CIB, ICln
β Myosin-X
β1 Talin, filamin, α-actinin, paxillin, ILK, FAK, skelemin, melusin, MIBP, ICAP-1, CD98
Dab1, X11a, EPS8, EB-1, GAPCenA, tensin, merlin, phospholipase Cγ (PLCγ), Nckinteracting
kinase (NIK), 14-3-3β, Kindlin-2
β2 Talin, filamin, α-actinin, FAK, cytohesin-1, cytohesin-3, Rack-1, JAB1, Dab1, Dok-1,
ILK Rack-1,
β3 Talin, filamin, paxillin, FAK, GRb2, Shc, ERK1, myosin, skelemin, β3-endonexin,
CD98, Pyk2, Numb, Dab1, Dab2, X11a, X11g, Lin10, JIP, EPS8, RGS12, CED6, EB1,
GAPCenA, tensin, IRS-1, Dok-1, ILK, Syk, ZAP-70, Kindlin-2
β4 F-actin, plectin, p27(BBP), ERBIN, 12-lipoxygenase
β5 Talin, Rack-1, p21-activated kinase 1, Numb, Dab1, Dab2, EPS8, tensin, Dok-1, annexin-
V
β6 ERK-2
β7 Talin, filamin, WAIT-1, Shc, Numb, Dab1, Dab2, X11a, X11g, JIP, CED6, EB-1,
GAPCenA, tensin, Dok-1
β8 ?
(Adapted from Krissansen et al. 2006a)
18

The binding of integrins to their ligands leads to integrin clustering and changes in integrin-
avidity, and the recruitment of actin filaments through interactions of the cytoplasmic
domains with cytoskeletal proteins such as talin, vinculin, α-actinin and filamin (reviewed in
Krissansen et al. 2006a). Integrin activation first came to light from studies of integrin αIIbβ3
on platelets, where thrombin binding to its receptor led to increased fibrinogen binding
(reviewed in Kieffer et al. 1990; Phillips et al. 1991; Krissansen et al. 2006a). It was then
shown that deletions of the cytoplasmic domain of α- or β- tails resulted in constitutively
active integrins (O'Toole et al. 1991; Crowe et al. 1994). Over the years, there have been
extensive studies on integrin activation and signalling.
1.3.1 Models of integrin activation
As mentioned, it is thought that integrins in their inactive state are in a bent conformation.
The integrin must be extended to enable binding to its ligand some 200 Å away. Currently
there are two prevailing models of integrin activation, the switch-blade and deadbolt models.
Switch-blade model: In the switch-blade model, inside-out activation causes a separation of
the cytoplasmic and transmembrane domains of the two subunits (Kim et al. 2003b), causing
a jack-knife-like swing-out extension at the “knee” of the integrin β-subunit hybrid domain
which occurs concurrent with or after ligand binding (Figure 1.8). The swing-out motion has
been proposed to participate in the initiation of outside-in signalling induced by ligand
binding. This is supported by the calculated Stokes radius based on gel elution profiles which
increases from 56 Å (in 1 mM Ca 2+ ), to 60 Å (in Mn 2+ ), to 63 Å upon addition of cyclic RGD
tripeptide (Takagi et al. 2002). In addition, X-ray solution scattering, EM imaging and crystal
structures show an outward swing of the β-subunit hybrid domain from 45˚ to 80˚ (Mould et
al. 2003b; Takagi et al. 2003; Xiao et al. 2004).
19

Figure 1.8 Switch blade model of integrin activation
A cartoon depiction of the switch blade model of integrin activation. The different domains of the integrin α and
β-subunits are shown in different colours. The integrin α-subunit is shown without (A), and with the α subunit Idomain
(B). The illustrations depict the conformations of the inactive state in which the head piece is bent
towards the plasma membrane (left), the extended conformation with a closed headpiece (middle), and the active
state with an open headpiece for ligand binding (right). The dotted lines show the flexibility of the integrin βsubunit
stalk region. (Adapted from Luo et al. 2007).
Deadbolt model: The deadbolt model of integrin activation was coined by taking account of
the discrepancies in the switchblade model (Arnaout et al. 2007), such as the ability of
integrins in the bent integrin conformation to bind to ligand (Xiong et al. 2003). In the
deadbolt model, the site of ligand-binding is thought to be tightly bound (deadbolt) when in
an inactive form. When integrins are activated the head region does not extend out straight in
response to straightening of the “knee” region, but rather changes in the transmembrane angle
disengage the hybrid domain from the I-domain or the β-subunit I-like-domain (Figure 1.9).
20

The β-terminal domain (β-TD) releases the β-subunit I-like-domain which is responsible for
ligand binding, allowing it to undergo a subtle conformational change and adopt a high-
affinity ligand-binding state. Extension of the receptor at both the genu domain of the α-
subunit and the interface between the integrin epidermal growth factor-1 and IEGF-2 domains
of the β subunit is proposed to occur after ligand-binding in this model (Arnaout et al. 2007).
This is supported by the ability of the bent integrin to bind its ligands in solution, not by leg
straightening, but through the Mn 2+ -induced shift of the ADMIDAS metal ion coordination to
a ligand bound state, causing tertiary changes as seen in the active state (Mould et al. 2003a).
In support, deletion of integrin transmembrane and cytoplasmic segments leads to partial
disengagement of the deadbolt (Humphries 2000). Furthermore, recent cryoelectron
tomography of integrin αIIbβ3 revealed membrane heights remained the same after Mn 2+
activation (Ye et al. 2008).
Figure 1.9 Deadbolt model of integrin activation
A cartoon representation of the deadbolt model of integrin activation. The α-subunit without an I-domain is
shown in the upper diagram, and with an I domain in the lower diagram. (A) and (D) denote the inactive integrin
conformation, while (B) and (E) shows the release of the deadbolt to an active state, and (C) indicates a ligand
bound state. (Adapted from Arnaout et al. 2007).
21

1.3.2 Integrin regulation by inside-out signalling
Inside-out integrin signalling refers to the transmission of signals from within the cell to the
extracellular domain of the integrin, affecting the affinity of integrins for their ligands
(reviewed in Hynes 2002). This form of regulation is precisely controlled in time and space,
as exemplified in platelet aggregation and leukocyte transmigration (reviewed in Hynes
2002). The switching from low- to high-affinity states is rapid, occurring in subsecond time
frames, and is reversible in less then a minute. Leukocyte integrins generally exist in a lowaffinity
state, and are activated intracellularly by signals from within the cell in response to
extracellular chemicals and factors (Lollo et al. 1993; Constantin et al. 2000), or mechanical
stress (Zwartz et al. 2004).
As mentioned above, integrin α- and β-subunit cytoplasmic domains contain conserved
sequence motifs which interact with intracellular proteins, and are important for integrin
regulation and function. Sequence motifs within the α- and β- subunits that are recognized by
intracellular signalling proteins have been mapped (Figure 1.10).
The GFF(R/K)/R motif in the α-subunits is recognised by calreticulin, and appears to be
essential for subunit association and stability (Leung-Hagesteijn et al. 1994). Calreticulin
transiently interacts with integrins during cell attachment and spreading. There are many
cytoplasmic proteins that interact with the β-subunit, as discussed below.
22

Figure 1.10 Sequence alignments of the human integrin β subunit (a), and α subunit (b) cytoplasmic
domains.
Comparison of the primary sequences of the cytoplasmic domains of the integrin α and β subunits. Highly
conserved amino acid residues are highlighted, and provided as a consensus below the alignments. (Adapted
from Krissansen et al. 2006b).
Intracellular ligands of the integrin β-subunit
This thesis will attempt to summarise the features of some important intracellular binding
partners for the integrin β-subunit, with specific attention to binding partners relevant to the
aims of the thesis. The current model of integrin activation places the binding of the
cytoskeletal protein talin to the integrin β-subunit cytoplasmic domain as the common last
step in the pathway of inside-out signalling (Tadokoro et al. 2003; Tanentzapf et al. 2006).
Talin: The cytoskeletal adaptor protein talin is critical for integrin activation (Tadokoro et al.
2003; Tanentzapf et al. 2006). Talin consists of a large C-terminal rod domain and an Nterminal
FERM (band 4.1, ezrin, radixin, and moesin) domain with three subdomains F1, F2,
and F3 (Garcia-Alvarez et al. 2003). Talin plays a crucial role in regulating integrin affinity.
The binding of the talin head region to the integrin cytoplasmic domain causes the
dissociation of the α- and β- cytoplasmic domains and induces conformational changes in the
23

extracellular regions to increase affinity for ligand (Tadokoro et al. 2003). In addition, talin is
essential in the early coupling of integrins to the cytoskeleton following the binding of
integrins to their extracellular ligands (Jiang et al. 2003). Talin reinforces the integrin-
cytoskeleton linkage through recruitment of other cytoskeletal proteins such as paxillin,
vinculin, α-actinin, tensin, and zyxin (Giannone et al. 2003). Talin depletion studies revealed
that talin is not important in the initial signalling processes required for spreading of cells, but
rather it provides an important mechanical linkage between the ligand-bound integrin and the
actin cytoskeletal network (Zhang et al. 2008a).
Talin binds to the central NPxY motif of several integrin β subunit cytoplasmic domains
(Calderwood et al. 2002). Using structure-based mutagenesis, the talin F3 domain was shown
to complex with the membrane proximal region of the cytoplasmic tail of the β3 integrin
subunit (Wegener et al. 2007). This result led to a proposed model of talin-induced integrin
activation. In this model, chemokine or growth factor receptor activation of cells leads to the
activation of the small G-protein Rap1 by protein kinase C (PKC) or Rap1-GEF, which is
then recruited to the plasma membrane. RIAM (Rap1–GTP-interacting adapter molecule)
then associates with talin, freeing talin from its autoinhibitory interactions and rendering it
available for binding to integrins (Lafuente et al. 2004; Han et al. 2006). Other mediators of
talin activation include phosphatidylinositol 4,5-bisphosphate (PIP2; Martel et al. 2001) and
calpain (Yan et al. 2001). The F3 subdomain of talin, which resembles a phosphotyrosine-
binding domain, binds to the NPxY motif in the membrane distal region of integrin β subunit
cytoplasmic domains. While still maintaining an interaction with the membrane distal region,
talin then engages the membrane proximal portion, which destabilises the salt bridge between
the α- and β-subunits, and stabilises the helical structure of the membrane proximal region.
This changes the conformation of the transmembrane helix causing a separation or reorientation
of the integrin tails leading to integrin activation. It should be noted, however, that
the exact role of the salt bridge in vivo has been questioned from the results of genetic studies
of β1 integrins in mice, in which mutations of the aspartic acid to alanine in the membrane
proximal region had no apparent function under physiological conditions (Czuchra et al.
2006). Talin binding to integrin β-subunits is thought to be the final step leading to integrin
activation, but exactly how this step is regulated still remains to be solved. It is possible that
phosphorylation (Tapley et al. 1989), proteolysis (Yan et al. 2001) and phosphoinositide
binding (Martel et al. 2001) all play roles.
24

Cytoplasmic proteins
Other cytoplasmic proteins that influence integrin activation are described briefly below.
Kindlin-2: Kindlin-2 (mitogen-inducible gene-2, MIG-2), another adaptor protein which
possesses a FERM domain, has recently been shown to bind to β1 and β3 integrins to enhance
talin-mediated integrin activation (Shi et al. 2007; Montanez et al. 2008). Kindlin-2 binds to
the membrane distal NxxY motif of integrin β subunits through its PTB site. Loss of kindlin-
2 results in peri-implantation lethality and impaired integrin activation in mice and embryonic
stem cells respectively. Kindlin-2-deficiency also prevents the formation of multiple focal
adhesions (FAs) and recruitment of integrin-linked kinase (ILK) to integrin adhesion sites
(Dowling et al. 2008; Montanez et al. 2008).
Filamin: Filamin is an actin binding protein widely expressed in animal cells, which is a
potent actin filament cross-linker (Stossel et al. 2001). Filamin subunits of 280 kDa form noncovalently
associated dimers. Filamin contains an actin-binding domain at its N-terminus
followed by 24 Ig-like domains (IgFLN1-24). It was found that IgFLNa21 binds to the β7
integrin subunit cytoplasmic domain. Filamin binds to the same region as talin within the
integrin β-subunit cytoplasmic domain. It is thought that filamin acts as a negative regulator
of talin-induced integrin activation, which may explain the known inhibitory role of filamin
on cell migration (Calderwood et al. 2001). Threonine phosphorylation at aa 758 in the triple
threonine motif (TTT) immediately downstream of the central NPxY motif of β2 integrins
prevents filamin binding. In contrast, threonine phosphorylation enables the molecular
adaptor protein 14-3-3 to bind to the β2 subunit cytoplasmic domain (Kiema et al. 2006;
Takala et al. 2008). This suggests that phosphorylation of threonine 758 may provide a
molecular switch for filamin and 14-3-3 binding.
Migfilin: Migfilin possesses three LIM domains at its C-terminus, a central proline-rich
region, and an N-terminal portion which lacks identifiable structural domains (Tu et al. 2003).
The LIM domains mediate binding to kindlin-2 and are required for localisation to focal
adhesions (Tu et al. 2003). Migfilin is thought to provide a link between the actin
cytoskeleton and integrin-extracellular matrix contact sites. Migfilin binds preferentially to
the filamin IgFLNa21 domain, similarly to the β7 integrin-filamin (IgFLNa21) interaction,
suggesting it may provide a mechanism for switching between different integrin-cytoskeleton
linkages (Lad et al. 2008).
25

Dok1: Dok1 was found to bind weakly to the central NPxY in the β3 subunit cytoplasmic
domain, where the affinity of interaction was strongly increased by phosphorylation of
tyrosine residue Y747 (Oxley et al. 2008). Phosphorylation of Y747 inhibited talin binding,
suggesting that phosphorylation at this position can act as a switch by reversing the
preference for talin to Dok1 to bind the β3 subunit cytoplasmic domain (Oxley et al. 2008).
Paxillin: Paxillin is an adaptor protein found in focal adhesions, which is highly tyrosine
phosphorylated (Schaller et al. 1995b). It regulates focal adhesion dynamics and cell
migration . Paxillin binds to the tyrosine kinases focal adhesion kinase (FAK) and src, as well
as other cytoskeletal proteins such as vinculin (Turner 1998; Wade et al. 2006). It binds with
high affinity to the α4 integrin subunit cytoplasmic domain (Liu et al. 1999; Liu et al. 2000).
α-actinin: α-actinin is an actin binding protein consisting of two rod-shaped anti-parallel 100
kDa monomers (Ylanne et al. 2001). It has actin-binding motifs at either ends of the rod
enabling α-actinin to cross-link actin filaments into bundles (Ylanne et al. 2001). α-actinin
binds to the integrin β1 subunit cytoplasmic domain in vitro (Otey et al. 1990) and binds to
activated β2 integrins, but not to inactivated integrins in vivo (Pavalko et al. 1993).
Vinculin: Vinculin is an adaptor protein that is a key regulator of FAs (Zamir et al. 2001;
Ziegler et al. 2006). Vinculin is comprised of three domains, namely an N-terminal head, a
flexible proline-rich hinge (neck), and a C-terminal tail domain (Winkler et al. 1996).
Intramolecular interaction between the head and tail domains constrain vinculin in an inactive
conformation (Bakolitsa et al. 2004). Upon recruitment to FAs, vinculin switches to an open
active conformation allowing direct interactions with other cytoskeletal proteins such as the
binding of talin and α-actinin to the head domain, and actin and paxillin to the tail domain
(Zamir et al. 2001; Ziegler et al. 2006). The interaction of the N-terminal head of vinculin
with talin drives the clustering of integrins in cell-matrix adhesions. It also leads to the
recruitment of paxillin by a mechanism that is independent of the paxillin binding site of
vinculin. The binding of actin to the tail of vinculin is a major link in the interaction of focal
adhesions with the actin cytoskeleton (Humphries et al. 2007).
Radixin: Radixin is another FERM domain-containing protein that activates integrins (Tang
et al. 2007). Radixin enhances the adhesive activity of the integrin αMβ2, by binding to a
membrane proximal region (possibly the LxxLxxYRRF sequence) of the β2 subunit (Tang et
al. 2007).
26

PTB proteins: Several PTB proteins are known to bind to the integrin β-subunit cytoplasmic
domain with high affinity, including syk and Numb, however none of them are able to
activate integrins (Calderwood et al. 2002). The PTB-domain containing protein tensin binds
to the β subunit cytoplasmic domain in a talin-like manner, but with lower affinity
(McCleverty et al. 2007). Many other PTB proteins interact with the NPXY motif in β-
subunits including Shc, Dab1, Dab2, X11α, X11γ, CeLin10, JIP, EPS8, RGS12, CED6, EB-1
and GAPCenA, IRS-1 and ICAP-1, as listed in Table 1.3 (Krissansen et al. 2006a). It is
possible that one or a number of the latter proteins can activate or regulate integrins.
1.3.3 Intracellular ligands of the α-subunit
So far this thesis has focussed largely on intracellular ligands that interact with the
cytoplasmic domain of the β-subunit. However, it should be noted that the integrin α-subunit
cytoplasmic domain is not a passive player in integrin activation and/or signalling. As can be
seen in Table 1.3, the integrin α-subunit has several intracellular ligands. Of note,
interactions between talin and the αIIb cytoplasmic domain have been described (Knezevic et
al. 1996). Calcium and integrin binding protein 1 (CIB1) interacts with the membrane
proximal region of the αIIb cytoplasmic domain and interferes with talin binding (Yamniuk et
al. 2006), suggesting it acts as a potential negative regulator of talin induced-integrin
activation. The integrin α4 subunit cytoplasmic domain has been showed to bind to paxillin
(Liu et al. 1999), which allows talin to bind indirectly to the cytoplasmic domain. Spatial α4
phosphorylation at the leading edge of cell migration limits the interaction of α4 with paxillin.
However the α4-paxillin complex at the side and trailing edge can recruit ADP-ribosylation
factor GTPase-activating protein (Arf-GAP) which inhibits Rac and blocks lamellipodia
formation (Nishiya et al. 2005).
Certain integrins including α1β1, α5β1, and αvβ3 can signal through the Fyn/Shc pathway
(Wary et al. 1996; Guo et al. 2004). The α-subunits of theses integrins are coupled to Fyn
through association with the oligomeric transmembrane protein caveolin-1 (Wary et al. 1996;
Guo et al. 2004). Upon integrin ligand-binding, Fyn is activated and subsequently recruits and
phosphorylates Shc at Tyr317, leading to Grb2-SOS complex binding and the activation of
the Ras-Raf-MEK-Erk pathway which promotes cell cycle progression (Wary et al. 1996;
Guo et al. 2004).
27

1.3.4 Integrin outside-in signalling
Changes in integrin avidity (clustering) complement changes in integrin affinity, and are
critical for cell signalling (Hato et al. 1998). Integrin clustering, which is RhoA-dependent,
takes place within seconds when induced by chemokines (reviewed in Hynes 2002). It leads
to the activation of signalling cascades and recruitment of multiprotein complexes to FAs
(reviewed in Hynes 2002). FA formation at the cytoplasmic face of the plasma membrane
connects integrins through adaptor proteins to the actin cytoskeleton, as well as receptor
tyrosine kinases (RTKs).
Integrin-mediated adhesion controls multiple aspects of cellular behaviour. It can trigger
calcium influx and an increase in intracellular pH, activate tyrosine and serine/threonine
protein kinases, regulate the activity of the Rho family of small GTPases, activate inositol
lipid metabolism, and trigger gene expression leading to cytokine and metalloprotease
production (reviewed in Krissansen et al. 2006a).
A prominent biochemical event in integrin outside-in signalling is protein tyrosine
phosphorylation due to activation of the src and FAK family of protein kinases (Arias-
Salgado et al. 2003). Src and its inhibitor Csk are constitutively bound to the C-terminus of
the integrin β subunit cytoplasmic domain. Ligand-binding induces src activation by
dissociation of Csk leading to src transphosphorylation in clustered integrins and/or
recruitment of tyrosine phosphatases such as receptor tyrosine phosphatase alpha (RPTPα), or
PTP-1B (von Wichert et al. 2003b; Arias-Salgado et al. 2005). Src-induced tyrosine
phosphorylation of phosphatidylinositol (4) phosphate 5 kinase type Iγ (PIPKIγ) at tyrosine
644 increases its affinity for the talin head domain, which dissociates the integrin β-tails from
the talin F3 domain (Ling et al. 2003). PIP2, a lipid second messenger, is generated that coactivates
a number of focal adhesion proteins, including vinculin and talin. An increase in
PIP2 may enhance talin-integrin association and displace PIPKIγ from talin, leading to a
reduction in PIP2, suggesting a self-limiting dynamic process. Integrins may also activate Syk
in focal complexes (Woodside et al. 2001). Syk binds directly to the last 4 amino acids of the
C-terminal portion of the β3 tail. It binds tensin, converting focal adhesions into fibrillar
adhesions (Woodside et al. 2001). Src can phosphorylate and activate FAK and p130Cas,
where the latter is essential in converting mechanical force into biochemical signals (Sawada
et al. 2006). This creates a binding site for the adaptor protein Crk either through the
association of an activator of Ras, son of sevenless (SOS), or through the association of Rap
28

guanine nucleotide exchange factor (GEF) 1 (also known as C3G) which subsequently
activates Erk and Raf respectively (Sawada et al. 2006). The adaptor protein Nck is
stimulated upon integrin-mediated adhesion, leading to association with p130Cas which can
lead to Erk activation. In other possible pathways, the protein tyrosine phosphatase Shp2 is
recruited, which downregulates FAK and thereby permits the incorporation of α-actinin into
focal adhesions (von Wichert et al. 2003a).
FAK is autophosphorylated at Tyr397 upon integrin-mediated adhesion, which creates a
binding site for Src (Schlaepfer et al. 1998). Src subsequently phosphorylates FAK at Tyr925,
creating a binding site for the adapter protein Grb2 that links FAK to SOS, a guanine
nucleotide exchange factor for Ras which activates the Ras-Raf-MEK-ERK pathway
(Schlaepfer et al. 1998). In addition, the adaptor protein Shc also binds to the phosphorylated
Tyr397 residue of FAK. Shc is then phosphorylated by FAK and Src and provides an
alternate link to Grb2.
Phosphoinositide 3-kinases (PI-3-kinase) binds to phosphorylated FAK at Tyr397 upon
integrin-mediated adhesion (Chen et al. 1996). Active PI-3-kinase may activate Erk through
its protein kinase activity or through SOS and phosphatidylinositol-3,4,5-trisphosphate (PIP3;
Chen et al. 1996). PI-3-kinase is recruited to the plasma membrane by Cbl binding (Ojaniemi
et al. 1997; Zell et al. 1998; Feigelson et al. 2001), which can stimulate ubiquitin-mediated
turnover of both Src-family kinases and PI-3-kinase itself (Fang et al. 2001). PI-3-kinase is
activated by integrins in a FAK and src independent pathway, resulting in the
phosphorylation of Akt, the generation of PIP2 and PIP3, and the recruitment of signalling
cascade proteins (Velling et al. 2004).
The serine/threonine (ser/thr) kinase ILK binds directly to and may phosphorylate integrin β1
and β3 subunit cytoplasmic domains (Legate et al. 2006). ILK binds to the LIM adaptor
proteins PINCH-1, and PINCH-2 (Tu et al. 1999). It links the actin binding proteins α, β, and
γ parvins to the integrin β cytoplasmic domain, hence linking integrins to the cytoskeleton
and signalling pathways (Legate et al. 2006). The assembly of an ILK-PINCH-parvin
complex is essential for the localisation of FAs (Zhang et al. 2002), while the binding of ILK
with PINCH stabilises these proteins and protects them from proteasome-mediated
degradation (Fukuda et al. 2003; Legate et al. 2006).
29

1.3.5 Force sensing and focal adhesions
Movement of focal adhesion proteins has been visualised by total internal reflection
fluorescence microscopy of fluorescently tagged proteins (Brown et al. 2006; Hu et al. 2007).
These studies found that active cells display vigorous retrograde movement of the cell
membrane resulting from continuous pulling of myosin II while ECM-integrin bonds remain
stationary. The transmission of motion from F-actin to proteins in the focal adhesion (FAK,
paxillin, zyxin) to integrin decreased. The actin binding protein α-actinin displayed the
greatest motion, due to its late incorporation into focal adhesions. Talin and vinculin were
only partially coupled to the F-actin motion which prevents transmission of forces from
actomyosin contractions to the ECM through integrins (Evans et al. 2007).
As mentioned above, p130Cas is a primary sensor and transducer of externally applied force
into biochemical signals (Sawada et al. 2006). p130Cas binds to FAK through the N-terminal
src homology (SH)3 domain, possibly linking the β-integrin cytoplasmic domain and/or α-
integrin cytoplasmic domain through paxillin. Mechanical extension of the central domain in
p130Cas enhances tyrosine phosphorylation by src, which leads to the association of Crk and
C3G, leading to Rap1 activation and ERK signalling (Tamada et al. 2004; Defilippi et al.
2006).
The small GTPases Rac and RhoA also play a role in integrin activation (Nobes et al. 1995;
Laudanna et al. 1996). Both Rac1 and its downstream effector molecule, RhoA, induce the
adhesion of the α4β7 integrin to its physiological ligand MAdCAM-1 (Zhang et al. 1999).
The Rho-related guanosine triphosphatase, Cdc42, induces filopodia, whereas Rac induces
lamellipodia, and Rho induces focal adhesions and associated stress fibers (Hall 1998).
Therefore each of these Rho-related proteins interact with multiple downstream effectors to
control the actin cytoskeleton.
1.3.6 Integrin crosstalk
To add to the complexity of integrin signalling, the signalling cascades mediated by integrins
are also linked to those of other receptors including growth factor receptors, receptor tyrosine
kinases (RTKs), cytokine receptors, and ion channels (Schwartz 2001). The signalling
pathways downstream of growth factor receptors and integrins can synergize. For instance,
the combined effects of cell adhesion and growth factors can transform a weak transient
activation of Erk to a strong and sustained Erk activity (Schwartz 2001). Integrin-mediated
30

adhesion leads to the clustering and transactivation of several RTKs, including platelet-
derived growth factor receptor, epidermal growth factor receptor, Ron, Met, and vascular
endothelial growth factor receptor. This transactivation of RTKs can occur in the absence of
their cognate growth factors (Schwartz 2001).
Crosstalk can also occur between different integrin receptors. Integrins bound to a particular
ligand can actively suppress the activation of other integrins on the same cell (Orr et al.
2006). For instance, α2β1 bound to its ligand suppressed the α5β1 and αvβ3 integrins through
PKA, and ligated α5β1 suppressed α2β1 through PKCα (Orr et al. 2006). In contrast,
activation of one integrin can lead to the activation of another integrin. For instance, the
binding of T-cells to VCAM-1 via integrin α4β1 activates β2 integrin function (Hyduk et al.
2002), while binding of αLβ2 to ICAM-1 will activate α4β1 (Porter et al. 1997).
1.4. Integrins and immunological synapses, unique signalling structures
1.4.1 Synapses formed between communicating immune cells
The immunological synapse (IS) is a supramacromolecular structure that forms at the
membrane interface between a lymphoid effector cell and an APC or target cell through
which information passes. The IS enables sustained signalling and T-cell activation, which
can be stable for many hours (Sims et al. 2002). Formation of an IS is a multistep process,
such that a nascent or immature IS initially forms and then develops into a mature IS,
otherwise referred to as a supramolecular activation complex, (SMAC). α4β1 is initially
engaged in the central region of the nascent SMAC (cSMAC; Figure 1.11A), and the T-cell
receptors (TCRs) are in the peripheral SMAC (pSMAC). In the mature IS, the localisation of
the α4β1 integrins and TCR are reversed, such that integrins form a ring around the centrally
located signalling molecules. Talin remains associated with the active form of α4β1 within
the pSMAC ring (Monks et al. 1998), while the TCR and its associating proteins such as
CD2, CD28, PKC, lck, Fyn, CD4 and CD8 are localised in the cSMAC (Lin et al. 2005).
Integrin α4β1-mediated formation of the pSMAC enhances the accumulation of TCR
complexes and the exclusion of the phosphatase CD45 from the cSMAC (Graf et al. 2007),
and thereby α4β1 helps to organize the proteins present in the IS. CD45 inactivates lck by
dephosphorylation of Y394 which reduces TCR signalling within the microclusters, hence its
exclusion from the cSMAC serves an important purpose.
31

Figure 1.11 Models of the immunological synapse and kinapse
(A) The model of an IS comprised of three concentric zones. The cSMAC (central SMAC) in the center contains
concentrated TCRs where signaling is terminated. The pSMAC (peripheral SMAC) which surrounds the
cSMAC contains LFA-1 and talin. The dSMAC (distal SMAC) is a second outer ring which contains the
phosphatase CD45 to prevent dephosphorylation of key kinases. (B) A polarized migrating cell is divided into
three regions, the lamellipodium at the leading edge, the focal zone in the mid section, and the uropod at the
trailing edge. The migrating cell forms transient synapses or kinapses at the cell surface. Signalling
microclusters form in the dSMAC and move through the pSMAC to the cSMAC, corresponding to the three
regions of a polarized cell. LFA-1 which is expressed at different levels in each of the three zones varies in its
affinity for ligand depending on the particular zone, as indicated. (Figure is adapted from information published
by Evans et al. 2009, and Dustin 2008a).
The roles of the different substructures of the SMAC are still somewhat controversial. It was
initially proposed that TCR signalling occurred within the cSMAC, however at least one
study suggests that the cSMAC is the site where TCRs are downregulated (Lee et al. 2003).
Sustained TCR signalling is thought to take place in transient microclusters that form at the
outside edges of pSMAC, also known as distal SMAC, or dSMAC (Campi et al. 2005; Varma
et al. 2006). The microclusters only transmit signals while they are being transported through
the pSMAC to the cSMAC (Campi et al. 2005; Varma et al. 2006). However, it is not known
whether all IS form pSMAC and cSMAC, and it is possible that in T-cell signalling the
formation of these SMACs is not a physiological requirement. Improved resolution of
membrane structures by in vivo microscopy will be required to unravel the substructures of
SMAC in vivo (Lin et al. 2005).
32

1.4.2 Synapses termed kinapses are formed on migrating T cells
T cells are highly motile when scanning APCs. Synapses are formed at the surface of
migratory T cells, which have been termed kinapses (Dustin 2008a). Migrating T cells are
polarized and travel in a particular direction towards a chemoattractant. They are speedsters
travelling at speeds of 5 to 40 μm/minute. In contrast, fibroblasts move at approximately 15
μm/hour (reviewed by Evans et al. 2009). The distribution of αLβ2 (LFA-1) on the surface of
migrating T cells is not uniform. LFA-1 is present at low levels at the leading edge and at
high levels at the rear of the cell (Smith et al. 2005). A migrating T cell can be separated into
three different zones with a leading lamellipodium which expresses low levels of intermediate
affinity LFA-1, a mid-cell focal zone which expresses higher levels of high affinity LFA-1,
and a trailing edge uropod with the highest expression of LFA-1 of unknown affinity (Figure
1.11B; reviewed by Evans et al. 2009).
The lamellipodial membrane rapidly protrudes and retracts to rapidly scan its target, and
therefore expresses intermediate-affinity LFA-1 (reviewed by Evans et al. 2009). The focal
zone provides firm attachment, which requires high affinity LFA-1 (Figure 1.11B; reviewed
by Evans et al. 2009). The uropod at the rear of the cell anchors the cells to a substrate while
the front leading edge is able to scan its surroundings (reviewed by Evans et al. 2009). Dustin
proposed that kinapses contain a dSMAC at the leading edge of the cell, pSMAC at the mid
region and a cSMAC at the uropod of the polarized cell Figure 1.11B (Dustin 2008a).
1.5. Tyrosine kinases and cell signalling
As mentioned above, tyrosine kinases play an important role in integrin activation and
signalling. This thesis largely focusses on two tyrosine kinase families, namely the FAK and
src families.
1.5.1 The focal adhesion kinase (FAK) family
FAK is a 125 kDa non-receptor protein tyrosine kinase, first identified as a phosphorylated
protein whose expression became increased after v-src transformation of chicken embryo
cells (Schaller et al. 1992). FAK is ubiquitously expressed in virtually all tissues and cell
types, and plays a crucial role in mediating signal transduction pathways initiated either at the
sites of cell attachment or through growth factor receptors. Hence, FAK plays an important
role in cell attachment, migration, invasion, proliferation and survival. It is involved in
33

important processes in cell, tissue, and organ structuring, functioning and remodelling
(reviewed in van Nimwegen et al. 2007). In addition, FAK is essential during the early stages
of embryonic development as FAK-null mice embryos die at day 8.5 (Ilic et al. 1995).
Conversely, increased FAK signalling may result in uncontrolled proliferation, survival and
migration of cells (reviewed in van Nimwegen et al. 2007). FAK exists as various isoforms
due to alternative splicing and usage of alternative promoters (Schaller et al. 1993). The most
well known isoform lacks the catalytic domain, and is called FAK related non-kinase (FRNK;
Schaller et al. 1993). FRNK competes with FAK for localisation at focal adhesions. It acts as
an inhibitor of FAK due to the lack of a kinase domain.
The only other FAK family member is proline-rich tyrosine kinase 2 (PYK2), also called cell
adhesion kinase β (CAKβ), related adhesion focal tyrosine kinase (RAFTK), or calciumdependent
proteins-tyrosine kinase (CADTK; reviewed in van Nimwegen et al. 2007). PYK2
is slightly smaller then FAK, at 112 kDa, and its highest expression levels are in cells of the
nervous system (reviewed in van Nimwegen et al. 2007).
FAK structure and signalling
There is more then 90% aa similarity between the human, chicken, mouse and frog
homologues of FAK. FAK has a central kinase domain flanked by a large N-terminal region
containing a FERM domain, and a C-terminal domain containing a focal adhesion targeting
(FAT) sequence (Figure 1.12; reviewed in van Nimwegen et al. 2007).
Figure 1.12 Structural features and binding partners of FAK
Structural features of FAK include an N-terminal region containing a FERM domain, a central kinase domain,
and a C-terminal focal adhesion targeting (FAT) site. There are various tyrosine phosphorylation sites for
autophosphorylation and activation. The known interacting sites for other cytoplasmic signalling proteins are
indicated. (Figure adapted from van Nimwegen et al. 2007).
34

Integrin clustering leads to autophosphorylation of FAK on tyrosine 397 (Y397), which
increases the catalytic activity of FAK (Calalb et al. 1995). The autophosphorylation of Y397
leads to the binding of src, p85, shc, or Grb7 via their src-homology 2 (SH2) domains. The
kinase domain contains two tyrosines at aa positions 576 and 577 (Y577 and Y577,
respectively), which are phosphorylated in response to src binding and induce the full
activation of FAK (Calalb et al. 1995). The kinase domain of FAK phosphorylates several
focal adhesion associated proteins such as paxillin, Grb2, and p130CAS (Thomas et al. 1999).
The FERM domain mediates protein-proteins interactions, where the most well known
partner is the integrin β subunit cytoplasmic domain (Cooper et al. 2003). The FERM domain
also functions as a regulator of FAK activity, as it can inhibit the autophosphorylation of
Y397, which is necessary for FAK activation (Cooper et al. 2003). Upon integrin clustering,
the FERM domain binds to the integrin β-subunit cytoplasmic tail, which releases the FERM-
mediated autoinhibiton of FAK, and allows autophosphorylation of Y397, and activation of
FAK (Cooper et al. 2003). Conversely, the FERM domain can also interact with full-length
FAK, optimise its kinase activity, and promote FAK signalling (Dunty et al. 2004).
The FAT sequence of FAK is required for the localisation of FAK to focal adhesions, as the
fusion of the FAT sequence with other proteins is sufficient for their localisation to focal
adhesions (Hildebrand et al. 1993). The FAT domain has also been shown to interact with
integrin β subunit cytoplasmic domains. However, whether the interaction is direct or via
other integrin-associated proteins like talin, and paxillin remains unclear (Schaller et al.
1995b). Phosphorylation of FAK residues tyrosine 861 (Y861) and/or tyrosine 925 (Y925)
early during cancer cell adhesion by association with the src family kinase (SFK) member,
fyn, helps FAK translocate from lipid rafts in which SFKs usually reside, and leads to the
activation of Akt-mediated signalling (Baillat et al. 2008).
FAK has been implicated in a variety of biological processes, including cell survival,
proliferation, migration, invasion, and angiogenesis, through various signalling cascades
summarised in Table 1.4. In respect of cell survival, apoptotic proteins such as Bad, GSK3
and p53 are inactivated by FAK either directly as with p53, or indirectly through signalling
pathways such as the PI-3 kinase pathway as with Bad and GSK3 (Datta et al. 1997; Sonoda
et al. 1999; Almeida et al. 2000; Golubovskaya et al. 2005). Overexpression of FAK induces
the up-regulation of Cyclin D1 and Cyclin D3, causing accelerated cell cycle transition from
the G1 phase to the S phase (Sonoda et al. 2000; Yamamoto et al. 2003).
35

Table 1.4 Overview of the proteins/pathways that are involved in FAK-mediated signalling
Biological
Proteins of FAK-mediated signalling cascades
process
Survival FAK→PI-3 kinase → PKB → Bad → GSK3 (Sonoda et al. 1999)
FAK→p53 (Golubovskaya et al. 2005)
FAK→ Src→p130CAS → Ras → → JNK (Almeida et al. 2000)
Proliferation FAK→Grb2 → MAPK → Cyclin D1 (Sonoda et al. 2000)
FAK→PKC/PI-3 kinase → Rb → Cyclin D3 (Yamamoto et al. 2003)
Migration FAK↔Calpains (Giannone et al. 2004)
FAK→Src/p130CAS → MAPK (Cary et al. 1998)
FAK→Src/p130CAS/PI-3 kinase → MAPK (Reiske et al. 1999)
GSK3/PP1→ regulate FAK Ser 722 phosphorylation (Bianchi et al. 2005)
(Adapted from van Nimwegen et al. 2007)
FAK is both an activator and a substrate of calpains (Giannone et al. 2004). The interplay
between FAK and calpains is important in the disassembly of focal adhesions at the rear of
cells during cell migration (Giannone et al. 2004). As mentioned above, FAK also activates
p130CAS through src, which leads to the activation of the mitogen-activated protein kinases
(MAPK) pathway and cell migration (Cary et al. 1998; Reiske et al. 1999). Phosphorylation
of FAK on serine 722 (S722) through the competing actions of the serine/threonine kinase
GSK3, and the protein phosphatase PP1 serves to regulate cell migration (Bianchi et al.
2005).
1.5.2 Src kinase family (SFK)
The non-receptor tyrosine kinase src is important in many aspects of cellular physiology
(Sandilands et al. 2008) . The src kinase family (SFK) consists of nine members, namely c-
src, c-yes, c-fyn, fgr, lyn, hck, lck, blk and yrc (Sandilands et al. 2008). They share a similar
structure, with molecular masses ranging from 52-62 kDa. They are each comprised of 6
functional domains: a myristylation domain, which enables plasma membrane interactions; a
unique domain; SH2 and SH3 domains for binding to other proteins; a kinase domain for
phosphorylation of tyrosines which also contains an autophosphorylation site at tyrosine 416
(Y416); and a C-terminal regulatory region which contains a phosphorylation site at tyrosine
527 (Y527) which is crucial for regulating the tyrosine kinase activity of src (Sandilands et al.
2008). V-src is a constitutively active form of src, which lacks several amino acids at the Cterminus,
notably Y527 (Y527; reviewed by Martin 2001).
36

Src is negatively regulated by phosphorylation of Y527, which is catalysed by the tyrosine
kinase Csk (c-Src terminal kinase; Cooper et al. 1986; Okada et al. 1989). Phosphorylation of
Y527 confines src in an inactive state (Roussel et al. 1991). The SH2 domain of src interacts
with Y527 (Roussel et al. 1991) allowing the SH3 domain of src to become engaged with the
SH2 kinase linker region such that Y416 in the kinase region becomes unphosphorylated
(Figure 1.13). The crystal structure of src showed that the SH2 and SH3 domains were
positioned at the back of the kinase domain, and interactions between these domains locked
the molecule in an inactive state (Xu et al. 1997).
Figure 1.13 Structural features and activation of src
A cartoon picture depicting the features of src and its active and inactive conformations. In the inactive
conformation on the left-hand side, the SH2 domain is bound to the phosphorylated tyrosine residue 527 (Y527)
and bends back towards the kinase domain. The SH3 domain is likewise bound to the kinase domain. Upon
activation, tyrosine residue 416 (Y416) is phosphorylated, and tyrosine residue 527 is dephosphorylated as
shown in the middle drawing. Activated src is extended with a phosphorylated tyrosine residue at 416 as shown
in the right-hand cartoon. The essential phosphorylation sites for activation are indicated. (Adapted from Martin
2001).
Most wild-type c-src is localised at the perinuclear regions of cells, where it directly
associates with the microtubule-organising centre and co-localises with endosomal markers
(Kaplan et al. 1992). However, active c-src is found in focal adhesions near the plasma
membrane (Kaplan et al. 1994). This change in localisation of src is due to endosomal
trafficking of src, which is regulated by Rho GTPases in concert with the actin cytoskeleton
(Sandilands et al. 2008).
37

Upon activation, src is autophosphorylated at Y416 (Y419 in humans), and dephosphorylated
at Y527 by protein tyrosine phosphatases (PTPs; Pallen 2003). The most likely candidate is
the transmembrane phosphatase PTPα (Pallen 2003), as ptpα-/- cells were found to have
decreased src and fyn kinase activity, which correlated with enhanced tyrosine
phosphorylation of the negative regulatory domain of src (Ponniah et al. 1999). Furthermore,
overexpression of PTPα leads to increased tyrosine phosphorylation of focal adhesion
associated proteins in cells, and was shown to activate src in vivo (den Hertog et al. 1993;
Harder et al. 1998; Zheng et al. 2000). Other tyrosine phosphatases that might regulate src in
response to cell adhesion include PTP1B and Shp2. Overexpression of a catalytically
defective mutant of PTP1B in mouse L cells reduced src activity, and PTP1B purified from
breast cancer cells was able to dephosphorylate a src peptide phosphorylated on Y527
(Arregui et al. 1998; Bjorge et al. 2000). shp2-/- cells deficient in Shp2 were found to have
increased numbers of immature focal adhesions, and displayed defective phosphorylation of
focal adhesion-associated proteins upon cell adhesion (Oh et al. 1999).
Src and cancer: Many human tumours exhibit elevated src kinase activity (Irby et al. 2000),
including human mammary carcinomas, colon cancer and pancreatic cancer (Playford et al.
2004). The increased activity of src in cancers has been proposed to be due to tyrosine
phosphatase-mediated dephosphorylation of the C-terminal Y527, an increase in src protein
levels, altered src protein stability, an increase in upstream RTKs, or a loss of key proteins
that negatively regulate src.
1.5.3 Integrin signalling, FAK and src
As mentioned briefly above, integrin cytoplasmic domains have no intrinsic kinase activity,
therefore the signals initiated by integrin-ECM interactions are transduced into cells through
association of the cytoplasmic domains with intracellular signalling proteins, in particular
non-receptor tyrosine kinases such as FAK and SFKs (Mitra et al. 2006).
Integrin clustering recruits FAK via its C-terminal FAT domain to integrin-associated
proteins such as talin and paxillin, or through its FERM domain to the integrin β cytoplasmic
tail (Mitra et al. 2006). The latter interactions are thought to be required to stably localise and
autophosphorylate FAK at the focal adhesions (Mitra et al. 2006). As mentioned above, the
binding of the FERM domain to the integrin β cytoplasmic tail releases the autoinhibiton of
FAK, and triggers the autophosphorylation of FAK on Y397. The autophosphorylation of
38

Y397 creates a high affinity binding site for the SH2 domain of SFKs, and the binding of src
leads to conformational activation of src. Src then transphosphorylates FAK on residues Y576
and Y577 within the kinase domain activation loop, and on residues Y861 and Y925 within
the FAK C-terminal domain. The FAK-src complex acts to control cell shape and focal
contact turnover events during cell motility (Mitra et al. 2005). The phosphorylation of Y925
creates a Grb2 binding site, thereby linking FAK to the MAP kinase pathway (Schlaepfer et
al. 1994). The FAK-src complex phosphorylates the adaptor proteins paxillin at residues
tyrosine 31 (Y31) and tyrosine 118 (Y118), and p130Cas within the Cas substrate domain.
This creates binding sites for the SH2 domain of the adaptor protein Crk. The overexpression
of Crk can lead to p130Cas-dependent activation of FAK (Iwahara et al. 2004). The
overexpression of p130Cas can promote enhanced tyrosine phosphorylation of FAK and
paxillin (Brabek et al. 2004). The association of p130Cas with src may preferentially trap src
in the active state (Nasertorabi et al. 2006).
Src can also be activated by integrins via other pathways, as briefly mentioned above. Src
binds directly to the β3 integrin cytoplasmic tail through its SH3 domain, and it was proposed
that this primes src for maximal activation during integrin clustering (Arias-Salgado et al.
2003; Shattil 2005). α4β1 can activate src independently of FAK, and rescue the motility
defects of FAK-null fibroblasts through increased phosphorylation of p130Cas (Hsia et al.
2005).
p130Cas-Crk signalling is negatively regulated by the Abl RTK (Holcomb et al. 2006). Abl is
activated by integrin clustering, and localises to focal adhesions (Holcomb et al. 2006). It
phosphorylates Crk at Y221 and prevents Crk from binding to p130Cas (Holcomb et al.
2006). Other proteins found to regulate p130Cas function include p130Cas associated protein
(p140Cap; Di Stefano et al. 2004), and the LIM protein Ajuba (Pratt et al. 2005).
1.6. Heat shock proteins
Heat shock proteins (Hsps) are a superfamily of highly conserved molecular chaperones
which are induced in plants, yeast, bacterial and mammalian cells in response to sub-lethal
physiological and environmental stresses (reviewed in Daugaardet al., 2007). Heat shock
proteins were first identified as inducible proteins whose expression was induced by elevated
temperatures, however it became clear that Hsp synthesis was also initiated by many
chemical and physical stress stimuli (Lindquist et al. 1988). Physiological events such as
39

antigen presentation, cell growth, differentiation, development and aging can also induce Hsp
synthesis (Calderwood et al. 2006).
Hsps protect cells from stress by assisting in protein folding and stabilisation, and they help to
sequester damaged proteins for degradation (reviewed in Daugaardet al., 2007). They are
overexpressed in a wide range of cancers, and have been implicated in cancer growth by
promoting tumour cell proliferation and inhibiting cellular death pathways.
Extracellular and membrane-bound Hsps have important immunological roles (reviewed in
Daugaardet al., 2007). In particular they stimulate the immune system, in part by presenting
antigenic peptides. Hsp70 and Hsp90 families are frequently located on the plasma membrane
of tumour cells (reviewed in Daugaardet al., 2007). In addition, elevated levels of Hsp70 and
Hsp90 have been detected in the medium of tumour cell lines and the serum of cancer
patients.
Mammalian hsps have been divided into 5 different subfamilies according to their molecular
weights, namely Hsp20, Hsp60, Hsp70, Hsp90 and Hsp100 (reviewed in Daugaardet al.,
2007). This thesis will primarily concentrate on the Hsp70 family of heat shock proteins and
their role in integrin regulation/function.
1.6.1 Hsp70 family
The human Hsp70 family consists of at least 8 different members, encoded by different
genes, which differ in amino acid sequence, expression and stress induction (Table 1.5;
reviewed in Daugaardet al., 2007). Hsp70 proteins mainly reside in the cytosol and nucleus,
however some are confined to the lumen of the endoplasmic reticulum (ER; Hsp70-5), and
the mitochondrial matrix (Hsp70-9; Daugaard et al. 2007). The different compartmental
localisation of the Hsp70 members suggests they may display specificity for client proteins,
or serve other chaperone-independent functions. Three members of the Hsp70 family, namely
Hsp70-1a, Hsp70-1b and Hsp70-6 are stress-inducible, whereas the other members Hsc70,
Hsp70-1t, Hsp70-2, Hsp70-5 and Hsp70-9 are not stress-inducible (reviewed in Daugaardet
al., 2007). Hsp70-1a and Hsp70-1b may function primarily in cellular stress situations, while
the others may perform tissue-specific and housekeeping functions. Hsp70 family members
are involved in a large range of processes, including the folding of newly synthesized
proteins, the transport of proteins across membranes, the refolding of misfolded and
40

aggregated proteins, and the control of activity of regulatory proteins (reviewed in
Daugaardet al., 2007).
Table 1.5 Essential features of members of the human Hsp70 family
Protein
Hsp70-1a
Hsp70-1b
Alternative
names
Hsp70, Hsp72,
Hsp70-1
Hsp70, Hsp72,
Hsp70-1
Homology to
Hsp70-1a (%)
Locus Localization
100 HSPA1A 6p21.3
99 HSPA1B 6p21.3
Hsp70-1t Hsp70-hom 91 HSPA1L 6p21.3
41
Cellular
localization Stress-induced
Cytosol,
Nucleus, Yes
Lysosomes
Cytosol,
Nucleus, Yes
Lysosomes
Cytosol,
No
Nucleus
Cytosol,
No
Nucleus
Hsp70-2
Hsp70-3,
HspA2
84 HSPA2 14q24.1
Hsp70-5 Bib, Grp78 64 HSPA5 9q33-q34.1 ER No
Hsp70-6 Hsp70B′ 85 HSPA6 1cen-qter
Cytosol,
Nucleus
Yes
Hsc70 a Hsp70-8,
Hsp73
Grp75,
86 HSPA8 11q23.3-q25
Cytosol,
Nucleus
No
Hsp70-9 mtHsp75,
Mortalin
52 HSPA9 5q31.1 Mitochondria No
a
A 54 kDa Hsc70 splice variant (NM_153201) has been reported, but its functional significance remains
unclear. (Adapted from Daugaard et al. 2007).
The Hsp70 family, like other Hsps, show a high level of conservation in their amino acid
sequences and domain structures (reviewed in Daugaardet al., 2007). They all have a
conserved ATPase domain, a central protease sensitive site, a peptide binding domain, and a
G/P-rich C-terminal region which contains an EEVD-motif that binds co-chaperones and
other Hsps (Figure 1.14). The various conserved domains enable Hsp70 proteins to bind and
release, in an ATP-dependent manner, extended hydrophobic amino acids exposed by
incorrectly folded globular proteins (Bukau et al. 2006).

Figure 1.14 Domain structure of human hsp70 family members
A cartoon picture of the human hsp70 family members showing their different structural and functional
domains. (Adapted from Daugaard et al. 2007).
The importance of the Hsp70 family is illustrated by the phenotypes of Hsp70 knockout mice
(Table 1.6). Knockout of the genes of different family members produces a variety of
phenotypes ranging from embryonic lethality, to viable and fertile mice that are susceptible to
various stressors such as UV and heat, and predisposed to develop disease, including
pancreatitis and infections (reviewed in Daugaard et al. 2007). The range of phenotypes
illustrates the various functions of different Hsp70 family members.
Hsp70 is expressed by tumour cells, and is associated with chemotherapeutic resistance in the
case of leukemias (Sliutz et al. 1996) and breast cancers (Vargas-Roig et al. 1998). Inhibition
of Hsp70 by the heat shock protein inhibitor KNK-437 was shown to reduce the adhesion of
myeloma cells to fibronectin (FN) or stromal cells, causing them to undergo apoptosis, and
sensitizing them to chemotherapeutic agents (Nimmanapalli et al. 2008).
This thesis will concentrate on the Hsp70-1(a-b), Hsc70, and Hsp-9 proteins, hence a fuller
description of each of these Hsps is provided below.
42

Table 1.6 Phenotypes of Hsp70 knockout mice
Protein
Gene
locus
Chromosomal
localization
Analogous
human gene
43
Phenotype
Hsp70.1 Hspa1a 17 B1 HSPA1A
Viable and fertile. Susceptible to UV,
osmotic stress, ischemia, TNF, pancreatitis
and heat.
Hsp70.3 Hspa1b 17 B1 HSPA1B Viable and fertile. Susceptible to heat.
Hsp70.1 +
Hsp70.3
Hspa1a +
Hspa1b
17 B1
HSPA1A +
HSPA1B
Viable and fertile. Susceptible to radiation
and sepsis. Increased genomic instability.
Hsp70.2 Hspa2 17 B1 HSPA2
Viable and females fertile. Meiotic defects in
male germ cells.
Hsc70 Hspa8 9 A5.1 HSPA8 Not applicable. Knockout cells are non-viable
Bip, GRP78 Hspa5 2 B HSPA5 Lethal at embryonic day 3.5
(Adapted from Daugaard et al. 2007)
Hsp70-1a and Hsp70-1b
Hsp70-1a and Hsp70-1b (Hsp70-1) are encoded by the genes HSPA1A and HSPA1B,
respectively, which are closely related, sharing 99% aa identity (reviewed in Daugaard et al.
2007). They are the major stress-inducible members of the Hsp70 family. Basal levels of
mRNAs encoding Hsp70-1a and Hsp70-1b are detectable at a similar level in many tissues
and cell types, however HSPA1A expression is dramatically higher in blood (Su et al. 2004).
In normal conditions, Hsp70-1 proteins are expressed in a cell type and cell cycle dependent
manner. However, during stress conditions, the hsp70-1 genes are activated by the binding of
a stress inducible transcription factor, heat shock factor 1 (HSF1), to upstream regulatory
regions called heat shock elements (HSE; reviewed in Daugaard et al. 2007). Stress-induced
Hsp70-1 enables the cell to cope with denatured protein aggregates, and hence confers
protection against protein damage caused by heat, ischemia, and oxidative stress (Li et al.
1991; Bellmann et al. 1996; Chong et al. 1998).
Knockout of the murine Hsp70-1 homologues Hsp70.1 and Hsp70.3 produces mice that are
viable and fertile, but which however are more sensitive to pancreatitis, UV light (epidermis),
osmotic stress (renal medulla), and brain ischemia (Table 1.6; reviewed in Daugaardet al.,
2007).
Hsp70-1 is abundantly expressed in malignant tumours of various origins, where its
expression correlates with increased cell proliferation, poor differentiation, and lymph node
metastases (Mosser et al. 2004).

Hsc70 (Hsp70-8)
Hsc70 is encoded by the gene HSPA8A (Table 1.5) and is expressed in most tissues (Su et al.
2004). It is 86% identical to Hsp70-1a, and is an important housekeeping chaperone which is
involved in the folding of nascent polypeptides, protein translocation across membranes,
prevention of protein aggregation under stress conditions, and disassembly of clathrin-coated
vesicles (Bukau et al. 2006). Hsc70 is essential for cell survival as gene knockout of Hsc70 is
lethal (Table 1.6), and RNA interference (RNAi)-based knock-down of Hsc70 results in
massive cell death (Rohde et al. 2005). Hsc70 participates in cytokine-mediated, posttranscriptional,
co-chaperone-dependent, regulation of the Bcl-2 family member Bim in
human blood cells (Matsui et al. 2007).
Hsp70-9
Hsp70-9, which is encoded by the gene HSPA9 gene (Table 1.5), is 52% identical to Hsp70-
1a but is not inducible by stress. Hsp70-9 is localised to the mitochondrial lumen where it
assists in protein folding after transmembrane transport.
1.7. Integrins and disease
Integrins are implicated in many human diseases, including inflammation, thrombosis,
tumour metastasis and infections, and hence are being considered as major targets for
therapeutic intervention (Horwitz 1997). Knockout of integrin genes in mice has shown
integrins are essential for normal embryonic development (Bouvard et al. 2001). A number of
diseases are associated with corresponding mutations of human integrin genes (Table
1.7Error! Reference source not found.). Gene knockout studies have identified critical roles
for integrins in development (β1 integrins), vasculogenesis (αV integrins), lymphangiogenesis
(α9β1), thrombus formation (αIIbβ3), the integrity of the skin (α6β4), and immune responses
(β2 and β7 integrins; Takada et al. 2007).
44

Table 1.7 Ablation of integrin genes in mice and their resulting phenotypes
Gene Phenotype
α1 V, F Increased collagen synthesis, reduced tumour vascularisation
α2 V, F Reduced branching morphogenesis and platelet adhesion
α3 L, birth Defects in kidneys, lungs, and cerebral cortex; skin blistering
α4 L, E11–E14 Defects in chorioallantois fusion and cardiac development
α5 L, E10 Defects in extraembryonic and embryonic vascular development
α6 L, birth Defects in cerebral cortex and retina; skin blistering
α7 V, F Muscular dystrophy
α8 L+V/F Small or absent kidneys; inner ear defects
α9 L, perinatal Bilateral chylothorax
α10 V, F No apparent phenotype
α11 ... ...
αv L, E12–birth Defects in placenta and in CNS and GI blood vessels; cleft palate
αD V, F Reduced T-cell response and T-cell phenotypic changes
αL V, F Impaired leukocyte recruitment and tumour rejection
αM V, F Impaired phagocytosis and PMN apoptosis; obesity; mast cell
development
αX ... ...
αE V, F Inflammatory skin lesions
αIIb V, F Defective platelet aggregation
β1 L, E5.5 Inner cell mass deterioration
β2 V, F Impaired leukocyte recruitment; skin infections
β3 V, F Defective platelet aggregation; osteosclerosis
β4 L, perinatal Skin blistering
β5 V, F No apparent phenotype
β6 V, F Skin and lung inflammation and impaired lung fibrosis
β7 V, F Abnormal Peyer’s patches; decreased number of intraepithelial
lymphocytes
β8 L, E12–birth Defects in placenta and in CNS and GI blood vessels; cleft palate
V, indicates viable; F, fertile; L, lethal; L+V/F, mutations that disrupt development but
also allow survival in a fraction of mice; CNS, central nervous system; GI,
gastrointestinal; and PMN, polymorphonuclear neutrophil. (Adapted from Bouvard et al.
2001; Takada et al. 2007)
1.7.1 Hereditary disease
Gene mutations or polymorphisms of integrin subunits have been associated with various
hereditary diseases. The lack of expression of the α6 integrin subunit in humans results in
junctional epidermolysis bullosa with pyloric or duodenal atresia (Pulkkinen et al. 1997).
Similarly β4 mutations cause generalized skin blistering and aplasia cutis (Vidal et al. 1995).
Mutations in the α7 gene gives rise to congenital myopathy and forms of congenital muscular
dystrophy (Hayashi et al. 1998), while the down regulation of expression of α7β1 contributes
to the pathology of congenital laminin deficiencies (Vachon et al. 1997).
Defective platelet aggregation arising from mutations in the αIIb and β3 genes is the cause of
the inherited bleeding disorder Glanzmann thrombasthenia (Derrick et al. 2001). Low platelet
α2β1 densities due to the polymorphic C807T allele of the α2 subunit of the integrin α2β1
45

leads to an increased tendency to bleed. In contrast, the polymorphic C807T/G873A allele
causes high receptor expression which may contribute to an increased risk of stroke in
younger people (reviewed in Carlsson et al. 1999; Krissansen et al. 2006a).
Leukocyte adhesion deficiency syndrome (LAD), is a hereditary immunodeficiency disease
with high morbidity and mortality due to mutations in the β2 gene (Kishimoto et al. 1987).
1.7.2 Inflammatory disease
Integrins have been implicated in a variety of inflammatory diseases, as listed below. Several
anti-integrin therapeutic reagents are currently being developed to treat these diseases.
Multiple sclerosis: Blockade of the α4, β2, and β7 integrin pathways in mice leads to
successful remission of different forms of experimental autoimmune encephalomyelitis
(EAE), an animal model for multiple sclerosis (Kanwar et al. 2000; Bullard et al. 2005).
Asthma: Blockade of integrin α4 expression prevents the transmigration of eosinophils into
the lungs, and the development of bronchial hyper-responsiveness to agonists in animal
models (reviewed in Krissansen et al. 2006a). Blocking αLβ2-ICAM-1 pathways also has a
protective effect (Taylor et al. 1997; Krissansen et al. 2006a).
Arthritis: Several integrins and their ligands are expressed in the arthritic joint tissue,
including αLβ2, αMβ2, ICAM-1, α4β1 and α4β7 on cells in the synovial infiltrate, αLβ2,
αMβ2 and ICAM-1 on the synovial lining layer, and VCAM-1 on endothelial cells (reviewed
by Krissansen et al. 2006a).
1.7.3 Other diseases
Integrins have also been implicated in a variety of other diseases, including cancer, hepatitis,
skin diseases, Graves disease, glomerulonephritis, muscular dystrophy, sepsis, emphysema,
osteoporosis, wound healing, cardiovascular disease, atherosclerosis, and osteoporosis
(reviewed by Krissansen et al. 2006a).
46

1.7.4 β7 integrins and disease
The β7 integrins are involved in the following diseases:
Graft versus host disease (GVHD): β7 -/- donor T cells caused less GVHD morbidity and
mortality than wild-type (WT) donor T cells in a murine hematopoietic stem cell
transplantation model because of selectively decreased T-cell infiltration of the liver and
intestines (Waldman et al. 2006).
Inflammation and cancer: Polycyclic aromatic hydrocarbons (PAHs) are a major class of
chemical pollutants to which humans are widely exposed, that are generated by the burning of
fossil fuels or wood (Burchiel 1999). They are notably found in diesel exhaust particles,
cigarette smoke, charcoal barbecued foods, and industrial waste byproducts (Burchiel 1999).
PAHs were found to upregulate the expression of β7 integrins in human macrophages through
both the aryl hydrocarbon receptor (AhR) and the basic leucine zipper transcription factor cmaf
(Monteiro et al. 2007). This may contribute to the inflammatory effects PAHs have by
altering macrophage homing to epithelial tissues (Haskill et al. 1992). The carcinogenic
effects PAHs may be mediated in part by overexpression of c-maf/β7, which increases the
development of myelomas (Hurt et al. 2004) and T-lymphomas (Morito et al. 2006).
Human immunodeficiency virus (HIV): Recently, β7 integrins have been identified as playing
a new role in HIV infection. It was suggested that HIV-1 is able to mediate depletion of gut
CD4+ T-cells in part by binding to the integrin α4β7, which helps direct T-cells to the gut
(Arthos et al. 2008). HIV-1 envelope glycoprotein 120 (gp120) was found to bind to α4β7
independently of the principle HIV receptor CD4, and antibodies against α4β7 could block
the binding of gp120 to α4β7 (Arthos et al. 2008). The binding of HIV-1 to α4β7 induced the
activation of LFA-1, which has a key role in immune cell interactions.
Diabetes: Mucosa-associated lymphocytes expressing high levels of β7 integrins associate
and accumulate in pancreatic islets during insulitis in the nonobese diabetic (NOD) mouse
model of type 1 diabetes (Hanninen et al. 1996). Antibody blockade of α4 and β7 integrins
and their ligand MAdCAM-1 successfully interrupted the development of diabetes
development in NOD mice (Kommajosyula et al. 2001).
47

Crohn’s disease and colitis: In the Tnf(DeltaARE) mouse model of Crohn's-like
inflammatory bowel disease, development of intestinal inflammation was found to be
critically dependent on β7 integrin-mediated T-lymphocyte recruitment (Apostolaki et al.
2008). Furthermore, a mAb against αEβ7 prevents immunization-induced colitis in IL-2 -/-
mice (Ludviksson et al. 1999).
Therapeutic agents based on α4β7
A specific barbituric acid-based inhibitor that blocks α4β7-MAdCAM-1 interactions, but
spares α4β1-VCAM-1 interactions was recently reported (Harriman et al. 2008). Small
interference RNA (siRNA) against cyclin D1 specifically directed to leukocytes expressing
β7 integrin shows promise for the treatment of inflammatory bowel disease (Peer et al. 2008).
Monoclonal antibodies (mAb) are being developed to treat Crohn’s disease and ulcerative
colitis, and nonpeptide chemicals are being developed to treat inflammatory bowel disease
and multiple sclerosis (Hehlgans et al. 2007). In addition, there are several drugs currently
being developed and in clinical trials that specifically target α4β7, such as Vedolizumab, a
humanised monoclonal antibody for treatment of inflammatory bowel disease (Soler et al.
2009).
Clinical examples of therapeutic agents targeting integrins
Examples of drugs in the clinic are; Tysabri (Natalizumab), a humanised monoclonal
antibody against α4 subunit is used for therapy for multiple sclerosis (Sheremata et al. 1999)
and Crohn’s disease (Ghosh et al. 2003), and Efelizumab, a humanised anti-αL (CD11a)
monoclonal antibody is used for treating psoriasis and tissue rejection (Dedrick et al. 2002;
Schon 2008).
48

1.8. Aims of this thesis
Integrins signal through their relatively short cytoplasmic domains by binding to key
signalling molecules and cytoskeletal elements that change the affinity of an integrin for its
ligand, or induce clustering and changes in avidity. The β7 integrins are leukocyte-specific
integrins that play central roles in gut mucosal immunity, but also contribute to the
progression of an array of chronic inflammatory diseases. The host laboratory discovered a
cell adhesion regulatory domain (CARD) within the cytoplasmic domain of the β7 subunit
that regulates β7 integrin-mediated cell adhesion. Further investigation of this domain, and
identification of intracellular ligands for the β7 cytoplasmic domain, may provide insights
into signalling pathways controlling β7 integrin-mediated cell adhesion. Armed with this
information, it might be possible to devise therapeutic interventions to prevent β7 integrin-
mediated inflammatory disease.
The broad aim of this thesis was to identify β7 integrin signalling pathways and the molecules
involved. One of the specific objectives was to investigate structure-function relationships of
the β7 CARD by identifying residues that are essential for function. Another objective was to
identify intracellular signalling molecules that interact with the CARD, and the β7
cytoplasmic domain, and determine whether they influence α4β7-mediated adhesion of T
cells.
49

Chapter 2. Materials and Methods
Section 1 – Materials
2.1. Chemicals, reagents, buffers and media
2.1.1 Suppliers
Abcam Inc., (Cambridge, MA, USA)
Alpha Diagnostic (San Antonio, TX, USA)
American Type Culture Collection, ATCC (Manassas, VA, USA)
Amersham Bioscience – now GE Healthcare (Buckingham, United Kingdom)
AppliChem (Darmstadt, Germany)
Bangs Laboratories Inc. (Fishers, IN, USA)
BD Biosciences (Franklin Lakes, NJ, USA)
BDH chemicals (Poole, United Kingdom)
Bio-Rad Laboratories (Hercules, CA, USA)
Biosource – now supplied by Invitrogen
Biovision (Mountain View, CA, USA)
Calbiochem – now supplied by Merck
Cell Signaling Technology (Danvers, MA, USA)
Chemicon International – now supplied by Millipore Corp.
Cytoskeleton Inc. (Denver, CO, USA)
DIFCO – now supplied by BD Biosciences
Eppendorf AG (Hamburg, Germany)
GE Healthcare (Chalfont St. Giles, United Kingdom)
GibcoBRL/Life Technologies – now supplied by Invitrogen
Immunochemistry Technologies, LLC. (Bloomington, MN, USA)
Invitrogen (Carlsbad, CA, USA)
Merck KGaA. (Darmstadt, Germany)
50

Millipore Corp. (Bedford, MA, USA)
Mimotopes Pty Ltd. (Melbourne, Australia)
Molecular Probes – now supplied by Invitrogen
Nunc – now part of Thermo Fisher Scientific
Peptide 2.0 Inc. (Chantilly, VA, USA)
Pierce (Rockford, IL, USA)
Pharmacia Biotech (Piscataway, NJ, USA)
Progen Biotechnik GmbH (Heidelberg, Germany)
ProSpec-Tany TechnoGene Ltd (Rehovot, Israel)
Promega Corp. (Madison, WI, USA)
Qiagen Inc. (Hilden, Germany)
R&D Systems (Minneapolis, MN, USA)
Roche Applied Science (Roche; Mannheim, Germany)
Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA)
Scharlau Chemie S.A. (Barcelona, Spain)
SERVA Electrophoresis (Heidelberg, Germany)
Sigma-Aldrich (St. Louis, MO, USA)
Thermo Fisher Scientific Inc. (Waltham, MA, USA)
Upstate Cell Signaling Solutions – now supplied by Millipore
USB Corporation. (Cleveland, OH, USA)
United States Biological (Swampscott, MA, USA)
Vector laboratories (Peterborough, United Kingdom)
51

2.1.2 Chemicals/Molecular biology reagents
All chemicals were purchased from; AppliChem, BDH chemicals, Bio-Rad Laboratories,
Invitrogen, Scharlau Chemie S.A., or Sigma-Aldrich, unless otherwise stated.
Molecular biology reagents:
ABTS – Vector Labs, cat#SK4100
Agarose LE – Roche Diagnostic, cat #1685678
Alkaline phosphatase – Roche Diagnostic, cat #713023
Ampicillin – Roche Diagnostic, cat #835269
Aprotinin – Sigma-Aldrich, cat #A6103
Aquacide II – Calbiochem, cat #17851
Bovine serum albumin (BSA) – Gibco, cat #30036-578
Bromophenol blue – BDH chemicals, cat #200152E
CMFDA – Invitrogen, cat#C7025
Complete Mini EDTA-free caspase inhibitors – Roche Diagnostic, cat #11836171001
Coomassie Brilliant Blue R250 – DIFCO, cat#5128-13
Cyanogen bromide-activated Sepharose 4B – Amersham Bioscience, cat#17 0430 01
Deoxynucleoside triphosphate set – Roche, cat #1969064
Dialysis tubing (cellulose membrane) – Sigma-Aldrich, cat #D-9277
Dimethyl sulfoxide (DMSO) – Sigma-Aldrich, cat #D-5879
DL-Dithiothreitol (DTT) – Sigma-Aldrich, cat #D0632-10G
DNA ladder, 1 kb plus – Invitrogen, cat #10787-026
Glutathione, reduced – Sigma-Aldrich, cat #G-4251
Hybond-C Extra nitrocellulose membrane – Amersham Bioscience, cat #RPN303E
Hybond-P PVDF membrane – Amersham Bioscience, cat #RPN303F
Kodak BioMax film – Kodak Company, cat #1651454
LB Broth Base – Invitrogen, cat #1270-052
Leupeptin – Sigma-Aldrich, cat#L9783
Lipofectamine-2000 reagent – Invitrogen, cat #11668-019
Lysozyme – Roche Diagnostic, cat #107255
Magnetic beads (Dynabeads® M-280 Streptavidin) – Invitrogen, cat#112-05D
2-β-mercaptoethanol – Sigma-Aldrich, cat #M-7522
Methylene blue – USB, Cat#19220
Pepstatin A – Sigma-Aldrich, cat#P5318
Peptone 140 – GibcoBRL, cat #30392-021
Phenylmethylsulphonyl fluoride (PMSF) – Sigma-Aldrich, cat#P7626
Phorbol-12-myristate 13-acetate (PMA) – Sigma-Aldrich, cat#P8139
PhosSTOP phosphatase inhibitor cocktail – Roche Applied Science, cat#04906845001
Precision Plus protein standard (All Blue) – Bio-Rad Laboratories, cat #161-0373
52

Precision Plus protein standard (Unstained) – Bio-Rad Laboratories, cat #161-0363
Protease inhibitor cocktail - Roche Applied Science, cat #11836145001
Protein A coated polystyrene microspheres (0.95μm) – Bangs Laboratories, cat#CP02N
Protein G Sepharose 4B – Sigma-Aldrich, cat#P3296
Random oligonucleotide primers – Invitrogen, cat #48190011
RNase A – Sigma-Aldrich, cat #R4875
Rubidium chloride – Sigma-Aldrich, cat #R-2252
SuperScriptTM First-strand synthesis system - Invitrogen, cat #11904-018
SuperScriptTM RNase H- reverse transcriptase – Invitrogen, cat #18064-014
T4 DNA ligase – Roche Diagnostic, cat #10481220001
Triton X-100 – BDH chemicals, cat #306324N
TRIzol reagent – Invitrogen, cat #15596-018
Yeast extract – Merck, cat #1.03753
2.1.3 Buffers/Solutions
Buffer/Solution Composition
AGE buffer (10x; DNA Loading
Buffer)
0.25% Bromophenol Blue
0.1% Sodium dodecyl sulphate (SDS)
100 mM Tris(hydroxymethyl)methylamine (Tris)-Cl
100 mM Ethylenediaminetetra-actetic acid disodium salt
(EDTA)
50% (v/v) Glycerol
pH 8.0
Agarose gel (1%) 1% (w/v)Agarose in deionised water
Antibiotics:
Ampicillin (Sigma-Aldrich) was used at a final concentration of
100 μg/mL
Geneticin (G-418 sulphate; Gibco, Invitrogen)
Hygromycin B (Invitrogen)
EDTA 0.5 M EDTA pH 8.0
LB agar 10% (w/v) Bacto-peptone
5% (w/v) Bacto-yeast extract
10% (w/v) Sodium chloride (NaCl)
1.5% (w/v) Agar
LB medium 10% (w/v) Bacto-peptone
5% (w/v) Bacto-yeast extract
10% (w/v) NaCl
4% Paraformaldehyde 4% (w/v) Paraformaldehyde made in PBS, pH 7.2
Phosphate Buffered Saline (1X; 137 mM NaCl
PBS)
2.7 mM Potassium chloride (KCl)
1.4 mM Potassium dihydrogen orthophosphate (KH2PO4)
4.3 mM Disodium hydrogen orthophosphate (Na2HPO4.7H2O)
pH 7.4
STE buffer
10 mM Tris-Cl pH 8
1 mM EDTA
50 mM NaCl
TAE (50x)
2 M Tris
1 M Acetic acid
0.2 M EDTA
pH 8.2
53

Cell culture solutions:
AlF4 - 10mM Sodium fluoride (NaF)
40µM Aluminium chloride (AlCl3)
Magnesium solution 200 mM Magnesium chloride (MgCl2)
200 mM Calcium chloride (CaCl2)
Manganese solution 200 mM Manganese Chloride (MnCl2)
200 mM CaCl2
HBSS buffer w/o Ca/Mg (1x) 5.36 mM KCl
0.44 mM KH2PO4
0.14 mM NaCl
0.34 mM Na2HPO4.2H2O
5.55 mM D-glucose
10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) solution
pH 6.5
Methylene blue 0.1% (w/v) Methylene blue in water
Solutions to prepare competent cells:
Psi broth 5% (w/v) Bacto-yeast extract
20% (w/v) Bacto-peptone
5% (w/v) Magnesium sulphate (MgSO4)
water to 1L, pH to 7.6 with potassium hydroxide (KOH)
TfbI
30 mM Potassium acetate
100 mM Rubidium chloride (RbCl)
10 mM CaCl2
50 mM MnCl2
15% (v/v) Glycerol
pH to 5.8 with dilute acetic acid
TfbII
10 mM 3-[N-Morpholino] propane sulphonic acid (MOPS)
75 mM CaCl2
10 mM RbCl
15% (v/v) Glycerol
pH to 6.5 with dilute NaOH
Western blot solutions:
TTBS 102.7 mM NaCl
24.7 mM Tris-Cl
2.7 mM KCl,
pH 8.0
0.1% Tween-20
Transfer buffer 48 mM Tris
39 mM Glycine
0.037% SDS
20% v/v Methanol
Blocking buffer TBS-T containing 5% (w/v) non-fat milk powder, or 2% ECL
advanced blocking solution (Amersham)
Western blot buffer TBS-T containing 3% (w/v) non-fat milk powder, or 2% ECL
advanced blocking solution (Amersham)
2-ME stripping solution 62.5 mM Tris pH 7.2
100 mM 2-β-mercaptoethanol
2% (w/v) SDS
High-pH-stripping solution 0.2 M NaOH
54

Phosphorylation assay solutions:
Cell lysis buffer
10 mM Tris pH 7.4
50 mM NaCl
50 mM NaF
1 mM Sodium orthovanadate (Na3VO4)
300 μg/mL PMSF
1% NP-40
Protease inhibitors:
20 μg/mL Aprotinin
10 μg/mL Leupeptin
10 μg/mL Pepstatin A
Or 1 x Complete EDTA-free protease inhibitor cocktail (Roche)
Phosphatase inhibitors:
1 x PhosSTOP phosphatase inhibitor cocktail (Roche)
Kinase buffer 10 mM Tris pH 7.4
50 mM NaCl
5 mM MgCl2
5 mM MnCl2
0.1 mM Na3VO4
Plasmid preparation solutions:
Solution I (GET)
Solution II (NaOH/SDS)
Solution III (Potassium acetate)
SDS-PAGE solutions:
50 mM Glucose
10 mM EDTA
25 mM Tris-Cl (pH 8)
0.2 M NaOH
1% (w/v) SDS
3 M Potassium acetate
11.4% (v/v) Acetic acid
pH 5.2
30% Acrylamide mixture 30% (w/v) Acrylamide
0.2% Bis-acrylamide
2 x Crosslinker acrylamide 48% (w/v) Acrylamide
3.0% (w/v) Bis-acrylamide
Separating Tris-glycine buffer 1.5 M Tris-Cl pH 8.8
Stacking Tris-glycine buffer 0.5 M Tris-Cl pH 6.8
Separating/spacing Tris-tricine 3 M Tris base
buffer
0.3% SDS
pH 8.9 with HCl
Stacking Tris-tricine buffer 1 M Tris-HCl pH 6.8
10% SDS 10% (w/v) SDS
10% APS 10% (w/v) Ammonium persulfate
SDS-PAGE Tris-glycine running 274.4 mM Tris
buffer (10x)
2 M Glycine
10% (w/v) SDS
Cathode 10x buffer
1 M Tris base
1 M Tricine, 1% w/v SDS
Anode 10x buffer 2 M Tris-Cl pH 8.9
SDS-PAGE reducing loading 10% (w/v) SDS
buffer (5x)
30% (w/v) Glycine
360 mM Tris-Cl pH 6.8
0.5 M DTT
0.03% Bromophenol blue
55

SDS-PAGE staining solutions:
Coomassie blue staining solutions
Fixing solution 25% (v/v) Propanol
10% (v/v) Acetic acid
Staining solution (Coomassie blue 0.006% Coomassie brilliant blue R-250
stain)
40% (v/v) Methanol
10% (v/v) Acetic acid
Destaining solution 50% (v/v) Methanol
10% (v/v) Acetic acid
Silver Staining solutions
Fixation solution 40% (v/v) Ethanol
10% (v/v) Acetic acid
Sensitizing solution 30% (v/v) Ethanol
0.125% Glutaraldehyde
0.2% Sodium thiosulphate
6.8% (w/v) Sodium acetate
(make in fume hood)
Silver reaction solution 0.25% Silver nitrate
0.04% Formaldehyde
(make in fume hood)
Developing solution 2.5% (w/v) Sodium carbonate
0.02% Formaldehyde
(make in fume hood)
Stopping solution 1.5% (w/v) EDTA
Preserving solution 10% (v/v) Glycerol
2.1.4 Tissue culture reagents
RPMI 1640 / Advanced RPMI 1640 (23400-062 / 12633-012, GibcoBRL ® / Invitrogen)
Dulbecco's Modified Eagle's Medium (DMEM) / Advanced DMEM (30-2002 /12491-015,
GibcoBRL ® / Invitrogen)
Sf-900 II SFM (10902088, GibcoBRL ® / Invitrogen)
Penicillin, streptomycin, glutamine (PSG; 10378016, GibcoBRL ® / Invitrogen) - 2 mM
glutamine, 50 μg/mL penicillin, 50 μg/mL streptomycin.
2-β-mercaptoethanol (M-7522, Sigma-Aldrich) was used at a final concentration of 0.05 mM.
Fetal calf serum / fetal bovine serum (FCS; GibcoBRL ® / Invitrogen) was heat-inactivated for
30 min at 56°C before use. It was added to culture media at a final concentration of 10%
(v/v).
Recombinant human tumour growth factor β1 (TGF-β1; 100-21B, Peprotech) was kept as a
stock solution at 50 µg/mL in PBS containing 2 mg/mL of albumin, and stored at -20 °C.
56

2.2. Molecular biology reagents
2.2.1 Mammalian cell lines
TK-1 (CRL-2396 , ATCC ® ) – Mouse CD8+ T lymphoma cell line from the AKR/CUM
mouse strain which expresses high levels of α4β7, but no β1 integrins. Cultured in RPMI-
1640 supplemented with 10% FCS, PSG, 2 ME at 37°C in 5% CO2.
MTC-1 (kindly donated by Dr P Kilshaw, AFRC Babraham Institute, Cambridge, UK) – T
cell hybridoma which expresses high levels of αEβ7. Cultured in RPMI-1640 medium
supplemented with 10% FCS, PSG,, and 5 ng/ml TGF-β1 at 37°C in 5% CO2.
HEK-293T (CRL-1573 , ATCC ® ) – Human epithelial kidney cell line transformed with the
SV40 large T-antigen. Cultured in DMEM, containing 10% FCS, and PSG at 37°C in 10%
CO2.
H9 (HTB-176 , ATCC ® ) – Human T cell lymphoma. Cultured in RPMI, containing 10%
FCS, and PSG at 37°C in 5% CO2.
CHO-K1 (CRL-9618 , ATCC ® ) – Chinese hamster ovary cells. Cultured in DMEM,
containing 10% FCS, and PSG at 37°C in 10% CO2.
C2C12 (CRL-1772 , ATCC ® ) – Mouse myoblast cells. Cultured in DMEM, containing 10%
FCS, and PSG at 37°C in 10% CO2.
2.2.2 Insect cell line
Sf9 (CRL-1711 , ATCC ® ) – Spodoptera frugiperda (fall armyworm) epithelial ovary cells.
Grown in Sf-900 II SFM.
2.2.3 Bacterial cell line
DH5α (Stratagene): This is a derivative of E.coli K12 (hsd R17, supE44, rec A1, endA1,
lacZM5) which provided a source of competent cells for DNA subcloning.
57

2.2.4 DNA cloning vectors
pGEM ® -T (A3600, Promega): The pGEM-T vector system was used for cloning PCR
products.
pGEX-2T/2TK (27-4801-01/27-4587-01, GE Healthcare Life Sciences): The pGEX plasmids
are designed for inducible, high-level expression of genes or gene fragments as fusions with
Schistosoma japonicum 26kDa glutathione S-transferase (GST).
pcDNA3.1/Hygro © (V870-20, Invitrogen): pcDNA3.1 is derived from the pcDNA vector and
is designed for high-level stable and transient expression of proteins in mammalian host cells,
using the hygromycin resistance gene for selection.
pcDNA TM 6/V5 (V220-01, Invitrogen): pcDNA6/V5 vector is derived from pcDNA3.1(+) and
is designed for high-level stable and transient expression and detection of recombinant
proteins in mammalian host cells. The vector contains the blasticidin resistance gene from
Aspergillus terreus for cell selection.
pIRES2-EGFP (6029-1, BD Biosciences): pIRES2-EGFP contains an internal ribosome entry
site of the encephalomyocarditis virus between the multiple cloning site (MCS) and the
enhanced green fluorescent protein (EGFP) allowing the translation of both the gene of
interest and the EGFP gene.
2.2.5 Enzymes/isotopes
Restriction enzymes: BamHI, BglII, EcoRI, EcoRV, HindIII, KpnI, NheI, NotI, SacI, XbaI,
and XhoI. All restriction enzymes were purchased from Roche Diagnostics.
T4-DNA ligase: Manufactured by Promega.
Expand High Fidelity PCR System DNA polymerase: From Roche Diagnostics.
Ribonuclease A (RNase A): Manufactured by Sigma-Aldrich, and resuspended at a stock
concentration of 10 μg/mL in 10 mM Tris (pH 7.5) and 15 mM NaCl. The solution was
heated at 100°C for 15 min to inactivate contaminating DNase activity, and aliquots were
stored at –20°C.
Isotopes: 32 P-γATP: [γ 32 P] – [adenosine 5’-triphosphate]-[tetra(triethylammonium) salt] from
Amersham Biosciences.
58

2.2.6 Antibodies
Antigen recognised Company or source Dilution used
β-actin ab8227, Abcam 1:1000
E-cadherin BTA3, R & D Systems 1:200
E-cadherin ECCD-1, R & D Systems 25 μg/mL
FAK PC314, Calbiochem 1:200
Hsp90 (N-17) sc-1055, Santa Cruz Biotechnology 1:200
Hsp70 OBT1688, AbD Serotec 1:200
integrin α4 (PS/2) Produced from hybridoma cells by host 1:200
laboratory (Purchased from ATCC)
integrin α4 120-65984, Sapphire Biosciences 1:200
integrin α-M290 Produced from hybridoma cells by host
integrin-αE (R-15)
laboratory (Kindly donated by Dr P Kilshaw,
AFRC Babraham Institute, Cambridge, UK)
sc-6607, Santa Cruz Biotechnology 1:200
Integrin-α4β7 (DAKT32) Produced from hybridoma cells by host 1:100
laboratory (Purchased from ATCC)
integrin-β7 (Fib504) Produced from hybridoma cells by host 1:100
integrin-β7-cytoplasmic domain
laboratory (Purchased from ATCC)
Produced by host laboratory (Krissansen et al. 1:400
1992)
integrin-β7-extracellular domain AF4669, R & D Systems 1:1000
lck sc-13, Santa Cruz Biotechnology 1:200
MAdCAM-1 (MECA357) Produced by host laboratory 25 μg/mL
phosphotyrosine Ab-4, Calbiochem 1:200
src ABsrc.1, Oncor
src 2109, Cell Signaling Technology 1:200
src 2110, Cell Signaling Technology 1:200
Secondary antibodies:
Goat anti-human-Fc-IgG A0170, Sigma-Aldrich 1:1000
Goat anti-mouse IgG peroxidase
conjugated
A4416, Sigma-Aldrich 1:80,000
Goat anti-rabbit IgG peroxidase
conjugated
A0545, Sigma-Aldrich 1:10,000
Mouse anti-goat/sheep IgG A9452, Sigma-Aldrich 1:80,000
peroxidase conjugated
Alexa Fluor® 488 goat anti-rat
IgG
A-11006, Molecular Probes
1:250
Alexa Fluor® 594 goat antirabbit
IgG
A-11012, Molecular Probes 1:250
Alexa Fluor® 594 donkey antigoat
IgG
A-11058, Molecular Probes
1:250
Other Antibodies:
Mouse ascites Produced by host laboratory
Rat-IgG I8015, Sigma-Aldrich
59

2.2.7 PCR oligonucleotide primers
All oligonucleotide primers were purchased from Invitrogen.
Primers for integrin α4 cloning:
CC28 primer: 5’ AAGCTTGATATCATGGCTTGGGAAGCGAGG 3’
CC29 primer: 5’ AAACACTCTTCCTTCCTCTC 3’
Primers for integrin β7 cloning:
CC24 primer: 5’ GGATCCGAGCTCCTGGGGATGTGTGCGA 3’
CC25 primer: 5’ GATTTCCACGGAGAGCGGGTAAGCCAGGAC 3’
CC26 primer: 5’ GTCCTGGCTTACCGGCTCTCGGTGGAAATC 3’
CC27 primer: 5’ GAATTCGCGGCCAGAGAGTGGGACTG 3’
2.2.8 cDNA for cloning/protein expression
GST-β7 cytoplasmic domain construct: A pGEX-2T construct encoding the cytoplasmic
domain of the β7 subunit was obtained from Dr Andrew Lazarovits, Imperial Cancer
Research Fund, London, UK.
pGEX-2T vectors encoding mutated forms of the β7 subunit cytoplasmic domain were
constructed by Dr Klaus Lehnert of the host laboratory (The University of Auckland).
α4 full-length DNA construct in pCDM8 kindly donated by Professor B. Seed (Harvard
Medical School Massachusetts General Hospital, MA, USA).
β7 full-length DNA construct in pCDM8 constructed by Dr Euphemia Leung of the host
laboratory (The University of Auckland).
60

2.2.9 Recombinant proteins
Name Companies
FAK PKA-312, ProSpec; PHO3145, BioSource
src PKA-317, ProSpec; S5439, Sigma-Aldrich
p56lck (lck) L2792, Sigma-Aldrich
Hsp70a HSP-170, ProSpec
Filamin 62006, Progen
Paxillin P3113-85, United States Biological
α-actinin AT01, Cytoskeleton Inc.
2.2.10 Inhibitors of intracellular signalling pathways
PD98059 – C16H13NO3, 2′-Amino-3′-methoxyflavone (Cat. No. 513000, Calbiochem) –
Inhibitor of MAP kinase kinase (MEK).
SB203580 – C21H16N3OSF, 4-(4-Fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)1H-
imidazole, (Cat. No. 559389, Calbiochem) – Inhibitor of p38 MAP kinase.
JNK-I – C168H293N67O42, (L)-HIV-TAT48-57-PP-JBD20, (L)-JNKI1, c-Jun NH2-terminal kinase
H-GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQDT-NH2, SAPK Inhibitor I (Cat. No.
420116, Calbiochem) – Blocks c-Jun NH2-terminal kinase (JNK) signalling.
JNK-II – C14H8N2O, Anthra[1,9-cd]pyrazol-6(2H)-one, 1,9-pyrazoloanthrone, SAPK
Inhibitor II, SP600125 (Cat. No. 420119, Calbiochem) – Inhibitor of c-Jun N-terminal kinase
(JNK).
Radicicol – C18H17ClO6 (Cat. No. 553400, Calbiochem) – Inhibits v-src kinase activity.
AG99 – C10H8N2O3, a-Cyano-(3,4-dihydroxy)cinnamide, Tyrphostin A46, Tyrphostin B40
(Cat. No. 658430, Calbiochem) – Inhibits epidermal growth factor (EGF) receptor tyrosine
kinase.
PP2 – C15H16ClN5, AG 1879, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4d]pyrimidine
(Cat. No. 529576, Calbiochem) – Inhibitor of the src family of protein tyrosine
kinases. Inhibits lck, fyn, hck, and src.
Damnacanthal – C16H10O5, 3-Hydroxy-1-methoxyanthraquinone-2-aldehyde (Cat. No.
251650, Calbiochem) – Inhibitor of the tyrosine kinase lck.
61

ML-7 – C15H17IN2O2S . HCl, 1-(5-Iodonaphthalene-1-sulfonyl)homopiperazine-HCl (Cat. No.
345834 , Calbiochem) – Inhibitor of myosin light chain kinase (MLCK).
Genistein – C15H10O5, 4′,5,7-Trihydroxyisoflavone (Cat. No. 345834, Calbiochem) – Inhibits
protein tyrosine kinases.
KNK-437 – C13H11NO4, Heat shock protein inhibitor 1, N-Formyl-3,4-methylenedioxybenzylidine-γ-butyrolactam
(Cat. No. 373260, Calbiochem) –Inhibits heat shock proteins,
including HSP70, Hsp72, and HSP105.
2.2.11 Peptides
Biotin-rrrrrrrrr-pptdqsrpvqpflnlttprkpr-OH (Mimotopes) – used as a negative control peptide
for poly-arginine containing peptides, it is an inverse sequence of part of the mitogen-
activated protein kinase 8 interacting protein 1 (157-176 aa)
Biotin-APTLPPAWQPFLK-OH (Mimotopes) – used as a negative control peptide for non-
poly-arginine containing peptides, it is part of the baculoviral IAP repeat-containing 5 protein
(3-15 aa)
Biotin-GGYDRREY-OH (GGYDRREY; Mimotopes)
Biotin-rrrrrrrrr-YDRREY-OH (YDRREY; Mimotopes)
Biotin-rrrrrrrrr-YDRRE-OH (YDRRE; Mimotopes)
Biotin-rrrrrrrrr-DRREY-OH (DRREY; Mimotopes)
Biotin-rrrrrrrrr-YDGGEY-OH (YDGGEY; Mimotopes)
Biotin-rrrrrrrrr-YEEEEY-OH (YEEEEY; Mimotopes)
Biotin-rrrrrrrrr-YDRGGGGREY-OH (YDRGGGGREY; Mimotopes)
Biotin-rrrrrrrrr-xDRREx-OH (xDRREx; Mimotopes)
Biotin-rrrrrrrr-YDRREYGYDRREYGYDRREYGYRDDEY-OH (pYDRREY; Mimotopes)
Biotin-rrrrrrrr-FDRREFGFDRREFGFDRREFGFDRREF-OH (pFDRREF; Mimotopes)
62

5-6-FAM-rrrrrrrrr-YDRREY-OH (FAM-YDRREY; Mimotopes)
Biotin-rrrrrrrrr-SILQEENRRDSWSYI-OH (Mimotopes) – α4-integrin-paxillin binding site.
Biotin-RLSVEIYDRREYSRFEKEQQQLNWKQDSNPLYKSAITTTINPRFQEADSPTL-
OH (Synthesized by Chemistry Department, The University of Auckland) – full length β7
subunit cytoplasmic domain
Biotin-rrrrrrrr-NPLY-OH (NPLY; Peptide 2.0)
Polymer of glutamate and tyrosine (4:1), (PolyEY, Sigma-Aldrich)
63

Section 2 – Methods
2.3. Molecular biology techniques
2.3.1 Preparation of competent cells
DH5α E. coli were inoculated into Psi broth and cultured at 37°C overnight. One millilitre of
overnight culture was inoculated into 100 mL of Psi Broth, and incubated at 37°C with
aeration to an optical density of 0.48 at 550 nm. The culture was then placed on ice for 15
min, and cells pelleted at 5,000 × g (Sorvall) for 5 min. The pellet was resuspended in 0.4
volume of TfbI, and placed on ice for a further 15 min. Cells were pelleted again at 5,000 × g
for 5 min, resuspended in 0.04 volume of TfbII, and placed on ice for a further 15 min.
Aliquots of 100 μL were snap-frozen in ethanol-dry ice, and stored at –80°C until needed.
2.3.2 Small scale preparation of plasmid DNA (miniprep)
DH5α E. coli that were transformed with the desired plasmid were grown overnight
(approximately 16 hrs) in 5 mL of LB medium containing 100 μg/mL of ampicillin (amp). A
1.5 mL aliquot of culture was centrifuged for 1 min at 12,000 × g. The pellet was resuspended
in 100 μL of GTE buffer (solution 1) by vortexing, and left for 5 min at room temperature
(RT). The cells were lysed by the addition of 200 μL of lysis solution (solution 2) with gentle
inversion 5 times, and left to stand for a further 5 min. The solution was neutralised with the
addition of 150 μL of ice-cold potassium acetate (solution 3) and mixed immediately by
repeated inversion. The mixture was centrifuged for 10 min at maximum speed (12,000 × g)
on a bench top centrifuge and the supernatant transferred into 500 μL of chloroform (CHCl3),
followed by vortexing. The mixture was centrifuged a further 10 min at 12,000 × g, and 80-
90% of the top aqueous phase was transferred into a new tube containing 1 mL of 100%
ethanol. This mixture was then vortexed, and centrifuged for 5 min at 12,000 × g. The pellet
was washed once with 70% ethanol, air-dried at 37°C, then re-suspended in 30 μL of sterile
water containing RNase (10 μL/mL), and stored at –20°C until needed. Plasmids to be
subjected to DNA sequencing were purified using a Qiagen ® Rapid Plasmid Mini Kit
according to the manufacturer’s instructions. All DNA sequencing was carried out at the
DNA sequencing facility in the School of Biological Sciences, Faculty of Science, University
of Auckland.
64

2.3.3 Large scale preparation of plasmid DNA
Large scale plasmid preparations were carried out using either a midiprep kit from Invitrogen,
or by centrifugation through caesium chloride gradients.
Large scale preparation of plasmid DNA with the midiprep kit
Purification of plasmids was carried out using the PureLink HiPure Midiprep kit
(Invitrogen) by following the manufacturer’s instructions. All required buffers were provided
with the kit. The following is a brief description of the method. DH5α E. coli containing the
desired plasmid was cultured overnight in LB media at 37°C. The bacteria were pelleted and
resuspended in 4 mL of Resuspension Buffer containing RNase, and then lysed by the
addition of 4 mL of Lysis Buffer with gentle inversion. The solution was neutralised using 4
mL of Precipitation Buffer and the mixture was centrifuged at 12,000 × g for 10 min at RT.
The soluble fraction was passed through the provided columns and washed twice with 10 mL
of Wash Buffer, and the DNA was eluted with 5 mL of Elution Buffer. The DNA was
precipitated with the addition of 3.5 mL of isopropanol and pelleted at 15,000 × g for 30 min
at 4°C. The pellet was further washed with 3 mL of 70% ethanol and re-pelleted at 15,000 × g
for 30 min at 4°C. The pellet was air-dried for 10 min at 37°C and the DNA pellet was
resuspended in 200 µL of water and stored at -20°C until needed.
Large scale preparation of plasmid DNA with caesium chloride
Bacteria transformed with a desired plasmid were cultured overnight in LB medium with 100
μg/ml of amp. Five millilitres of the resulting culture was inoculated into 500 ml of fresh LB
medium containing 100 μg/ml amp. The culture was incubated overnight at 37°C with
vigorous shaking. The bacteria were collected by centrifugation at 12,000 × g, and then
resuspended in 40 ml of GTE buffer (solution 1). The bacteria were lysed by the addition of
80 ml of 0.2 M NaOH/1% SDS (solution 2) with gentle inversion. The solution was
neutralized by the addition of 40 ml of ice-cold potassium acetate (solution 3), mixed
thoroughly by inversion, and placed on ice for 10 min. The mixture was centrifuged at 12,000
× g for 20 min at 4°C. The supernatant was transferred to a new bottle, and 90 ml of
isopropanol was added followed by centrifugation at 12,000 × g for 20 min at 15°C. The
pellet was washed with 40 ml of 70% ethanol followed by centrifugation at 12,000 × g for 5
min at 4°C. The plasmid DNA was resuspended in 4 ml of water and mixed with 4.5 g of
caesium chloride. To the mixture was added 100 μl of ethidium bromide solution (stock 10
mg/ml) and 51 μl of Triton-X100 (1:100 stock). The mixture was transferred into an
65

ultracentrifuge tube, and centrifuged for 22 hrs at 78,000 rpm (40,000 × g) at 20°C under
vacuum. The plasmid DNA band, which was coloured a faint pink, was harvested and mixed
with 2x volume of saturated-butanol. The mixture was shaken and left standing until two
layers formed. The pink DNA layer containing traces of ethidium bromide was removed and
saturated-butanol was again added followed by shaking. The pink DNA layer was harvested,
and the process was repeated until the solvent phase became clear. The aqueous phase was
transferred to a clean tube and mixed with 7.5 ml of 100% ethanol. The mixture was
centrifuged at 12,000 × g for 20 min, the supernatant was removed, and the pellet was washed
with 7.5 ml of 70% ethanol and air-dried. The plasmid pellet was resuspended in sterile water
and stored at -80°C.
2.3.4 Linearization and dephosphorylation of plasmid vectors
Plasmid vectors were linearised by digestion with appropriate enzymes for 2 hrs at 37°C,
unless otherwise stated. The linearised plasmids were then de-phosphorylated by adding 2 μL
of alkaline phosphatase to a reaction volume of 50 μL and further incubated at 37°C for 45
min. The DNA was electrophoresed on 0.8-1% agarose gels, and the desired DNA band(s)
was excised. The DNA was isolated from the gel slice using a Wizard ® DNA clean-up kit
(Promega) by following the manufacturer’s instructions.
2.3.5 Isolation of plasmid inserts
DNA inserts were liberated from plasmids by restriction enzyme digestion with appropriate
enzymes and temperatures for 2 hrs. The DNA inserts were electrophoresed on 1-2% agarose
gels, and the desired DNA fragment(s) excised, and purified using Wizard ® DNA clean-up kit
(Promega) following manufacturer’s instructions.
2.3.6 Agarose gel electrophoresis
DNA electrophoresis was carried out in 1x TAE solution using 0.8-2% agarose depending on
the expected size of the desired DNA fragments. Electrophoresis was carried out at 50 V for
1-2 hrs. Ethidium bromide (2 μL of 10 mg/mL) was added to the TAE solution
(approximately 50 mL) after electrophoresis and gently mixed on a platform shaker for 10-15
min. The gels were viewed using an EagleEye © UV light gel imager (Bio-Rad), and
photographs were taken.
66

2.3.7 Ligation of DNA inserts into vectors
Ligation reactions were mixed as shown in the following table.
Reaction Mixture Background control Reaction (1:3) Reaction (1:6)
10x Buffer 1 μL 1 μL 1 μL
Vector 5 μg 5 μg 5 μg
DNA insert - 15 μg 30 μg
T4 DNA ligase - 1 μL 1 μL
Water to 10 μL 10 μL 10 μL
A background control was carried out in which insert and ligase were omitted, to determine
whether the vector was linearised and dephosphorylated properly. Two ligation reactions
using different ratios of vector to insert were carried out. Where two inserts were to be ligated
into one vector, the overall insert concentration was kept at 30 μg/10 μl with a ratio of
approximately 2:1 (smaller : larger) for the DNA fragments to be ligated. All reaction
mixtures were left at either 4°C overnight or at 16°C for 8 hrs.
2.3.8 Transformation
Frozen competent DH5α cells were thawed on 30% ice-water. A 50 μL aliquot of the
competent cells was mixed gently with 5-10 μL of ligation mixture, and incubated on ice-
water for a 15 min. The cell-ligation mixture was then heat-shocked for 45 s at 42°C, then
placed back on ice-water immediately for a further 5 min. The mixture was then added to 1
mL of LB medium (without antibiotics) and incubated at 37°C with aeration for 45 min. The
cells were pelleted at 5,000 × g for 5 min and most of the LB medium was removed leaving
100 μL. The cells were resuspended in the 100 μL solution and spread-out on LB-agar plates
containing antibiotics, and grown overnight at 37°C.
2.3.9 RNA isolation
Cells to be used for RNA isolation were washed and pelleted. An aliquot of 10 7 cells was
incubated with 1 mL of TRIzol reagent for 5 min at RT. The solution was vortexed for 15 s
with the addition of chloroform (0.2 ml per 1 ml of TRIzol), and then incubated for a further
15 s at RT. The mixture was centrifuged at 12,000 × g for 15 min at 4°C, and the aqueous
phase was collected and dispensed into a fresh tube. Propanol (0.5 ml per 1 ml of TRIzol) was
added and the tube was inverted several times in order to mix the solutions. The mixture was
centrifuged at 12,000 × g for 10 min at 4°C and the supernatant discarded. The pellet was
67

washed once with 75% (v/v) ethanol (diluted in DEPC-treated water) and air-dried. The RNA
pellet was resuspended in DEPC-treated water and stored at -80°C.
2.3.10 Synthesis of cDNA by reverse transcriptase
Complementary DNA was synthesized from mRNA using the SuperScript TM First-Strand
Synthesis System from Invitrogen. Total RNA (1-3 μg) was mixed with 1 μl of dNTPs (10
mM), 1 μl of random oligos (50 ng/μl) and DEPC-treated water in a 13 μl reaction mixture.
The mixture was incubated at 65°C for 5 min, quick-chilled on ice, and 2 μl of 10X reverse
transcriptase (RT) buffer, 4 μl of MgCl2 (25 mM), and 2 μl of DTT (0.1 M) were added. The
reaction mixture was incubated at 25°C for 2 min and 1 μl (50 units) of SuperScript TM RT
was added, and the mixture incubated for a further 10 min at 25°C, then at 42°C for 50 min.
The reaction was terminated by heating at 70°C for 15 min, and 1 U of RNase H was added
followed by incubation at 37°C for 20 min before storage at -80°C.
2.3.11 Polymerase chain reaction (PCR)
A polymerase chain reaction was prepared by mixing 15 ng of template DNA or 2 μL of
cDNA from RT of mRNA, 2 mM dNTPs, 1x PCR reaction buffer, 0.1 μM of specific forward
and reverse primers (synthesized by Invitrogen), 2 U High fidelity DNA polymerase (Roche)
or 2 U of Taq polymerase (supplied by Professor John Fraser, Department of Molecular
Medicine & Pathology, The University of Auckland) in sterile MilliQ grade water. The
reactions were carried out in a DNA thermal cycler (PE Applied Biosystems, Gene Amp ®
PCR system 9700). The cycling program consisted of 25 cycles with the following steps: a
denaturation temperature of 95°C for 30 s, an annealing temperature of 50°C to 60°C for 30 s
depending on the annealing properties of the different PCR primer sets, and an elongation
temperature of 72°C for 30 s.
Overlapping PCR
Overlapping PCR was performed in order to join two separate fragments of DNA with
overlapping ends. The PCR was preformed as above with equal amounts of two separate
DNA fragments having overlapping complementary ends.
68

2.4. Protein chemistry
2.4.1 Production and purification of GST fusion proteins
An overnight bacterial culture (5 mL) containing a desired plasmid was inoculated into 500
mL of LB medium containing 100 μg/mL amp, and grown on a bacterial shaker at 37°C until
an optical density at 600 nm of between 0.6 and 0.8 was reached. Synthesis of fusion proteins
was induced by the addition of IPTG (0.4-0.8 mM), and the bacteria cultured for a further 2 to
3 hrs at 30°C or 37°C. The bacteria were collected by centrifugation at 3000 × g for 10 min at
4°C, washed once with PBS, and frozen at –80°C. The frozen pellet was thawed on ice and
resuspended in 10 mL of STE containing 1% (v/v) Triton X-100. Cells were lysed by the
addition of 1 % (w/v) lysozyme, and 10 mM DTT, and sonicated for 1 min. The sonicate was
then centrifuged at 15,000 × g for 10 min at 4°C, and the supernatant was transferred to a 50
ml tube. One millilitre of glutathione-agarose slurry was washed with several volumes of PBS
and added to the supernatant followed by incubation for 2 to 3 hrs on a rotating wheel at 4°C.
The beads and supernatant were transferred to a 10 ml disposable-mini column (Bio-Rad),
washed once with 10 mL of STE, and then with 50 ml of PBS. The beads were kept in PBS
with 50% glycerol containing 0.02% NaH3, and stored at 4°C.
2.4.2 Sodium dodecylsulphate polyacrylamide gel electrophoresis
Proteins were analysed under reducing Sodium dodecylsulphate polyacrylamide gel
electrophoresis (SDS-PAGE) unless otherwise stated. The protein samples were mixed with
an equal volume of SDS-PAGE reducing loading buffer and heated at 98°C for 5 min.
Samples (1-10 µg) were loaded into the wells of the polyacrylamide SDS-gel. Minielectrophoresis
tanks including the Hoefer Mighty Small SE250/SE260 mini vertical unit
(Amersham Bioscience) and the Mini Protean ® II Cell (Bio-Rad) were used. Two types of
SDS gels were employed: Tris-glycine gels for proteins with molecular weights greater then
20 kDa, and Tris-tricine gels for proteins with molecular weights less than 20 kDa. Trisglycine
gels were run using a Tris-glycine running buffer, whereas for Tris-tricine gels the
proteins were resolved using a cathode and anode buffer system. Electrophoresis was carried
out at a constant current of 20 mA per gel for approximately 2-6 hrs depending on
polyacrylamide composition of the gel. If required, gels were either stained with Coomassie
blue or silver stain, and dried using a gel drying system (Model 583 Gel Dryer, Bio-Rad;
DryEase Gel Drying System, Invitrogen) for storage or autoradiography.
69

Tris-glycine gels
Tris-glycine gels were prepared as follows for a 10% polyacrylamide SDS gel.
Separating gel (10 mL):
Water 4 mL
30% Acrylamide mixture 3.3 mL
Separating Tris-glycine buffer 2.5 mL
10% SDS 0.1 mL
10% APS 100 µL
TEMED 4 µL
Stacking gel (4 mL):
Water 2.7 mL
30% Acrylamide mixture 0.67 mL
Stacking Tris-glycine buffer 0.5 mL
10% SDS 40 µL
10% APS 40 µL
TEMED 4 µL
Tris-tricine gels
Tris-tricine gels (16% polyacrylamide) for the separation of low molecular weight proteins
were cast with a separating gel layer, a 1 cm spacing gel, and a stacking gel to form wells for
protein loading. Gels were made by mixing the following reagents:
Separating gel (30 mL):
Water 6.7 ml
Separating/spacing Tris-tricine buffer 10 ml
2 x Crosslinker acrylamide 10 ml
Glycerol 3.2 ml
TEMED 10 μl
10% APS 100 μl
Spacing gel (15 mL):
Water 6.95 ml
Separating/spacing Tris-tricine buffer 5 ml
30% Acylamide mixture 3 ml
TEMED 5 μl
10% APS 50 μl
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Stacking gel (15 mL):
Water 10.3 ml
Stacking Tris-tricine buffer 1.9 ml
30% Acrylamide mixture 2.5 ml
0.5M EDTA 150 μl
TEMED 7.5 μl
10% APS 150 μl
2.4.3 Staining of SDS gels
Coomassie blue staining
SDS gels were fixed with 10% (v/v) acetic acid for 10 min, and then stained with Coomassie
blue staining solution for 30 min with gentle agitation. The gel was then de-stained with destaining
solution under gentle agitation, with the presence of an absorbent sponge or tissue to
aid dye absorption, until bands were clearly visible.
Silver staining
Each silver staining solution was prepared freshly before use. The staining of SDS gels was
carried out at RT. SDS gels were fixed for 30 min with silver staining Fixation buffer,
followed by treatment with Sensitisation buffer for 30 min. The gels were washed with
MilliQ-grade water for at least 3 x 5 min, and silver-stained by incubation in Silver staining
solution for 20 min. Excess staining solution was removed and the gel washed again with
MilliQ-grade water for 2 x 1 min washes. Developing solution was added for 3 to 5 min until
the desired intensity of staining of proteins was obtained. Further development was
terminated by incubating the gel with Stop solution for 10 min. Gels were preserved by
placing in Preserving solution for at least 20 min.
2.4.4 Western blot analysis
Proteins separated by SDS-PAGE were transferred onto Hybond -C-extra nitrocellulose
membrane or Hybond -P PVDF membranes (Amersham Biosciences) using a Semi-Dry
Transfer Unit (Pharmacia Biotech). Three transfer buffer-soaked Whatman filter papers were
placed onto the unit, and overlaid with nitrocellulose / PVDF membrane. An acrylamide gel
was placed on top of the membrane, and covered by three more transfer buffer-soaked filter
papers. Transfer was carried out at 0.8 mA/cm 2 for 2 hrs. The membrane was then stained
with Ponceau S solution to confirm protein transfer, washed in water, and incubated in
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locking solution overnight. The membrane was then washed thrice in TTBS for 5 min each,
and incubated in Western blot solution containing the primary antibody for 1 – 2 hrs at RT or
overnight (16 hrs) at 4°C. The membrane was once again washed thrice in TTBS for 5 min,
and incubated with Western blot solution containing the secondary antibody for 1 hr. Finally,
the membrane was washed thrice for 5 min, and developed with Amersham ECL Plus TM or an
ECL Advance TM kit (Amersham Biosciences). Antibody reactivity was visualised using either
the FujiFilm LAS-3000 scanner (FUJIFILM Cooperation) or by exposure to Kodak BioMax
XAR film (165 1454, Eastman Kodak Company) and development using the Kodak M35 X-
OMAT processor (Eastman Kodak Company).
2.4.5 Stripping of nitrocellulose and PVDF membranes
Stripping of PVDF membranes by heat and detergent.
PVDF membranes were washed with deionised water, and incubated with blocking solution
for 1 hr at RT. They were again washed with deionised water, and incubated with 2-ME
stripping solution for 30 min at 50˚C.
Stripping of nitrocellulose membranes with high pH.
Nitrocellulose membranes were washed with deionised water, and incubated with blocking
solution for 1 hr at RT. Membranes were again washed with deionised water, and incubated
with high pH stripping solution for 10 min at RT. After stripping, the membranes were
washed with copious amounts of water for at least 30 min at RT before re-blocking with
blocking solution and antibody probing.
2.4.6 Kinase assays
Phosphorylation of GST-fusion proteins
TK-1 cells grown in RPMI were harvested, and 5x10 7 cells were lysed in 1 mL of cell lysis
buffer on ice for 10 min. The cell lysate was centrifuged at 12,000 × g for 15 to 20 min at
4°C. One hundred microlitres of supernatant was pre-cleared once with 10 μL of activated
GSH-Sepharose and twice with 10 μL of GST-2T-Sepharose for 30 min at 4°C. A 1 μL
aliquot of pre-cleared supernatant was mixed and incubated with 10 μL of GST-fusion
proteins coupled to Sepharose beads for 2 hrs at 4°C. GST-fusion protein-beads were then
washed thrice with ice-cold lysis buffer and once with kinase buffer. The beads were
suspended in 30 μL of kinase buffer, 1 μCi of 32 P γ-ATP was added, and the mixture was
72

incubated for 20 min at 30°C. Alternatively, 0.4 μg of recombinant kinase(s) was mixed with
10 μL of Sepharose beads in 30 μL of kinase buffer and 1 μCi of 32 P γ-ATP, and incubated
for 20 min at 30°C. The phosphorylated GST-fusion protein-Sepharose beads were washed
extensively with kinase buffer to remove all non-specific radioactivity. The amount of
phosphorylation was measured with a Wallac 1450 TRILUX Microbeta ® counter or
visualised by separation of proteins via SDS-PAGE followed by gel drying, and
autoradiography.
Phosphorylation of peptides
Biotinylated peptides were bound to either streptavidin-coated Sepharose (Sigma-Aldrich) or
magnetic beads (Dynal, Invitrogen) for 30 min at RT (1 mM peptide/mg of beads) and
resuspended in kinase binding buffer. Recombinant kinases (0.4 µg) or trace amounts of cell
lysate (1 μL of 3 x 10 7 cells/mL diluted 1:100) were added together with 1 µCi of 32 P-γATP
per 30 µL of reaction mixture, and incubated for 30 min at 30°C. Some assays used
autophorphoylated kinases prior to addition to peptides. Phosphorylation of peptides was
either determined by measurement with a Wallac 1450 TRILUX Microbeta ® counter, or
visualised by separation of proteins by Tris-tricine SDS-PAGE followed by autoradiography.
Assays to determine kinase binding to peptides:
Peptides immobilised on beads were mixed with recombinant kinases (0.4 μg) for 2 hrs at 4°C
in cell lysis buffer without detergent. The beads were then washed six times with lysis buffer
without detergent and twice with kinase buffer before the addition of 1 μCi of 32 P-γATP as
above.
Autophosphorylation of kinases:
Recombinant kinases were autophosphorylated by incubation of kinases (0.4 µg) with 1 µCi
of 32 P-γATP in 30 µL of kinases buffer, followed by incubation for 30 min at 30°C.
Assays to measure the binding of recombinant proteins to β7 subunit cytoplasmic
domain peptides:
Integrin β7 subunit cytoplasmic domain peptides immobilised on beads were mixed with
recombinant proteins (0.1 μg) and recombinant kinases (0.4 μg) for 2 hrs at 4°C in cell lysis
buffer without detergent. The beads were then washed six times with lysis buffer without
detergent and twice with kinase buffer before the addition of 1 μCi of 32 P-γATP as above.
73

2.4.7 Coupling of proteins and antibodies to Sepharose
Proteins and antibodies to be coupled to Sepharose were resuspended in PBS containing 0.5
M NaCl (coupling buffer). Freeze-dried cyanogen bromide-activated Sepharose beads
(Amersham Bioscience) were swelled using 1 mM HCl and washed for 15 min with 1 mM
HCl. About 5 mL of coupling solution containing 25 to 50 mg of protein was mixed with
each gram of freeze-dried powder. The mixture was rotated end-over-end overnight at 4°C.
The beads were washed with at least 5 volumes of coupling buffer to remove excess ligand.
Remaining active groups were blocked with 1 M ethanolamine for 2 hrs at 4°C. The beads
were then washed with three cycles of 0.1 M acetate buffer, pH 4.0 containing 0.5 M NaCl
followed by 0.1 M Tris-HCl, pH 8 containing 0.5 M NaCl. The beads were further washed
with 6 x 1 mL of PBS, and stored in PBS containing 0.02% NaH3 at 4°C until needed.
2.4.8 Immunoprecipitation / pull-down assay
Cells were lysed at a concentration of 1 x 10 8 cells/mL in cell lysis buffer for 10 min on ice.
The cell lysate was separated from insoluble material by centrifugation in a bench-top
centrifuge at 12,000 × g for 15 min at 4°C. One millilitre of cell lysate was precleared thrice
by mixing with 50 μl of Sepharose beads for 30 min at 4°C. For immunoprecipitation, one
mL of precleared cell lysate was incubated with 50 μL of antibody-Sepharose for 2 hrs at 4°C
with gentle inversion on a rotating platform. For the pull-down assay, one mL of precleared
cell lysate was incubated with 50 μL of peptide-Sepharose overnight at 4°C with gentle
inversion. The Sepharose beads were recovered by centrifugation at 1,000 × g for 5 min at
4°C. The beads were washed 6 times with 1 mL of PBS for 5 min at 4°C.
An alternative procedure was employed when detecting whether immunoprecipitated proteins
were phosphorylated. Cells were lysed in cell lysis buffer containing phosphatase inhibitors,
and the lysate was denatured immediately in the presence of SDS-PAGE loading dye without
bromophenol blue and SDS by heating at 95°C for 5 min. The denatured cell lysate was
incubated overnight at 4°C with antibody-Sepharose beads. The beads were washed 6 times
with 1 mL of PBS for 5 min at 4°C.
74

2.5. Cell biology
2.5.1 Cell culture
Suspension cells
Suspension cells: TK-1, H9, and MTC-1 cells were subcultured every 2 to 3 days at
approximately 2 x 10 5 cells/mL with fresh cell culture medium, and grown at 37°C in a
humidified atmosphere of 5% CO2.
Adherent cells
The adherent cells HEK-293T and CHO-K1 were cultured in DMEM cell culture medium at
37°C in a humidified atmosphere of 10% CO2. To subculture, the media was removed and
discarded, and the adherent cells were washed once with PBS. Trypsin-EDTA solution
(GibcoBRL) was added and the cells were observed by microscopy until the cell layer was
dispersed (5 to 10 min). Detached cells were collected and centrifuged at 1,000 × g for 5 min
at RT, and washed once with cell culture media. The cells were re-cultured in complete
medium at approximately 2 x 10 5 cells/mL, and the media renewed every 2-3 days.
2.5.2 Production of recombinant proteins
Spodoptera frugiperda Sf9 insect cells were grown and adapted to Sf900II SFM. They were
then infected with a baculovirus engineered to produce a particular Fc-tagged recombinant
protein, and cultured at 27°C for 5 days. The culture was centrifuged at 1,000 × g for 5 min at
RT, and the supernatant stored at -20°C in the presence of 0.02% NaH3 and 1.6 µg/mL
soybean trypsin inhibitor. The supernatant was thrice passed through a protein A-Sepharose
column at 4°C to isolate the recombinant Fc-tagged proteins. The column was washed
extensively with PBS and the recombinant proteins were eluted with 0.1 M citric acid pH 1.5,
followed by neutralisation with 0.2 M Tris-Cl (pH 8). The elutant was concentrated in a
dialysis membrane covered with aquacide II (Calbiochem). The resulting solution was
dialysed against PBS, and stored in the presence of 0.02% NaH3 at -80˚C at a concentration
of 1 mg/mL until needed.
75

2.5.3 Spleen cell isolation
Mice were sacrificed and the spleens removed and temporarily placed on ice in DMEM media
containing 10% FCS and PSG. The spleens were mashed through a fine gauze with a sterile
syringe plunger, and clusters of cells were broken up by passing the solution 4 to 5 times
through an 18G needle. The cells were centrifuged for 5 min at 1,000 × g at 4°C, and then
resuspended in 10 to 20 mL of PBS. The cell suspension was layered gently onto 15 to 20 mL
of Ficoll-Hypaque solution and centrifuged at 2,000 × g for 30 min at 20°C with no brake.
The buffy coat layer was transferred into a 50 mL tube, topped up with PBS and centrifuged
at 2,000 × g for 5 min. The cells were washed thrice with PBS, and cultured at 1 x 10 6
cells/mL in DMEM media containing 10% FCS, PSG, and 1 µg/mL of concanavalin A
(Sigma-Aldrich).
2.5.4 Cell adhesion assay
Sixteen well chamber slides (178599, Nunc) for assays using unlabelled cells, and 96- well
plates (F96 Maxisorp 439454, Nunc) for assays using fluorescein-labelled cells, were coated
overnight with 70 µL (per well) of recombinant MAdCAM-1-Fc (10 µg/mL) at 4°C. The
wells were washed thrice with PBS, and blocked at RT with 100% FBS for 2 hrs. They were
then washed once with PBS and twice with HBSS binding buffer immediately prior to the
addition of cells.
Cell assays using 96-well plates: Cells were harvested by centrifugation at 1,000 × g for 5
min. They were incubated with the cell tracker CMFDA (Invitrogen) in serum-free media at
37°C for 30 min. All cells were washed thrice with PBS and incubated with 5 mM EDTA in
PBS for 20 min. They were then washed once with PBS and twice with HBSS binding buffer.
The cells were placed into aliquots of 5 x 10 5 cells per 300 µL of HBSS binding buffer. They
were activated by the addition of the activators 2 mM Mn 2+ /Ca 2+ , AlF4 - , or 50 ng/mL of PMA
in HBSS binding buffer for 5 to 10 min at RT. Controls cells were left non-activated or
incubated with the non-activating cation control 2 mM Mg 2+ /Ca 2+ in HBSS binding buffer for
5 to 10 min at RT. Each aliquot of cells was then transferred into the ligand–coated well of a
96-well plate, and allowed to adhere to the ligand at RT for 30 min. The solution in the wells
was gently removed with a multi-channel pipette. Each well was gently filled with PBS and
sealed. The plate was centrifuged in an inverted position in a microplate carrier at 500 × g for
5 min, and the solution discarded by pipetting. This step was performed 3 times. The
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fluorescence of the bound cells was measured using a fluorescence microplate reader
(VICTOR 1420 multilabel counter from Wallac) at an excitation wavelength of 485 nm and
an absorbance wavelength of 535 nm.
Cell assays using 16-well chamber slides: Cells were harvested and activated as above, but
were not labelled with CMFDA. They were allowed to adhere to the ligand-coated slides at
RT for 30 min. The chambers were removed and the slide dipped thrice into PBS to remove
unbound cells. Bounds cells were fixed in a solution of 2% glutaraldehyde in PBS at 4°C
overnight. Fixed cells were dipped twice into PBS, twice into water, and stained with 0.1%
methylene blue for 5 min. Excess dye was removed by dipping cells twice into water, and
then once into each of 50%, 70% and 100% ethanol. The dyed cells were counted using a
microplate reader (µQuant, Bio-Tek instruments) at a wavelength of 595 nm.
Effect of cell treatments on cell adhesion
Treatment with inhibitory peptides:
For peptide blocking assays, aliquoted cells were incubated with peptides for 30 min in HBSS
binding buffer prior to the addition of activators, and then transferred into the wells.
Treatment with chemical inhibitors:
For testing the effects of chemical inhibitors, aliquoted cells were incubated with the
inhibitors for 3 hrs in HBSS binding buffer prior to the addition of activators, and then
transferred into the wells.
Heat shock treatment:
For heat shock treatment, cells were subjected to increased temperatures ranging from 37°C
to 41°C for up to 1 hr, and then allowed to recover overnight at 37°C before use.
Serum starvation:
For serum starvation, cells were cultured at 1 x 10 6 cells/mL in media containing decreasing
amounts of serum ranging from 10% to 2.5%, and grown overnight at 37°C before use.
77

2.5.5 Enzyme-linked soluble E-cadherin-Fc-mediated adhesion assay.
MTC-1 cells were grown in RPMI containing 10% FCS, PSG (penicillin, streptomycin,
glutamine), and 5 ng/ml of TGFβ1 to induce the expression of αEβ7. Cells were pelleted by
centrifugation at 1000 × g for 5 min, washed thrice with PBS then incubated in PBS
containing 5 mM EDTA for 20 min. The cells were again washed thrice with PBS, then
incubated with goat anti-human IgG (diluted 1:200) in PBS for 30 min at RT as a blocking
step to prevent cell binding to the Fc region of E-cadherin-Fc. The cells were washed thrice
with PBS, once with HBSS, and then resuspended in HBSS containing cell activators (final
volume 200 µL), and incubated for 5 min. Recombinant soluble E-cadherin-Fc/goat-anti-
human-Fc-HRP complex (1 μg of E-cadherin-Fc mixed with 1:1000 dilution of goat-anti-
human-Fc-HRP in a 100 µL volume and incubated at RT for 30 min) was added to the cells,
followed by incubation at RT for 30 min. The cells were washed thrice with PBS, and ABTS
substrate [2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), 100 µL per well] was added.
ABTS produces a water soluble green colour upon reaction with HRP. The colorimetric
enzyme reaction was left to develop for 10 to 20 min, and the amount of dye formed was
measured on a Bio-Tek µQuant TM Microplate Reader at 405 nm.
2.5.6 Cell transfection
HEK-293T cells were cultured in 6-well plates in DMEM + 10% FCS until they were 80 to
90% confluent, and then transfected with Lipofectamine 2000 (Invitrogen). To prepare the
transfection solution, 4 µg of DNA and 10 µL of Lipofectamine 2000 were each separately
mixed with 250 µL of OptiMEM media (Invitrogen). The DNA and Lipofectamine solutions
were incubated together at RT for 20 min, and then added dropwise to a well. The cells in the
well were mixed into the solution by agitating the plates, which were then incubated in a
humidified atmosphere of 10% CO2 at 37°C for 24 to 48 hrs.
2.5.7 Immunofluorescence staining
Cells were fixed with 4% paraformaldehyde for 10 min on ice, and permeabilised with PBS
containing 0.1% Tween-20. They were incubated with primary antibody for 60 min at RT or
overnight at 4°C, followed by washing thrice with PBS for 5 min at RT. Fluorescentconjugated
secondary antibody was added followed by incubation for 1 hr at RT, and then
washing with PBS for 5 min at RT. The cells were mounted with mounting media containing
DAPI (H-1200, Vector Laboratories). They were covered with a coverslip, with the edges
78

eing sealed with nail polish. The slides were examined by microscopy using a Nikon E600
microscope under epi-fluorescence, and further examined by confocal microscopy using a
Leica SP2 confocal microscope (Biomedical Imaging Research Unit, The University of
Auckland).
2.6. Phylogenetic analysis
Phylogenetic analysis was performed using the Ensembl project website
(http://www.ensembl.org). The site was searched for orthologues of the human integrin β7
gene (itgb7). The intracellular domains of the orthologues were compared by alignment of the
deduced protein sequences.
In addition, a blast search using the National Center for Biotechnology Information website
(http://www.ncbi.nlm.nih.gov/) was performed using the cytoplasmic domain sequence of the
human integrin β7 subunit.
2.7. Statistical analysis
Results were expressed as mean values ± standard deviation (SD), and a Student’s t-test was
used for evaluating statistical significance for comparison with control groups. A value less
than 0.05 (p < 0.05) indicated statistical significance. Each experiment was repeated at least
twice.
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Chapter 3. Results
3.1. Expression and cell adhesion properties of β7-integrins
To investigate the regulation of cell adhesion by the β7 integrins α4β7 and αEβ7, cells which
express high levels of the β7 integrins were required. Cultured cells which were known to
express high levels of α4β7 and αEβ7 were chosen for the study. The mouse thymic
lymphoma T cell line TK-1 expresses high levels of α4β7 (Ruegg et al. 1992). Importantly,
TK-1 cells do not express the integrin β1 subunit; hence they represent an ideal model cell
system in which the function of α4β7 can be examined in the absence of α4β1. The murine T
cell hybridoma MTC-1 expresses αEβ7 when grown in the presence of TGF-β (Roberts et al.
1993). Therefore the initial aim of the thesis was to characterize the expression of the β7
integrins on TK-1 and MTC-1 cells.
3.1.1 Confirmation of the expression of α4β7 and αEβ7 on TK-1 and MTC-1 cells
TGF-β1-stimulated MTC-1 cells express the integrin αEβ7
The initial experiment was to confirm the expression of αEβ7 by TGF-β-stimulated MTC-1
cells by using immunoblotting. MTC-1 cells were grown in the presence of 5 ng/mL of TGF-
β1 for three days. The cells were harvested, lysed and the total cellular lysate was resolved by
SDS-PAGE. The proteins were transferred to PVDF membrane and immunoblotted with
rabbit antibodies against the integrin αE and β7 subunits (Figure 3.1). The major bands
detected by the anti-β7 and anti-αE antibodies were 120 and 100 kDa in size, respectively,
confirming that MTC-1 cells express both the αE and β7 subunits. Minor bands that appeared
beneath the αE and β7 subunits were non-specific.
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Figure 3.1 Immunoblot analysis of an MTC-1 cell lysate with antibodies against the αE and β7 subunits
MTC-1 cells were grown in the presence of 5 ng/mL of TGF-β1 for three days. The cells were lysed in cell lysis
buffer at 1 x 10 8 cells/mL. The cell lysate (1 x 10 6 cells/lane) was resolved on an 8% polyacrylamide SDS-gel,
and proteins transferred onto a PVDF membrane. The PVDF membrane was immunoblotted with rabbit
antibodies against the integrin αE subunit (1:1000 dilution) and the β7 subunit (1:400 dilution) as indicated.
Indicated in the right-hand margin are the positions of the αE and β7 integrin subunits. The sizes of molecular
weight markers are shown in the left-hand margin. This experiment was repeated twice.
To further confirm the expression of αEβ7 on MTC-1 cells, MTC-1 cells were stained with a
fluorescently-conjugated antibody against the β7 subunit, and staining visualized by
fluorescence microscopy. MTC-1 cells grown in TGF-β were fixed and stained with the rat
mAb antibody Fib504 against the β7 integrin subunit. The Fib504 antibody was detected with
an Alexa Fluor (AF) 488-conjugated secondary antibody (green). The green staining indicates
the presence of the β7 integrin on MTC-1 cells (marked by yellow arrows), as shown in
Figure 3.2B. DAPI staining of the nuclei (blue) reveals all cells in the field of view. The
Fib504 and DAPI images were merged, revealing that some MTC-1 cells have little or no
detectable β7 integrin subunit expression, as indicated by the red arrows. Analysis of 5 fields
suggested that approximately 50% of TGF-β1-stimulated MTC-1 cells expressed αEβ7. As a
control, MTC-1 cells were stained with an isotype control rat antibody (rat-IgG; Figure
3.2A). The control antibody did not stain the cells green, indicating the absence of nonspecific
antibody binding or autofluorescence.
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Figure 3.2 MTC-1 cells immunostained with an anti-β7 antibody.
MTC-1 cells grown in the presence of 5 ng/mL of TGF-β1 were fixed with 4% paraformaldehyde (PFA) and
immunostained with either; (A) a control rat-IgG antibody (diluted 1:100), or (B) the rat Fib504 mAb against the
β7 integrin subunit (diluted 1:100). Antibody reactivity was detected with an Alexa Fluor (AF) 488-conjugated
secondary antibody (diluted 1:250), and visualised by epi-fluorescence microscopy. Pictures shown are:
antibody staining (left), where the green colour indicates positive antibody staining; DAPIi staining of the nuclei
(middle); and a merged image of antibody and DAPI staining (right), which reveals the antibody staining with
respect to the nucleus. The yellow arrows indicate cells expressing the integrin β7 subunit, and the red arrows
indicate cells that do not express the β7 subunit. The bar indicates the length of 20 µm. This experiment was
repeated three times.
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TK-1 cells express the integrin α4β7
TK-1 cells were immunostained with the DAKT32 antibody which recognises the murine
α4β7 complex, both to confirm that TK-1 cells express α4β7, and to test the activity of the
DAKT32 antibody. TK-1 cells were fixed and stained with either an isotype control antibody
(Figure 3.3, images 1 and 2) or the DAKT32 antibody (Figure 3.3, images 3 and 4), and
stained with DAPI to reveal antibody staining with respect to the nuclei. The antibodies were
detected with an AF-488-conjugated secondary antibody, and visualised by fluorescence
microscopy. The green staining indicates that almost all TK-1 cells express α4β7 (Figure 3.3,
images 3 and 4), whereas no cells were stained green by the control antibody (Figure 3.3,
images 1 and 2). This result confirmed that TK-1 cells express α4β7, and that the DATK32
antibody was useful for the subsequent studies.
Figure 3.3 TK-1 cells stained with the DATK32 antibody against the α4β7 complex.
TK-1 cells in suspension were fixed in 4% PFA and immunostained with; a rat IgG isotype control antibody
(diluted 1:100; images 1 and 2), or the DAKT32 mAb against the integrin α4β7 complex (diluted 1:100; images
3 and 4). Cell nuclei were stained blue with DAPI. Antibody reactivity was detected with an AF-488-conjugated
secondary antibody (diluted 1:250), and visualised by epi-fluorescence microscopy. The pictures shown are
merged images of the antibody staining in green, and the DAPI staining of the nuclei in blue. Images 1 and 3
were taken at 10 x magnification, whereas images 2 and 4 were taken at 40x magnification. The bar indicates the
length of 20 µm. This experiment was repeated twice.
83

3.1.2 Production of recombinant forms of E-cadherin and MAdCAM-1
Recombinant MAdCAM-1 and E-cadherin proteins, which are ligands for integrin α4β7 and
αEβ7 respectively, were produced to study β7 integrin-mediated cell adhesion. Expression
systems to produce both proteins had already been established in our laboratory.
Complementary DNAs encoding MAdCAM-1 and E-cadherin without the membrane
domains had been fused to sequences encoding the CH2/CH3 regions of the human IgG
antibody heavy chain Fc domain, and inserted into the pVL1393 baculovirus vector (Yang et
al. 1995; Berg et al. 1999b). The baculovirus expression vectors were able to produce
recombinant MAdCAM-1-Fc, and E-cadherin-Fc proteins containing C-terminal Fc tags
which facilitate purification of the recombinant proteins.
The baculovirus expression systems were used to produce the recombinant MAdCAM-1-Fc,
and E-cadherin-Fc proteins for the current study of β7 integrin-mediated cell adhesion.
Briefly, Sf9 insect cells adapted for growth in serum-free medium were infected with the
baculoviral vectors containing the cDNAs encoding MAdCAM-1-Fc and E-cadherin-Fc. The
recombinant E-cadherin-Fc and MAdCAM-1-Fc proteins produced and secreted into the cell
media were purified by passing the cell culture supernatant through a protein-G Sepharose
column. The recombinant proteins were eluted at low pH, and were resolved by SDS-PAGE.
Purified E-cadherin was resolved as two bands of approximately 115 and 130 kDa (Figure
3.4A), which represent the processed and unprocessed forms of E-cadherin with or without
the 18 kDa propeptide, respectively (Berg et al. 1999b). Purified MAdCAM-1 was resolved
as a band of approximately 75 kDa, which corresponds to the correct size of MAdCAM-1-Fc
(Figure 3.4B). The identity of the protein bands were confirmed by Western blotting with
antibodies against E-cadherin and MAdCAM-1 (data not shown).
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Figure 3.4 Analysis of purified recombinant E-cadherin-Fc and MAdCAM-1-Fc by SDS-PAGE.
Recombinant E-cadherin-Fc and MAdCAM-1-Fc were produced by a baculovirus expression system. One
microgram of each of the affinity-purified recombinant E-cadherin-Fc (A) and MAdCAM-1-Fc (B) proteins was
resolved on 8% and 10% polyacrylamide SDS-gels, respectively, and stained with Coomassie blue. Purified Ecadherin
yielded a major processed form and a minor unprocessed form. The sizes of molecular weight markers
are shown in the left-hand margin.
3.1.3 Comparison of activators of β7 integrin-mediated cell adhesion
Cell adhesion assays provide a useful tool for investigating the effects of chemical inhibitors
or small inhibitory peptides on pathways that control integrin-mediated cell adhesion. Cells
expressing the β7 integrins were activated with a panel of different activators of β7 integrinmediated
cell adhesion, and bound to plates coated with their respective recombinant ligands.
The integrin activation agents included the pan-activator Mn 2+ , the protein kinase C (PKC)
activator phorbol-12-myristate 13-acetate (PMA), and the G-protein activator AlF4 - .
85

Activation of α4β7-mediated T cell adhesion to MAdCAM-1
TK-1 cells cultured in normal culture media were harvested and labelled with the fluorescent
dye chloromethyl fluorescein diaacetate (CMFDA). The cells were left non-activated, or
treated with 2 mM Mn 2+ , AlF4 - or 50 ng/mL PMA, and allowed to bind to MAdCAM-1-
coated plates. TK-1 cells activated with Mn 2+ , AlF4 - and PMA displayed 2 to 3-fold increased
cell binding to immobilised MAdCAM-1-Fc compared to non-activated cells (Figure 3.5).
Mn 2+ appeared to be the best activator of cell adhesion.
Fluorescence (485/535nm)
100000
80000
60000
40000
20000
0
No activator Mn2+ AlF4- PMA
Activators
Figure 3.5 Activation of TK-1 cell adhesion to MAdCAM-1
TK-1 cells labelled with CMFDA were left non-activated, or activated with Mn 2+ , AlF4-, and PMA. Cells were
allowed to bind (5 x 10 5 cells/well of a 96 well plate) to MAdCAM-1-Fc coated wells at RT for 30 min. Bound
cells were detected using a fluorescence microplate reader with excitation and emission wavelengths of 485 and
535 nm, respectively. The increase in fluorescence detected correlates with increased cell adhesion. Data
represent the mean and SD of three separate wells. The experiment was performed in triplicate.
Activation of αEβ7-mediated T cell binding to E-cadherin
The expression of αEβ7 on MTC-1 cells was variable, as were the activation and subsequent
cell adhesion of MTC-1 cells to E-cadherin-coated plates (data not shown). Therefore the
MTC-1 cell adhesion assay was modified to bind E-cadherin in solution rather than on coated
surfaces, which produced more consistent data. Briefly, MTC-1 cells were harvested and
either left non-activated or activated in the presence of Mn 2+ , AlF4 - or PMA (5 - 10 mins).
Cells were mixed with recombinant E-cadherin-Fc which had previously been complexed
with a goat-anti-human-Fc antibody conjugated with HRP (E-cadherin-antibody complex).
Cells that bound to the E-cadherin-antibody complex were detected via HRP conversion of
86

the peroxidise substrate ABTS. The colormetric products were detected using a microplate
reader. Mn 2+ , AlF4 - and PMA each increased the binding of the E-cadherin-antibody complex
to MTC-1 cells by approximately 5-6 fold compared to non-activated control cells (Figure
3.6). A goat-anti-human-HRP antibody alone did not stain activated MTC-1 cells (data not
shown).
Absorbance (A405nm)
0.2
0.16
0.12
0.08
0.04
0
No activator Mn2+ AlF4- PMA
Activators
Figure 3.6 Activation of MTC-1 cell binding to E-cadherin
MTC-1 cells were cultured in complete growth media in the presence of 5 ng/mL of TGF-β1. The cells were
harvested and left non-activated or activated with, Mn 2+ , AlF4-, or PMA. Recombinant E-cadherin-Fc (1 µg)
was mixed with 100 μl of goat-anti-human-Fc antibody conjugated with HRP (diluted 1:1000) to form an “Ecadherin-antibody
complex”. The cells were allowed to bind to the E-cadherin-antibody complex at RT for 30
min. Unbound E-cadherin-antibody complexes were removed by washing the cells thrice with PBS. Cell binding
to the E-cadherin-antibody complex was measured by the addition of the peroxidase substrate ABTS, and
analysis in a microplate reader at a wavelength of 405 nm. The increase in absorbance detected correlated with
binding of the E-cadherin-antibody complex. Data represent the mean and SD of three wells. The experiment
was performed in triplicate.
Antibodies against the αE and β7 subunits block the binding of E-cadherin to MTC-1
cells
Antibodies against the αE and β7 subunits were introduced into the adhesion assay to confirm
that αEβ7 was responsible for mediating the binding of MTC-1 cells to the soluble E-
cadherin-antibody complex. MTC-1 cells were incubated with anti-α4 and anti-αE (M290
mAb) subunit antibodies at RT for 30 min prior to addition of the cell activators. AlF4 - was
chosen to activate MTC-1 cells as it stimulated the binding of the E-cadherin-antibody
87

complex the greatest (Figure 3.6). As controls, antibody was omitted (no Ab) and cells were
incubated with antibodies against the α4 subunit, and MAdCAM-1, which are not expressed
by MTC-1 cells. Equivalent levels of binding of the E-cadherin-antibody complex to MTC-1
cells were obtained with each of the latter controls (Figure 3.7). In contrast, binding of the E-
cadherin-antibody complex was significantly reduced by antibodies against the αE subunit (p
= 7.08 × 10 -4 ), β7 subunit (p = 1.09 × 10 -5 ), and E-cadherin (p = 1.61 × 10 -5 ; Figure 3.7).
Note that the adhesion assays were preformed in the presence of the antibodies, which
explains why the anti-cadherin antibody was inhibitory. The results indicate that MTC-1 cell
adhesion to the E-cadherin-antibody complex is specifically mediated by αEβ7.
Figure 3.7 Binding of E-cadherin to MTC-1 cells is specifically mediated by αEβ7.
MTC-1 cells grown in the presence of 5 ng/mL of TGF-β1 were incubated in the absence of antibody (no Ab), or
with antibodies against the α4 subunit (anti-α4), the αE- subunit (anti-αE), β7 subunit (anti-β7), E-cadherin
(anti-E-cadherin) and MAdCAM-1 (anti-MAdCAM-1); and then treated with AlF4 - . The soluble E-cadherin-
antibody complex (E-cadherin-Fc-anti-human-IgG-HRP) was added and cells incubated at RT for 30 min.
Unbound E-cadherin-antibody complexes were removed by washing the cells thrice with PBS. Cell binding to
the E-cadherin-antibody complex was measured by the addition of the peroxidase-substrate ABTS, and analysis
in a microplate reader at a wavelength of 405 nm. The increase in absorbance detected correlated with binding of
the E-cadherin-antibody complex. Data shown represent the mean and SD of three wells. *** denotes p-value <
0.001 compared to cell binding in the absence of antibody. The experiments were performed in triplicate.
88

3.2. The effect of pathway inhibitors on β7 integrin mediated cell adhesion
Integrin activation is the result of a cascade of intracellular signalling events which propagate
from the cytoplasm to the plasma membrane (inside-out), or vice-versa are initiated from
extracellular signals (outside-in), as introduced in Section 1.3. A panel of inhibitors of
various cellular kinases and cell signalling pathways were tested for their ability to prevent β7
integrin-mediated cell adhesion in order to identify intracellular pathways that regulate β7
integrin signalling. Genistein, a general protein tyrosine kinase (PTK) inhibitor (Akiyama et
al. 1987), was previously found to prevent α4β7-mediated adhesion of TK-1 cells to
MAdCAM-1 (Zhang et al. 1999). Here, genistein was tested for its affects on cell adhesion,
together with a panel of other kinase inhibitors including inhibitors of the mitogen-activated
protein kinase (MAPK) pathway, Jun N-terminal kinase (JNK), epidermal growth factor
receptor (EGFR) tyrosine kinase, the Src family of tyrosine kinases (SFKs), and myosin light
chain kinase (MLCK).
Cells were deactivated by EDTA metal chelation, pretreated with chemical inhibitors at 37°C
for 3 hrs in HBSS buffer, and either left unactivated (treated only with diluents used to
dissolve the activators) or activated with Mn 2+ or PMA. All cells were checked for viability
by trypan blue exclusion prior to assessing cell adhesion. Cells were tested for their ability to
bind to MAdCAM-1-coated plates. Disruption of β7 integrin-mediated cell adhesion by a
chemical inhibitor would implicate a particular pathway/kinase in β7 integrin signalling.
3.2.1 Genistein, a PTK inhibitor, inhibits the binding of TK-1 cells to MAdCAM-1
Genistein is a general inhibitor of PTKs which acts as a competitive inhibitor of ATP
(Akiyama et al. 1987). EGFR tyrosine kinase induces several signal transduction cascades
including the MAPK pathway, Akt pathway, and JNK phosphorylation, which lead to DNA
synthesis and cell proliferation (Oda et al. 2005). TK-1 cells were bound to MAdCAM-1coated
slides in the presence or absence of genistein at a concentration of 10 µM. Genistein
significantly (p = 5x10 -4 ) prevented the binding of TK-1 cells to MAdCAM-1 (Figure 3.8).
Cell binding was inhibited by 60%, being almost reduced to levels seen with unactivated TK-
1 cells. Genistein at 10 µM was subsequently used as positive control in the following celladhesion
assays.
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Absorbance (A495nm)
Figure 3.8 Effect of genistein on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were resuspended in HBSS with or without 10 µM genistein, and incubated at 37˚C for 3 hrs. The
cells were then either left non-activated or activated with Mn 2+ , as indicated. Cells were adhered to MAdCAM-
1-coated plates at RT for 30 min. The bound cells were fixed with glutaraldehyde, stained with methylene blue
and quantified using a microplate reader at a wavelength of 495 nm. Data shown represent the mean and SD of
three wells. *** denotes a p-value of < 0.001 compared to cell binding of activated control cells not treated with
genistein. The experiments were performed in triplicate.
3.2.2 Involvement the MAP kinase kinase (MEK) pathway in TK-1 cell binding to
MAdCAM-1
0.250
0.200
0.150
0.100
0.050
0.000
Non-activated Activated Activated +
genistein 10 μM
Activation/Inhibitor
Many integrin signalling pathways involve the activation of the mitogen-activated protein
kinase (MAPK) cascade, ras → raf → MEK → ERK, which regulates cell growth through
cell cycle progression (Giancotti et al. 1999). Therefore the involvement of the MEK pathway
in β7 integrin signalling was investigated.
PD98059
PD98059 is an inhibitor of MAP kinase kinase (Dudley et al. 1995). It inhibits the activation
of MAP kinase and the subsequent phosphorylation of MAP kinase substrates. Treatment of
PMA- and Mn 2+ -activated TK-1 cells with 1, 10 and 100 µM of PD98059 did not
significantly affect α4β7-mediated cell adhesion to MAdCAM-1 (Figure 3.9A and B). All
cells were viable as analyzed by trypan blue exclusion. In contrast, genistein inhibited both
PMA and Mn 2+ -induced adhesion.
90
***

Figure 3.9 Effect of PD98059 on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated with 0, 1, 10 or 100 µM PD98059 or 10 µM genistein at 37°C for 3 hrs. The cells
were activated with either PMA (A) or Mn 2+ (B), and adhered to MAdCAM-1-Fc-coated plates at RT for 30
min. PMA-activated TK-1 cells (A) were labelled with CMFDA prior to addition of the inhibitors, and cell
binding was analysed using a fluorescence microplate reader with excitation and emission wavelengths of 485
nm and 535 nm respectively. Mn 2+ -activated TK-1 cells (B) were fixed with glutaraldehyde, stained with
methylene blue, and cell binding was analysed using a microplate reader at a wavelength of 495 nm. Data
shown represent the mean and SD of three wells. The experiments were performed in triplicate.
91

SB203580
SB203580 is an inhibitor of p38 MAP kinase (Cuenda et al. 1995), the most well
characterised member of the MAP kinase family (Cuenda et al. 2007). p38 MAP kinase is
activated in response to inflammatory cytokines, endotoxins and osmotic stress (Cuenda et al.
2007). Treatment of PMA- (Figure 3.10A) and Mn 2+ -activated (Figure 3.10B) TK-1 cells
with SB203580 at a concentration of 1 to 100 µM did not significantly affect α4β7-mediated
cell adhesion to MAdCAM-1. In contrast, genistein inhibited both PMA and Mn 2+ -induced
cell adhesion. All cells were viable as analyzed by trypan blue exclusion.
Figure 3.10 Effect of SB203580 on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated with 0, 1, 10 and 100 µM SB203580 or 10 µM genistein at 37 °C for 3 hrs. The cells
were activated with either PMA (A) or Mn 2+ (B), and adhered to MAdCAM-1-Fc-coated plates at RT for 30
min. PMA-activated TK-1 cells (A) were labelled with CMFDA prior to addition of the inhibitors, and cell
binding was analysed using a fluorescence microplate reader with excitation and emission wavelengths of 485
nm and 535 nm respectively. Mn 2+ -activated TK-1 cells (B) were fixed with glutaraldehyde, stained with
methylene blue, and cell binding was analysed using a microplate reader at a wavelength of 495 nm. Data shown
represent the mean and SD of three wells. The experiments were performed in triplicate.
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3.2.3 Jun N-terminal kinase (JNK) pathway
JNK is a serine-directed protein kinase which is involved in the phosphorylation and
activation of the transcription factors c-jun and activating-transcription-factor-2 (ATF2;
Bonny et al. 2001). JNK is activated in response to inflammation, endotoxins, and
environmental stress, and mediates the expression of proinflammatory genes, cell
proliferation and apoptosis (Bonny et al. 2001). There are three isoforms, namely JNK1 and 2
which exhibit broad tissue expression profiles, and JNK3 which is expressed predominantly
in the central nervous system (Roy et al. 2008). Studies of animal models and humans have
revealed increased activation of JNK pathway in inflammatory bowel diseases (reviewed by
Roy et al. 2008). Therefore, the involvement of the JNK pathway in β7 integrin signalling
was investigated.
JNK inhibitor 1 (JNK-I-1)
JNK inhibitor 1 is a biologically active cell-permeable peptide consisting of a C-terminal
sequence derived from the JNK-binding domain (JBD) and an N-terminal peptide containing
the HIV-TAT48-57 sequence (Bonny et al. 2001). It blocks phosphorylation of the activation
domain of JNK, which prevents the activation of c-jun (Bonny et al. 2001). It has no effect on
the activity of either ERK 1/2 or p38. TK-1 cells were treated with JNK-I-1 at a concentration
ranging from 0.1 to 10 µM or with 10 µM genistein. All cells were viable as analyzed by
trypan blue exclusion. Treatment of PMA-activated TK-1 cells with 10 µM JNK-I-1
significantly (p = 2.43 × 10 -4 ) decreased cell adhesion to MAdCAM-1-Fc by 20%, whereas
lower concentrations of JNK-I-1 had no statistically significant affect (Figure 3.11A).
Treatment of Mn 2+ -activated TK-1 cells with 0.1, 1, and 10 µM JNK-I-1 slightly reduced cell
adhesion by 11% (p = 2.34 × 10 -3 ), 13% (p = 4.13 × 10 -4 ), and 11% (p = 1.75 × 10 -3 ),
respectively (Figure 3.11B). As before, genistein inhibited both PMA and Mn 2+ -induced cell
adhesion.
93

Figure 3.11 Effect of JNK-I-1 on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated with 0, 0.1, 1 and 10 µM JNK-I or 10 µM genistein at 37 °C for 3 hrs. The cells were
activated with either PMA (A) or Mn 2+ (B), and adhered to MAdCAM-1-Fc-coated plates at RT for 30 min.
PMA-activated TK-1 cells (A) were labelled with CMFDA prior to addition of the inhibitors, and cell binding
was analysed using a fluorescence microplate reader with excitation and emission wavelengths of 485 nm and
535 nm respectively. Mn 2+ -activated TK-1 cells (B) were fixed with glutaraldehyde, stained with methylene
blue, and cell binding was analysed using a microplate reader at a wavelength of 495 nm. ** denotes p-value of
< 0.01 and *** denotes p-value of < 0.001 compared to cell binding of activated control cells not treated with
inhibitor. Data shown represent the mean and SD of three wells. The experiments were performed in triplicate.
94

JNK inhibitor 2 (JNK-I-2)
JNK-I-2 inhibits the activities of JNK-1, JNK-2, and JNK-3 by competing for ATP (Bennett
et al. 2001). It inhibits the phosphorylation of c-jun and blocks cellular expression of the
cytokines IL-2, IFN-γ, and TNF-α, and the enzyme cycloxygenase-2 (COX-2; Bennett et al.
2001). JNK-I-2 also blocks IL-1-induced accumulation of phospho-jun and induction of c-Jun
transcription (Bennett et al. 2001). Treatment of PMA- (Figure 3.12A) and Mn 2+ -activated
(Figure 3.12B) TK-1 cells with JNK-I-2 at a concentration of 1 to 100 µM had no statistically
significant affect on α4β7-mediated cell adhesion to MAdCAM-1. All cells were viable as
assessed by trypan blue exclusion. As before, genistein inhibited both the PMA and Mn 2+ -
induced cell adhesion.
Figure 3.12 Effect of JNK-I-2 on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated with 0, 1, 10 and 100 µM JNK-I-2 or 10 µM of genistein at 37 °C for 3 hrs. The cells
were activated with either PMA (A) or Mn 2+ (B), and adhered to MAdCAM-1-Fc-coated plates at RT for 30
min. TK-1 cells were labelled with CMFDA prior to addition of the inhibitors, and cell binding was analysed
using a fluorescence microplate reader with excitation and emission wavelengths of 485 nm and 535 nm
respectively. Data shown represent the mean and standard deviation of three wells. The experiments were
performed in triplicate.
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3.2.4 Epidermal growth factor receptor (EGFR) tyrosine kinase
AG99
AG99 inhibits EGFR tyrosine kinase and EGF-dependent cell proliferation by inhibiting
EGFR phosphorylation, and the activities of ERK1 and ERK2. Treatment of PMA (Figure
3.13A) and Mn 2+ -activated (Figure 3.13B) TK-1 cells with AG99 at a concentration of 1 to
100 µM had no statistically significant affect on α4β7-mediated cell adhesion to MAdCAM-
1. All cells were viable as assessed by trypan blue exclusion. As before, genistein inhibited
both PMA and Mn 2+ -induced cell adhesion.
Figure 3.13 Effect of AG99 on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated with 0, 1, 10 and 100 µM AG99 or 10 µM of genistein at 37 °C for 3 hrs. The cells
were activated with either PMA (A) or Mn 2+ (B), and adhered to MAdCAM-1-Fc-coated plates at RT for 30
min. PMA-activated TK-1 cells (A) were labelled with CMFDA prior to addition of the inhibitors, and cell
binding was analysed using a fluorescence microplate reader with excitation and emission wavelengths of 485
nm and 535 nm respectively. Mn 2+ -activated TK-1 cells (B) were fixed with glutaraldehyde, stained with
methylene blue, and cell binding was analysed using a microplate reader at a wavelength of 495 nm. Data shown
represent the mean and SD of three wells. The experiments were performed in triplicate.
96

3.2.5 Src family of tyrosine kinases (SFK)
The Src family of tyrosine kinases has been implicated in controlling signal transduction
pathways downstream of a variety of cell-surface receptors including integrins (reviewed in
Section 1.5.2); therefore it was important to determine their contribution to α4β7-mediated
cell adhesion.
PP2
PP2 is a potent and selective inhibitor of SFKs, which inhibits lck, fyn, hck, and src (Hanke et
al. 1996). It does not significantly affect the activity of EGFR kinase, the non-receptor
tyrosine kinase JAK2, or the protein kinase ZAP-70 (Hanke et al. 1996). PP2 also inhibits the
activation of FAK and its phosphorylation on Y577 (Hanke et al. 1996), and inhibits anti-
CD3-stimulated tyrosine phosphorylation of human T cells (Hanke et al. 1996). Treatment of
PMA-activated (Figure 3.14A) TK-1 cells with 1 µM PP2 significantly (p = 2.9 × 10 -2 )
decreased cell adhesion to MAdCAM-1-Fc by 15%, whereas lower concentrations of PP2 had
no statistically significant affect. Treatment of Mn 2+ -activated TK-1 cells (Figure 3.14B)
with PP2 at 10 to 1000 nM did not significantly affect α4β7-mediated cell adhesion to
MAdCAM-1. All cells were viable as assessed by trypan blue exclusion. As before, genistein
inhibited both PMA and Mn 2+ -induced cell adhesion.
97

Figure 3.14 Effect of PP2 on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated in HBSS with 0, 10, 100 and 1000 nM PP2 or 10 µM of genistein at 37˚C for 3 hrs.
The cells were activated with either PMA (A) or Mn 2+ (B), and adhered to MAdCAM-1-Fc-coated plates at RT
for 30 min. TK-1 cells were labelled with CMFDA prior to addition of the inhibitors, and cell binding was
analysed using a fluorescence microplate reader with excitation and emission wavelengths of 485 nm and 535
nm respectively. Data shown represent the mean and SD of three wells. * denotes a p-value < 0.05 compared to
cell binding of activated control cells not treated with inhibitor. The experiments were performed in triplicate.
Damnacanthal
Damnacanthal is a selective inhibitor of the tyrosine kinase lck (Faltynek et al. 1995). It
inhibits lck autophosphorylation and phosphorylation of exogenous peptide by lck (Faltynek
et al. 1995). It exhibits 7 to 20-fold greater selectivity for lck over src and fyn (Faltynek et al.
1995). TK-1 cells were treated with damnacanthal at concentrations ranging from 1 to 100
nM. All cells treated with 1 and 10 nM damnacanthal remained viable for the duration of the
experiment as determined by trypan blue exclusion. In contrast, 100 nM damnacanthal
induced approximately 50% cell death, hence the results using this concentration were
excluded from statistical analysis. Treatment of PMA-activated TK-1 cells (Figure 3.15A)
with 1 and 10 nM damnacanthal had no statistically significant affect on α4β7-mediated cell
adhesion to MAdCAM-1. In contrast, treatment of Mn 2+ -activated TK-1 cells (Figure 3.15B)
with 1 and 10 nM damnacanthal significantly reduced cell adhesion to MAdCAM-1 by 3% (p
= 1.47 × 10 -2 ) and 23% (p = 2.65 × 10 -4 ), respectively.
98

Figure 3.15 Effect of damnacanthal on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated with 0, 1, 10 and 100 µM damnacanthal or 10 µM of genistein at 37 °C for 3 hrs. The
cells were activated with either PMA (A) or Mn 2+ (B), and adhered to MAdCAM-1-Fc-coated plates at RT for
30 min. PMA-activated TK-1 cells (A) were labelled with CMFDA prior to addition of the inhibitors, and cell
binding was analysed using a fluorescence microplate reader with excitation and emission wavelengths of 485
nm and 535 nm respectively. Mn 2+ -activated TK-1 cells (B) were fixed with glutaraldehyde, stained with
methylene blue, and cell binding was analysed using a microplate reader at a wavelength of 495 nm. Data shown
represent the mean and SD of three wells. The experiments were performed in triplicate. # denotes 50% cell
death as assessed by trypan blue exclusion. * denotes a p-value of < 0.05 and *** denotes a p-value of < 0.001
compared to cell binding of activated control cells not treated with inhibitor.
Radicicol
Radicicol is a protein tyrosine kinase inhibitor, which inhibits v-src kinase activity
(Chanmugam et al. 1995). It also disrupts K-Ras-activated signalling pathways by selectively
depleting Raf kinase, and inhibiting tyrosine phosphorylation of lyn (Chanmugam et al.
1995). It suppresses the transformation of NIH/3T3 cells by diverse oncogenes such as src,
ras, and mos, in part by blocking the key signal transduction intermediates MAP kinase and
GAP-associated p62 (Soga et al. 1998). Treatment of PMA- (Figure 3.16A) and Mn 2+ -
activated (Figure 3.16B) TK-1 cells with radicicol at a concentration of 0.3 to 30 µM had no
statistically significant affect on α4β7-mediated cell adhesion to MAdCAM-1. All cells were
viable as assessed by trypan blue exclusion. As before, genistein inhibited both PMA and
Mn 2+ -induced cell adhesion.
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Figure 3.16 Effect of radicicol on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated with 0, 0.3, 3 or 30µM radicicol or 10 µM of genistein at 37 °C for 3 hrs. The cells
were activated with either PMA (A) or Mn 2+ (B), and adhered to MAdCAM-1-Fc-coated plates at RT for 30
min. TK-1 cells were labelled with CMFDA prior to addition of the inhibitors, and cell binding was analysed
using a fluorescence microplate reader with excitation and emission wavelengths of 485 nm and 535 nm
respectively. Data shown represent the mean and SD of three wells. The experiments were performed in
triplicate
3.2.6 Myosin light chain kinase (MLCK)
MLCK is a serine/threonine protein kinase that phosphorylates myosin II light chain (Smith et
al. 2003). Phosphorylation of myosin enables myosin to bind to actin filaments and allows
cell contraction to begin (Smith et al. 2003). T cell migration on ICAM-1 mediated by LFA-1
was found to involve MLCK activity, where MLCK was required for cell attachment and
movement of the cell at the leading edge (Smith et al. 2003).
ML-7
ML-7 is a selective inhibitor of myosin light chain kinase, which also inhibits protein kinase
A and protein kinase C, but at much higher concentrations (Saitoh et al. 1987). TK-1 cells
were treated with 0.3, 25, and 50 µM ML-7. Those treated with 0.3 and 25 µM ML-7
remained viable for the duration of the experiment, whereas those treated with 50 µM ML-7
showed more than 70% cell death as assessed by trypan blue exclusion and hence were
100

excluded from statistical analysis. Treatment of PMA-activated TK-1 cells with 0.3 and 25
µM ML-7 (Figure 3.17A) significantly decreased α4β7-mediated cell adhesion to
MAdCAM-1 by 11% (p = 4.88 × 10 -3 ) and 30% (p = 2.24 × 10 -5 ), respectively. Treatment of
Mn 2+ -activated TK-1 cells with 25 µM ML-7 (Figure 3.17B) significantly decreased α4β7-
mediated cell adhesion to MAdCAM-1 by 34% (p = 1.64 × 10 -4 ), whereas statistical
significance was not achieved with the lower concentration. As before, genistein inhibited
both PMA and Mn 2+ -induced cell adhesion.
Figure 3.17 Effect of ML-7 on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated with 0, 0.3, 25 and 50 µM ML-7 or 10 µM of genistein at 37 °C for 3 hrs. The cells
were activated with either PMA (A) or Mn 2+ (B), and adhered to MAdCAM-1-Fc-coated plates at RT for 30
min. PMA-activated TK-1 cells (A) were labelled with CMFDA prior to addition of the inhibitors, and cell
binding was analysed using a fluorescence microplate reader with excitation and emission wavelengths of 485
nm and 535 nm respectively. Mn 2+ -activated TK-1 cells (B) were fixed with glutaraldehyde, stained with
methylene blue, and cell binding was analysed using a microplate reader at a wavelength of 495 nm. Data shown
represent the mean and SD of three wells. The experiments were performed in triplicate. # denotes cells with
greater then 70% cell death by trypan blue exclusion. *** denotes a p-value < 0.001 which signifies statistical
significance when compared to cell binding of activated cells not treated with inhibitor.
101

3.3. Properties of a cell adhesion regulatory domain in the cytoplasmic tail
of the integrin β7 subunit
α4β7-mediated cell adhesion is regulated in part via the integrin β subunit cytoplasmic
domain. Previously, our laboratory employed cell-permeable peptide technology to scan the
β7 integrin cytoplasmic domain for cell adhesion regulatory domains (CARDs). Short
peptides encompassing the 52 aa sequence of the β7 cytoplasmic domain were synthesized by
fusion to a cell-permeable poly-arginine (r9) sequence. The peptides were tested for their
ability to interfere with α4β7-mediated cell adhesion. This approach led to the identification
of the 6 aa motif YDRREY which is proposed to serve as a CARD for β7 integrins (Figure
3.18; Krissansen et al. 2006b). The focus turned to an investigation of the YDDREY motif to
determine whether components of the above β7 integrin regulatory pathway(s) identified by
chemical inhibition might interact with the YDRREY peptide. The initial aim was to identify
critical residues in the β7 integrin CARD involved in β7 integrin regulation. Peptides fused
N-terminally to the poly-arginine cell permeable peptide were used in all the following
experiments, unless otherwise stated.
731 741 751 761 771 781
| | | | | |
β7 K LSVEIYDRRE YSRFEKEQQQ LNWKQDSNPL YKSAITTTIN PRFQEADSPT L
Figure 3.18 The amino acid sequence of the human β7 integrin cytoplasmic domain
The amino acid sequence of the integrin β7 subunit cytoplasmic domain with the potential cell adhesion
regulatory domain (CARD) highlighted, and the conserved NPLY motif underlined.
3.3.1 A cell-permeable YDRREY peptide inhibits β7 integrin-mediated adhesion to
MAdCAM-1 and E-cadherin
α4β7-mediated adhesion to MAdCAM-1
TK-1 cells were incubated with a synthetic, biotinylated, cell-permeable YDRREY peptide
and tested for adhesion to MAdCAM-1 to confirm that the YDRREY motif sequence has the
properties of a CARD. Peptides were biotinylated so that peptide uptake by cells could be
confirmed by staining cells with fluoresceinated streptavidin (data not shown). TK-1 cells
were incubated with 0, 25, 50 and 100 µM biotin-r9-YDRREY-OH (YDRREY) peptide for
30 min, and then activated with Mn 2+ and added to plates coated with MAdCAM-1-Fc. All
102

cells were confirmed to be > 98% viable as assessed by trypan blue exclusion. Bound cells
were fixed and stained with methylene blue and quantified in a microplate reader. Non-
activated cells were used as negative controls. Increasing concentrations of the YDRREY
peptide decreased TK-1 cell binding to MAdCAM-1 (Figure 3.19). YDDREY at 50 and 100
μM significantly decreased cell binding by 45% (p = 3.05 × 10 -3 ) and 68% (p = 2.1 × 10 -5 ),
respectively, compared to the binding of non-activated cells. The YDRREY peptide at 100
µM served as a positive control in subsequent experiments.
Figure 3.19 Effect of the YDRREY peptide on TK-1 cell adhesion to MAdCAM-1.
TK-1 cells were incubated in HBSS with 0, 25, 50 or 100 µM YDRREY peptide (green) at RT for 30 min, and
activated with Mn 2+ . Non-activated cells not treated with the YDRREY served as negative controls (blue bar).
Cells were adhered to MAdCAM-1-coated plates at RT for 30 min. The bound cells were fixed with
glutaraldehyde, stained with methylene blue and analysed using a microplate reader at a wavelength of 495 nm.
Data shown represent the mean and SD of three wells. The experiments were performed in triplicate. ** denotes
a p-value of < 0.01 and *** denotes a p-value of < 0.001, compared to cell binding of Mn 2+ -activated cells not
treated with the YDRREY peptide.
αEβ7-mediated adhesion to E-cadherin
The cell permeable peptide YDRREY was tested for its ability to disrupt αEβ7-mediated
adhesion to E-cadherin to confirm that the YDRREY motif is a CARD for both members of
the β7 integrin subfamily. TGF-β-differentiated MTC-1 cells were incubated with 0, 10, 50
µM YDRREY peptide for 30 min and activated with AlF4 - to bind to soluble E-cadherin-
antibody complexes (E-cadherin-Fc complexed with anti-human IgG-HRP; Figure 3.20).
Non-activated cells incubated with anti-human IgG-HRP served as negative controls. All
cells were confirmed to be viable as assessed by trypan blue exclusion. Treatment of AlF4 - -
activated MTC-1 cells with the YDRREY peptide at 10 µM and 50 µM significantly
103

decreased αEβ7-mediated adhesion to E-cadherin by 35% (p = 3.16 × 10 -5 ) and 70% (p = 2 ×
10 -7 ), respectively, compared to cell binding in the absence of the YDRREY peptide.
Figure 3.20 Effect of YDRREY peptide on MTC-1 cell adhesion to E-cadherin
TGF-β-differentiated MTC-1 cells were incubated in HBSS with 0, 10, or 50 µM biotin-r9-YDRREY-OH
peptide (green) at RT for 30 min. Cells were activated with AlF4 - and bound to the E-cadherin-complex at RT for
30 min. The E-cadherin-antibody complex was formed by incubating 1 µg of E-cadherin with of anti-human-
IgG-HRP (diluted 1:1000) at RT for 30 min. Control MTC-1 cells were stained with anti-human-IgG-HRP in the
absence of E-cadherin (No E-cadherin, blue bar). Cells bound to the E-cadherin-antibody complex were
visualised by the addition of ABTS, and analysed using a microplate reader at a wavelength of 405 nm. Data
shown represent the mean and SD of three wells. The experiments were performed in triplicate. *** denotes a pvalue
of < 0.001, compared to binding of the E-cadherin-antibody complex in the absence of YDRREY peptide.
3.3.2 Defining the features of the YDRREY peptide required to inhibit β7-mediated
adhesion
Various aa residues of the YDDREY peptide were substituted in order to identify key
residues and features of the peptide responsible for inhibiting α4β7 integrin-mediated cell
adhesion to MAdCAM-1. A previous study by the host laboratory had revealed that deletion
of either of the flanking tyrosines destroyed the activity of the peptide.
xDRREx, a phosphorylated form of YDRREY
Unphosphorylated tyrosine residues which flanked the YDRREY motif were replaced with
phosphorylated tyrosine residues to give the sequence xDRREx, where x represents a
phosphorylated tyrosine, to determine whether peptide phosphorylation affected the
inhibitory activity of the YDRREY peptide. TK-1 cells were incubated with 0, 25, 50 and 100
µM biotin-r9-xDRREx (xDRREx) peptide for 30 min, activated with Mn 2+ , and added to
MAdCAM-1-Fc-coated plates. Non-activated cells served as negative controls. All cells were
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confirmed viable as assessed by trypan blue exclusion. Treatment of Mn 2+ -activated TK-1
cells with the xDRREx peptide at 25, 50 and 100 µM decreased α4β7 integrin-mediated cell
adhesion to MAdCAM-1 by 14%, 27% (p = 1.47 × 10 -2 ), and 88% (3.07 × 10 -4 ), respectively,
compared to the binding of activated cells in the absence of peptide (Figure 3.21). The
xDRREx peptide at 100 µM prevented α4β7 integrin-mediated cell adhesion to the same
extent as the YDRREY peptide at 100 µM, suggesting that the phosphorylation state of the
flanking tyrosines does not adversely influence the activity of the CARD.
Figure 3.21 Effect of the phosphorylated xDRREx peptide on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated in HBSS with 0, 25, 50 or 100 µM xDRREx peptide (green) at RT for 30 min. The
cells were either left non-activated or activated with Mn 2+ . Non-activated cells not treated with the xDRREx
served as negative controls (blue bar). TK-1 cells incubated with the YDRREY peptide (purple bar) at 100 µM
served as positive controls. Cells were adhered to MAdCAM-1-Fc-coated plates at RT for 30 min. The bound
cells were fixed with glutaraldehyde, stained with methylene blue, and analysed using a microplate reader at a
wavelength of 495 nm. Data shown represent the mean and SD of three wells. The experiments were performed
in triplicate. * denotes a p-value < 0.05 and *** denotes a p-value of < 0.001; compared to cell binding of Mn 2+ -
activated cells not treated with the xDRREx peptide.
YDRGGGGREY
The two tyrosine residues flanking the YDRREY motif were found to be critical for the
ability of the YDRREY peptide to inhibit α4β7 integrin-mediated cell adhesion to
MAdCAM-1 (Krissansen et al. 2006b). However, as shown above, the state of
phosphorylation of the two flanking tyrosine residues did not seem to influence the activity of
the YDRREY peptide. The aim here was to determine whether the activity of the YDRREY
peptide was dependent on the proximity of the two flanking tyrosines. Four glycine residues
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were inserted into the middle of the YDRREY sequence between the arginine residues to
form the biotin-r8-YDRGGGGREY-OH (YDRGGGGREY) peptide. TK-1 cells were
incubated with 0, 25, 50 and 100 µM concentrations of the YDRGGGGREY peptide for 30
min, activated with Mn 2+ , and added to MAdCAM-1-Fc-coated plates. Non-activated cells
were used as negative controls. All cells were confirmed to be viable as assessed by trypan
blue exclusion. Treatment of TK-1 cells with increasing concentrations of the
YDRGGGGREY peptide decreased the amount of TK-1 cell binding to MAdCAM-1 (Figure
3.22). Treatment with 50 and 100 µM YDRGGGGREY peptide significantly decreased cell
adhesion by 23% (p = 1.46 × 10 -2 ) and 95% (p = 3.4 × 10 -6 ), respectively, compared to the
binding of activated cells in the absence of peptide. The YDRGGGGREY peptide at 100 µM
prevented α4β7 integrin-mediated cell adhesion to the same extent as the YDRREY peptide
at 100 µM. The results demonstrate that precise spacing of the flanking tyrosines may not be
a critical factor in the structure/function of the CARD. It is possible the flanking tyrosines act
independently.
Figure 3.22 Effect of the YDRGGGGREY peptide on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated in HBSS with 0, 25, 50 or 100 µM YDRGGGGREY peptide (green) at RT for 30
min. The cells were either left non-activated or activated with Mn 2+ . Non-activated cells not treated with the
YDRGGGGREY peptide served as negative controls (blue bar). TK-1 cells incubated with the YDRREY
peptide (purple bar) at 100 µM served as positive controls. Cells were adhered to MAdCAM-1-Fc-coated plates
at RT for 30 min. The bound cells were fixed with glutaraldehyde, stained with methylene blue, and analysed
using a microplate reader at a wavelength of 495 nm. Data shown represent the mean and SD of three wells. The
experiments were performed in triplicate. * denotes a p-value < 0.05 and *** denotes a p-value of < 0.001; when
compared to cell binding of Mn 2+ -activated cells not treated with the YDRGGGGREY peptide.
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YDGGEY
The two central positively charged arginine residues in the YDRREY motif were replaced by
neutral uncharged glycine residues, to determine whether the central arginines are important
for influencing β7 integrin mediated-cell adhesion. TK-1 cells were incubated with 0, 50, 100,
and 200 µM biotin-r9-YDGGEY (YDGGEY) peptide for 30 min, activated with Mn 2+ , and
added to MAdCAM-1-Fc-coated plates. Non-activated cells served as negative controls. All
cells were confirmed to be viable as assessed by trypan blue exclusion. Figure 3.23 shows
that treatment of TK-1 cells with up to 100 µM YDGGEY peptide had no significant affect
on the binding of TK-1 cells to MAdCAM-1, where 100 µM YDGGEY peptide inhibited
adhesion by just 10%. The peptide at the very high concentration of 200 µM significantly
reduced cell adhesion by 60% (p = 1.19 × 10 -3 ), but was not as effective as 100 µM YDRREY
peptide. The results suggest that the central arginines of the YDRREY peptide play an
influential role in the function of the CARD. The YDGGEY peptide was readily taken up by
cells (data not shown), ruling out the possibility that problems with cell-permeability
accounted for the lack of efficacy of the YDGGEY peptide.
Figure 3.23 Effect of the YDGGEY peptide on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated in HBSS with 0, 50, 100 or 200 µM YDGGEY peptide (green) at RT for 30 min. The
cells were either left non-activated or activated with Mn 2+ . Non-activated cells not treated with the YDGGEY
peptide served as negative controls (blue bar). TK-1 cells incubated with the YDRREY peptide (purple bar) at
100 µM served as positive controls. Cells were added to MAdCAM-1-Fc-coated plates at RT for 30 min. The
bound cells were fixed with glutaraldehyde, stained with methylene blue and analysed using a microplate reader
at a wavelength of 495 nm. Data shown represent the mean and SD of three wells. The experiments were
performed in triplicate. ** denotes a p-value of < 0.01; when compared to cell binding of Mn 2+ -activated cells
not treated with the YDGGEY peptide.
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YEEEY
The central core DRRE of the YDRREY motif was substituted with four glutamic acid
residues. Poly EY (4:1) is a substrate for many tyrosine kinases (Braun et al. 1984), hence
comparison of the YDRREY and YEEEEY peptides sought to investigate whether the
YDRREY peptide acts in a nonspecific manner as a general substrate sink by providing
phosphorylatable tyrosines, or whether the non-phosphorylatable central core was critical for
activity. TK-1 cells were incubated with 0, 50, 100, and 200 µM biotin-r9-YEEEEY
(YEEEEY) peptide for 30 min, activated with Mn 2+ , and added to MAdCAM-1-Fc-coated
plates. Non-activated cells served as negative controls. All cells were confirmed to be viable
as assessed by trypan blue exclusion. Figure 3.24 shows that treatment of TK-1 cells with up
to 100 µM YEEEEY peptide had no significant affect on the binding of TK-1 cells to
MAdCAM-1, where adhesion was inhibited by just 24% at 100 µM YEEEEY peptide. The
peptide at the very high concentration of 200 µM significantly reduced cell adhesion by 40%
(p = 5.29 × 10 -3 ), but was not as effective as 100 µM YDRREY peptide. The results confirm
that the central arginines in the central core of the YDRREY peptide do play an influential
role in the function of the CARD. The YDRREY peptide may be a preferential substrate for
tyrosine kinases that regulate the function of β7 integrins. The YEEEEY peptide was readily
taken up by cells (data not shown), ruling out the possibility that problems with cellpermeability
accounted for the lack of efficacy of the YEEEY peptide.
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Figure 3.24 Effect of the YEEEEY peptide on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated in HBSS with 0, 50, 100 or 200 µM YEEEEY peptide (green) at RT for 30 min. The
cells were either left non-activated or activated with Mn 2+ . Non-activated cells not treated with the YEEEEY
peptide served as negative controls (blue bar). TK-1 cells incubated with the YDRREY peptide (purple bar) at
100 µM served as positive controls. Cells were added to MAdCAM-1-Fc-coated plates at RT for 30 min. The
bound cells were fixed with glutaraldehyde, stained with methylene blue, and analysed using a microplate reader
at a wavelength of 495 nm. Data shown represent the mean and SD of three wells. The experiments were
performed in triplicate. ** denotes a p-value of < 0.01; compared to cell binding of Mn 2+ -activated cells not
treated with the YEEEEY peptide.
YDRREYGYDRREYGYDRREYGYDRREY
A polymer (4-mer) of the YDRREY motif YDRREYGYDRREYGYDRREYGYDRREY,
was synthesised and tested to determine whether multiple YDRREY motifs potentially
working in a multivalent fashion may more effectively inhibit TK-1 cell adhesion to
MAdCAM-1. TK-1 cells were incubated with 0, 25, 50 and 100 µM biotin-r8-
YDRREYGYDRREYGYDRREYGYDRREY (pYDRREY) peptide for 30 min, activated
with Mn 2+ , and added to MAdCAM-1-Fc-coated plates. Non-activated cells served as
negative controls. All cells were confirmed to be viable as assessed by trypan blue exclusion.
Increasing concentrations of the pYDRREY peptide decreased the amount of TK-1 cell
binding to MAdCAM-1 (Figure 3.25). Treatment with 25, 50 and 100 µM pYDRREY
peptide significantly decreased cell adhesion by 22% (p = 7 × 10 -3 ), 61% (p = 6.16 × 10 -4 ),
and 80% (p = 2.95 × 10 -4 ), respectively, compared to the binding of activated cells in the
absence of peptide. The pYDRREY peptide at 100 µM prevented α4β7 integrin-mediated cell
adhesion to the same extent as the YDRREY peptide at 100 µM, but was slightly more
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effective at 50 µM (refer to Figure 3.19). The pYDRREY peptide was used as a positive
control in subsequent experiments given its excellent inhibitory properties.
Figure 3.25 Effect of the pYDRREY peptide on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated in HBSS with 0, 25, 50 or 100 pYDRREY peptide (green) at RT for 30 min. The
cells were either left non-activated or activated with Mn 2+ . Non-activated cells not treated with the pYDRREY
peptide served as negative controls (blue bar). TK-1 cells incubated with the YDRREY peptide (purple bar) at
100 µM served as positive controls. Cells were added to MAdCAM-1-coated plates at RT for 30 min. The bound
cells were fixed with glutaraldehyde, stained with methylene blue and analysed using a microplate reader at a
wavelength of 495 nm. Data shown represent the mean and SD of three wells. The experiments were performed
in triplicate. ** denotes a p-value of < 0.01 and *** denotes a p-value of < 0.001; compared to cell binding of
Mn 2+ -activated cells not treated with the pYDRREY peptide.
FDRREFGFDRREFGFDRREFGFDRREF
As mentioned above, the flanking tyrosines of the YDRREY motif are important for the
function of the CARD. However, the central core sequence DRRE was also found to be
important for the activity of the YDRREY peptide (refer to Figure 3.24). Here, a polymer (4mer)
of the core sequence was tested for activity with the expectation that it may have
efficacy due to its multivalency. The flanking tyrosines in the 4-mer were substituted with
phenylalanine residues to give the sequence FDRREFGFDRREFGFDRREFGFDRREF. TK-
1 cells were incubated with 0, 25, 50 and 100 µM biotin-r8-
FDRREFGFDRREFGFDRREFGFDRREF (pFDRREF) peptide for 30 min, activated with
Mn 2+ , and added to MAdCAM-1-Fc-coated plates. Non-activated cells served as negative
controls. All cells were confirmed to be viable as assessed by trypan blue exclusion.
Increasing concentrations of the pFDRREF peptide decreased the amount of TK-1 cell
binding to MAdCAM-1 (Figure 3.26). Treatment with 50 µM and 100 µM pFDRREF
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peptide significantly reduced α4β7 integrin-mediated cell adhesion by 53% (p = 3.84 × 10 -3 )
and 82% (p = 5.19 × 10 -4 ), respectively. The pFDRREF peptide at 100 µM showed a similar
level of inhibition of cell adhesion as achieved with the YDRREY peptide at 100 µM. The
results suggest that the central core sequence in the absence of flanking tyrosines can be
rendered active when synthesized as a polymer to potentially increase the multivalency of
interaction.
Figure 3.26 Effect of the pFDRREF peptide on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated in HBSS with 0, 25, 50 or 100 pFDRREF peptide (green) at RT for 30 min. The cells
were either left non-activated or activated with Mn 2+ . Non-activated cells not treated with the pFDRREF peptide
served as negative controls (blue bar). TK-1 cells incubated with the YDRREY peptide (purple bar) at 100 µM
served as positive controls. Cells were added to MAdCAM-1-coated plates at RT for 30 min. The bound cells
were fixed with glutaraldehyde, stained with methylene blue and analysed using a microplate reader at a
wavelength of 495 nm. Data shown represent the mean and SD of three wells. The experiments were performed
in triplicate. *** denotes a p-value < 0.001; compared to cell binding of Mn 2+ -activated cells not treated with the
pFDRREF peptide.
3.3.3 Effect of NPLY on TK-1 cell adhesion
The β7 cytoplasmic domain contains three tyrosine residues, in addition to the two within the
YDRREY motif. The third tyrosine is present in the conserved central 758-NPLY-761 motif
(refer to Figure 3.18). Members of the laboratory had previously shown that cell-permeable
forms of the NPKF peptide from the cytoplasmic domain of the integrin β2 subunit were
capable of inhibiting αLβ2-mediated adhesion of T cells to ICAM-1 (unpublished results).
Hence, a cell-permeable form of the biotin-r8-NPLY (NPLY) peptide was synthesized and
tested for its ability to prevent α4β7-mediated adhesion of TK-1 cells to MAdCAM-1. TK-1
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cells were incubated with 0, 25, 50 and 100 µM NPLY peptide for 30 min, activated with
Mn 2+ , and added to MAdCAM-1-Fc-coated plates. Non-activated cells served as negative
controls. All cells were confirmed to be viable as assessed by trypan blue exclusion.
Increasing concentrations of the NPLY peptide decreased the amount of TK-1 cell binding to
MAdCAM-1 (Figure 3.27). Treatment with 50 and 100 µM NPLY peptide significantly
reduced α4β7-mediated cell adhesion by 21% (3.92 x 10 -3 ) and 72% (1.1 x 10 -3 ), respectively.
The NPLY peptide at 100 µM inhibited cell adhesion to a similar extent as that obtained with
the YDRREY peptide at 100 µM. Thus, the NPLY motif represents a second CARD within
the β7 subunit cytoplasmic domain that appears to be equally important for α4β7-mediated
cell adhesion.
Figure 3.27 Effect of the NPLY peptide on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated in HBSS with 0, 10, 50 or 100 NPLY peptide (green) at RT for 30 min. The cells
were either left non-activated or activated with Mn 2+ . Non-activated cells not treated with the NPLY peptide
served as negative controls (blue bar). TK-1 cells incubated with the YDRREY peptide (purple bar) at 100 µM
served as positive controls. Cells were added to MAdCAM-1-coated plates at RT for 30 min. The bound cells
were fixed with glutaraldehyde, stained with methylene blue and analysed using a microplate reader at a
wavelength of 495 nm. Data shown represent the mean and SD of three wells. The experiments were performed
in triplicate. ** denotes a p-value < 0.01; compared to cell binding of Mn 2+ -activated cells not treated with the
NPLY peptide.
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3.4. Tyrosine kinases interact with and phosphorylate the YDRREY motif
Integrin β subunit cytoplasmic domains do not possess intrinsic kinase activity. However they
contain several tyrosines residues that are potentially phosphorylatable (refer Figure 1.10).
The β1 subunit cytoplasmic domain contains two tyrosine residues at position 783 and 795
within the two conserved central and distal NPxY motifs, respectively. The β2 subunit
cytoplasmic domain contains only one tyrosine residue at position 736 within the β2
counterpart (SDLREY) of the β7 YDRREY motif. As mentioned above, the β7 subunit
cytoplasmic domain contains two tyrosine residues at positions 736 and 741 within the
YDRREY motif, and at position 761 within the central NPxY motif. The initial aim was to
determine whether these sites were phosphorylatable, with a particular interest in the
YDRREY motif of the β7 integrins.
3.4.1 Production of GST-β integrin cytoplasmic domain fusion proteins
Whilst the above experiments (Figure 3.21) suggested that the phosphorylation status of the
cell-permeable YDDREY peptide does not influence the activity of the peptide, it was
possible that the peptide was phosphorylated by an endogenous tyrosine(s) kinase after
uptake into cells. Phosphorylation of the YDRREY peptide would be expected to create SH2
binding domains for intracellular ligands (Krissansen et al. 2006b). The focus turned to
studying the phosphorylation status of tyrosine residues within the integrin β7 cytoplasmic
domain. Our laboratory had previously constructed vectors encoding GST-fusion proteins of
the β integrin subunit cytoplasmic domains. DNAs encoding the integrin β1, β2, and β7
cytoplasmic domains had been subcloned into the pGEX-2T vector. In addition to the native
form of the β7 cytoplasmic domain, the β7 cytoplasmic domain was mutated by substituting
phenylalanine residues for each of the tyrosines (Y736F, Y741F, Y736F+Y741F, and
Y761F), either alone or in combination. DNAs encoding the mutated variants were sub-
cloned into pGEX-2T. The pGEX-2T vectors containing the integrin β subunit sequences
were transformed into the DH5α strain of E.coli. Protein expression was induced with IPTG
for 180 min, and the bacteria were lysed with 1% Triton X-100. The soluble protein fractions
were purified by affinity chromatography using batch binding to glutathione (GSH)-
Sepharose. The purified GST-fusion proteins bound to GSH-Sepharose were kept in PBS in
the presence of sodium azide. The GST-fusion proteins bound to Sepharose were eluted with
reduced glutathione, resolved by SDS-PAGE under reducing conditions, and stained with
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Coomassie blue (Figure 3.28). The purification system yielded similar amounts (~ 1 mg/L of
bacterial culture) of each of the seven GST-fusion proteins. They migrated with a molecular
weight of ~30 kDa. GST protein alone with a molecular weight of ~25 kDa was also
produced and purified to serve as an experimental negative control (Figure 3.28, lane 3).
Figure 3.28 Production of GST-β subunit cytoplasmic domain fusion proteins, and analysis by SDS-PAGE
E. coli were transformed with GST-fusion constructs encoding integrin β-subunit cytoplasmic domains. Protein
expression was induced with 0.4 mM IPTG at 30°C for 180 min, and bacteria collected and lysed in 1% Triton
X-100. The soluble protein fraction was affinity-purified using GSH-Sepharose and eluted with reduced
glutathione. One microgram of each purified protein was analysed by SDS-PAGE under reducing conditions,
and stained with Coomassie blue. Lane 1, GST-β1 cytoplasmic domain; lane 2, GST-β2 cytoplasmic domain;
lane 3, GST; lane 4, GST-β7 cytoplasmic domain; lane 5, GST-β7 (Y736F) cytoplasmic domain; lane 6, GST-β7
(Y741F) cytoplasmic domain; lane 7, GST-β7 (Y736+41F) cytoplasmic domain; lane 8, GST-β7 (Y761F)
cytoplasmic domain. Molecular weights of marker proteins are shown in the left-hand margin in kDa.
3.4.2 Phosphorylation of GST-β7 cytoplasmic domain fusion proteins
Purified GST-β7 cytoplasmic domain fusion proteins bound to Sepharose beads were tested
for their ability to serve as substrates for tyrosine kinases present in TK-1 cells. They were
incubated with trace amounts of a TK-1 cellular lysate in the presence of 32 P-γATP. After
phosphorylation, the GST-fusion proteins bound to Sepharose beads were washed thoroughly,
and the amount of 32 P incorporation was analysed using a liquid scintillation counter (Figure
3.29). The level of phosphorylation corresponded to the number of tyrosine residues present
in each of the GST-β subunit cytoplasmic domains fusion proteins. GST-β1 with two tyrosine
residues had almost twice the level of 32 P incorporation (CPM 463,349) of GST-β2 with one
tyrosine residue (CPM 276,522). GST-β7 with three tyrosine residues showed the greatest
level of phosphorylation (CPM 651,641). Mutation of the β7 subunit tyrosine residues to
phenylalanines decreased the level of phosphorylation such that 32 P incorporation into Y736F
(CPM 412,924), and Y741F (CPM 315,989), was similar to that of GST-β1 with two tyrosine
residues. 32 P incorporation into Y736F+Y741F (CPM 56,794) was very low, suggesting that
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Y761 is poorly phosphorylated. In accord, substitution of Y761 with phenylalanine had little
affect on 32 P incorporation (CPM 565,109) into this cytoplasmic domain variant. This result
indicates that all the tyrosine residues in the cytoplasmic domains of the integrin β1, β2, and
β7 subunits are potentially phosphorylatable. However, Y761 within the NPxY CARD of the
β7 subunit cytoplasmic domain does not appear to be as readily phosphorylated as Y736 and
Y741 within the YDRREY CARD.
Figure 3.29 Phosphorylation of GST-β subunit cytoplasmic domain fusion proteins
Recombinant GST-β subunit cytoplasmic domain fusion proteins were incubated with trace amounts of a TK-1
cell lysate (3 x 10 7 cells/mL of lysis buffer, diluted 1:100) and 1 μCi 32 P-γATP. The amount of 32 P incorporated
was determined using a Wallac Trilux microbeta 1450 liquid scintillation counter, measured as counts per
minute (CPM). GST-fusion proteins included those containing the β1 (GST- β1), β2 (GST- β2), and β7 (GST-
β7) cytoplasmic domains, GST- β7 in which tyrosines 736 (Y736F), 741 (Y741F), 761 (Y761F) and both
tyrosines 736 and 741 (Y736F+Y741F) had been substituted for phenylalanines. A GST only control was
processed as for the GST-β subunit cytoplasmic domain fusion proteins to measure non-specific 32 P
incorporation which was subtracted from all the individual readings before plotting the bar graph. Data shown
represent the mean and SD of three wells. The experiments were performed in triplicate.
3.4.3 Tyrosine phosphorylation of the YDRREY peptide
The aim here was to employ a synthetic YDRREY peptide to confirm that Y736 and Y741
were phosphorylatable as suggested by the results obtained with the GST-β7 subunit
cytoplasmic domain fusion protein. A biotin-GGYDRREY peptide and an unrelated peptide
control (biotin-APTLPPAWQPFLK) were immobilised onto streptavidin-coated Sepharose
beads, and incubated with trace amounts of a TK-1 cell lysate and 32 P-γATP in an in vitro
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kinase assay. The in vitro kinase assay confirmed that the YDRREY peptide was
phosphorylatable, giving a two-fold increase in 32 P incorporation as compared to streptavidin-
coated Sepharose beads alone, or the unrelated peptide control (Figure 3.30).
Figure 3.30 The YDRREY peptide is phosphorylated by a tyrosine kinase(s) in a TK-1 cell lysate
A biotin-GGYDRREY peptide and a biotinylated unrelated control peptide (biotin-APTLPPAWQPFLK) were
each bound to streptavidin-Sepharose (1 µg /10 μL) at RT for 30 min. The peptide-Sepharose beads were
incubated with a TK-1 cell lysate diluted 1:100, and 1 µCi 32 P-γATP at 30˚C for 30 min. Sepharose beads treated
with the TK-1 cell lysate and 32 P-γATP (no peptide) served as a negative control. The amount of 32 P
incorporation (phosphorylation) was determined using the Wallac Trilux microbeta 1450 liquid scintillation
counter. Data shown represent the mean and SD of three wells. The experiments were performed in triplicate. **
denotes a p-value < 0.05 which signifies statistical significance, compared to Sepharose beads only (i.e. no
peptide).
3.4.4 Kinases in multiple cell types can phosphorylate the YDRREY motif
The YDRREY peptide (biotin-GGYDRREY; GGYDRREY) was phosphorylated by tyrosine
kinase(s) present in TK-1 cells. The question asked here was whether tyrosine kinases in
other unrelated cell types were also capable of phosphorylating the GGYDRREY peptide.
Cellular lysates tested included those prepared from TK-1 cells (positive control), a mouse
myoblast cell line (C2C12), human embryonic kidney cells (HEK-293T), and Chinese
hamster ovary cells (CHO-K1). Trace amounts of each cell lysate (1 µL of 1:100 dilution)
were incubated with the GGYDRREY peptide with the addition of 32 P-γATP, and the
incorporation of 32 P was analysed. Lysates of all cells tested were able to phosphorylate the
GGYDRREY peptide (Figure 3.31) with comparable ability, indicating that the tyrosine
kinase(s) able to phosphorylate the GGYDRREY peptide are not cell-type-specific.
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Figure 3.31 Kinases in multiple cell types can phosphorylate the YDRREY motif
The GGYDRREY peptide bound to streptavidin-coated Sepharose was incubated with lysates of TK-1, HEK-
293T, C2C12 and CHO-K1 cells, as indicated, in the presence of 1 µCi 32 P-γATP. The amount of 32 P
incorporation was determined using the Wallac Trilux microbeta 1450 liquid scintillation counter. Data shown
represent the mean and SD of three wells. The experiments were performed in triplicate.
3.4.5 Phosphorylation of the YDRREY peptide by FAK, src and lck
To identify the tyrosine kinase(s) that may be responsible for phosphorylating the YDRREY
peptide, several potential tyrosine kinase candidates, namely FAK, src, and p56lck (lck), were
chosen to study based on their involvement in integrin signalling. The recombinant kinases
were incubated with the GGYDRREY peptide immobilised either on streptavidin-Sepharose
or on streptavidin-magnetic beads, and subjected to an in vitro kinase assay. Increasing
amounts (0.1, 0.2 and 0.4 μg) of each of the recombinant kinases increased the level of
phosphorylation of the GGYDRREY peptide. The different kinases produced equivalent
levels of 32 P incorporation (Figure 3.32). This result shows that the kinases FAK, src, and lck
are able to phosphorylate the YDRREY sequence.
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Figure 3.32 Phosphorylation of the GGYDRREY peptide by recombinant FAK, src, and lck
Biotin-r9-GGYDRREY peptide immobilised on streptavidin-coated Sepharose beads was incubated with 0.1,
0.2, and 0.4 µg of recombinant FAK, src, or lck and 1 µCi 32 P-γATP at 30°C for 30 min. The amount of 32 P
incorporation was determined using the Wallac Trilux microbeta 1450 liquid scintillation counter. This
experiment was repeated twice.
Phosphorylation of the YDRREY peptide by recombinant FAK, src and lck was confirmed by
resolving the phosphorylated peptides by SDS-PAGE analysis. Two additional biotinylated
cell-permeable CARD peptides, biotin-r9-YDRRE (YDRRE) and biotin-r9-DRREY
(DRREY), were synthesized which lacked one of the flanking tyrosine residues to determine
whether the kinases phosphorylated both flanking tyrosines. The peptides and the intact
YDRREY peptide were immobilised on streptavidin-coated Sepharose beads and incubated
with recombinant FAK, src, and lck, in the presence of 32 P-γATP. The phosphorylated
peptides were resolved by SDS-PAGE, and the dried gel subjected to autoradiography
(Figure 3.33). The YDRREY, YDRRE and DRREY peptides were each phosphorylated by
FAK and lck. In contrast, src only phosphorylated the YDRREY and DRREY peptides. A
polyEY peptide, comprised of a 4:1 ratio of glutamic acid to tyrosine residues, was included
as a positive control. It was phosphorylated by FAK and src, but not by lck. A polymer of
arginine with an unrelated peptide (biotin-rrrrrrrrr-pptdqsrpvqpflnlttprkpr) not containing a
tyrosine phosphorylation site served as a negative control, and was not phosphorylated by the
kinases.
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Figure 3.33 Sequence recognition of the YDRREY peptide by FAK, src, and lck
Biotin-r9-YDRREY, biotin-r9-DRREY, biotin-r9-YDRRE, a positive control peptide polyEY, and a negative
control peptide r9 (biotin-rrrrrrrrr-pptdqsrpvqpflnlttprkpr) were immobilised on streptavidin-coated Sepharose.
The peptide-Sepharose beads were incubated with 0.4 µg of recombinant FAK, src, and lck in the presence of 1
µCi 32 P-γATP at 30°C for 30 min. The peptides were analysed on a 16% polyacrylamide tris-tricine SDS-gel.
The gel was dried and autoradiographed. Molecular weights of marker proteins are shown in the left-hand
margin in kDa. This experiment was repeated three times.
3.4.6 Direct binding of FAK and src to the YDRREY motif
Given that FAK, src and lck phosphorylate the YDRREY peptide, as shown above, it was
important to determine whether the kinases physically bind to the peptide. FAK had
previously been shown to bind the KLLMIIHDRREF sequence present in the tail of the
integrin β1 subunit (Schaller et al., 1995), which shares partial similarity with the YDRREY
motif. The pYDRREY, pFDRREF, and biotin-r9-pptdqsrpvqpflnlttprkpr (control) peptides
were immobilised on Sepharose beads, and mixed with recombinant FAK, src, and lck to
determine whether the kinases bind to the YDRREY sequence. The beads were thoroughly
washed, and subjected to an in vitro kinase assay. Phosphorylated forms of the peptides were
visualised by SDS-PAGE and exposure onto X-ray film. The premise was that in order for the
pYDRREY peptide to be phosphorylated the kinases must remain bound to the peptide on
washing. The pFDRREF and biotin-r9-control peptides do not contain a phosphorylation site,
hence served as negative controls. FAK and src phosphorylated the pYDRREY peptide after
extensive washing, suggesting that they had bound the peptide (Figure 3.34). In contrast, lck
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did not phosphorylate the pYDRREY peptide, and hence had not been retained by the
peptide. However, it is also possible lck bound the peptide but was inactivated by the peptide
and hence not detected. As expected no phosphopeptides were detected with the pFDRREF
and control-coated beads.
Figure 3.34 Analysis of the binding and phosphorylation of the YDRREY peptide by FAK, src, and lck
Control (biotin-rrrrrrrrr-pptdqsrpvqpflnlttprkpr), pYDRREY and pFDRREF peptides bound to streptavidincoated
Sepharose beads were mixed with 0.4 µg of recombinant FAK, lck, or src at 4°C for 2 hrs, and
extensively washed. The beads were resuspended in kinase buffer with the addition of 1 µCi 32 Pγ-ATP at 30°C
for 30 min for phosphorylation. After phosphorylation, the peptides were resolved on a 16% polyacrylamide tristricine
SDS-gel. The gel was dried, and exposed to Kodak X-ray film, and developed. A single pYDRREY
phosphopeptide was detected. Molecular weights of marker proteins are shown in the left-hand margin in kDa.
This experiment was repeated three times.
3.4.7 Binding of YDRREY peptide variants by FAK and src
Peptide variants of the YDRREY motif were employed to define the structural features
required for binding by FAK and src. YDRREY, pYDRREY, YEEEEY and YDGGEY
peptides were immobilised onto Sepharose beads and mixed with recombinant FAK or src.
The beads were extensively washed and subjected to an in vitro kinase assay. The peptides
were resolved by SDS-PAGE, and peptide phosphorylation was detected by exposure to X-
ray film. As shown in Figure 3.35, src phosphorylated the peptides YDRREY, pYDRREY,
YEEEEY and YDGGEY to similar extents. In contrast, FAK only weakly phosphorylated the
pYDRREY and YEEEEY peptides. This result suggests that the core region of the YDRREY
motif influences binding and/or phosphorylation by FAK, but has little effect on
binding/phosphorylation by src.
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Figure 3.35 FAK and src binding and phosphorylation of variants of the YDRREY peptide
YDRREY, pYDRREY, YEEEEY, and YDGGEY peptides bound to streptavidin-Sepharose beads were mixed
with 0.4 μg of either FAK or src at 4°C for 2 hrs and extensively washed. The beads were subjected to an in
vitro kinase assay, and phosphopeptides were analysed on a 16% polyacrylamide Tris-tricine SDS- gel, dried
and exposed to Kodak X-ray film, and developed. A single phosphopeptide of ~4 kDa was detected in each lane.
Molecular weights of marker proteins are shown in the left-hand margin in kDa. This experiment was repeated
twice.
3.4.8 Does src remain active after binding to the YDRREY peptide?
Src bound to the YDRREY peptide is still able to phosphorylate soluble unbound
YDRREY peptide
To further investigate the interaction between src and the YDRREY peptide, src was tested
for activity when bound to the YDRREY motif. Src was bound to the peptides YDRREY,
pYDRREY, pFDRREF, GGYDRREY and control peptide (biotin-rrrrrrrrrpptdqsrpvqpflnlttprkpr)
immobilised on magnetic beads. The beads were washed thoroughly
to remove unbound src, YDRREY peptide in solution was added or omitted, and the mixture
was subjected to an in vitro kinase assay. The phosphorylated peptides were analysed by
SDS-PAGE, as above. Src bound to each of the GGYDRREY, pFDRREF, pYDRREY and
YDRREY peptides immobilised on magnetic beads and remained able to phosphorylate the
added YDRREY peptide in solution (Figure 3.36). In the case of the immobilised pYDRREY
and YDRREY peptides, phosphopeptides of pYDRREY and YDRREY could be seen without
the addition of YDRREY in solution due to src phosphorylation of these peptides. In the case
of pYDRREY the YDRREY in solution and bead-immobilised (pYDRREY) were readily
distinguishable based on their differences in size. In the case of bead-immobilised YDRREY,
there was an increase in phosphopeptide due to the addition of soluble YDRREY peptide. Src
did not bind to the control peptide, and consequently the added YDRREY peptide in solution
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was not phosphorylated. YDRREY was directly phosphorylated by src and served as a
positive experimental control. The results demonstrate that src binds to the F/YDRREY/F
sequences, and remains fully active upon binding. The GGYDRREY peptide cannot be
resolved by Tris-tricine-SDS-PAGE (data not shown), therefore no phosphopeptides of
GGYDRREY were seen. However, soluble YDRREY was still phosphorylated by src that
was bound to the GGYDRREY peptide.
Figure 3.36 Src bound to the YDRREY motif remains active.
The GGYDRREY, pFDRREF, pYDRREY, YDRREY and control (biotin-rrrrrrrrr-pptdqsrpvqpflnlttprkpr)
peptides immobilised on magnetic beads were incubated with recombinant src (0.4 µg/ 10 µl of beads) at 4 °C
for 2 hrs. The beads were extensively washed to remove unbound src kinase. YDRREY peptide (0.2 μg) in
solution was added (+), or omitted (-), and the mixture subjected to an in vitro kinase assay at 30°C for 30 min.
The beads were centrifuged and the phosphorylated peptides in the supernatant were analysed on 16%
polyacrylamide Tris-tricine SDS-gels, as before. Soluble YDRREY directly phosphorylated by src served as a
positive experimental control. Molecular weights of marker proteins are shown in the left-hand margin in kDa.
This experiment was repeated twice.
Does src remain peptide-bound after phosphorylating the YDRREY peptide?
The above results suggest that src binds and phosphorylates the YDRREY peptide and
remains active once bound, but does src remain attached once it has phosphorylated the
peptide? Here this question was addressed. Autophosphorylated src was allowed to bind to
the YDRREY or xDRREx peptides immobilised on magnetic beads, which were washed
thoroughly to remove unbound kinases. Alternatively, unphosphorylated src was allowed to
bind to the peptides immobilised on magnetic beads, which were then subjected to an in vitro
kinase assay, and either left unwashed or washed extensively with PBS. Autophosphorylated
forms of src remaining on the beads were analysed by SDS-PAGE (Figure 3.37).
Autophosphorylated src (60 kDa) bound to both the YDRREY peptide (lane 1) and to the
xDRREx phosphopeptide (lane 4). Similarly unphosphorylated src bound to the YDRREY
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peptide and was able to be detected in its autophosphorylated form in the absence of washing
after it had phosphorylated the YDRREY peptide (lane 2), whereas in contrast
unphosphorylated src did not bind to xDRREx (lane 5). Once src had phosphorylated the
YDRREY peptide it was readily washed off the beads and could not be detected in its
autophosphorylated form after resolving bead-bound proteins by SDS-PAGE (lane 3 and 6).
Autophosphorylated src was included as a control for comparison (lane 7). The results
suggest that src binds and phosphorylates the YDRREY peptide, but having phosphorylated
the peptide it then detaches. Autophosphorylated and unphosphorylated forms of src are able
to bind to unphosphorylated forms of the YDRREY peptide. In contrast, only
autophosphorylated forms of src can bind to the phosphorylated YDRREY peptide.
Figure 3.37 Src binds to YDRREY but not to xDRREx
The peptides YDRREY and xDRREx immobilised on magnetic beads were incubated with 32 Pautophosphorylated
(lanes 1 and 4) and unphosphorylated src (lanes 2, 3, 5 and 6). The beads were washed
thoroughly to remove unbound src. The unphosphorylated src remaining bound to the beads was
autophosphorylated by the addition of 32 P-γATP in kinase buffer and incubation at 30°C for 30 min. The beads
were left unwashed (lanes 2 and 5) or washed thrice with PBS (lanes 3 and 6). Src remaining on the beads was
resolved on a 10% polyacrylamide SDS-gel, and autoradiographed. Lane 7, autophosphorylated src. The
position of phosphorylated src of 60 kDa is indicated in the right-hand margin. Molecular weights of marker
proteins are indicated in the left-hand margin in kDa. This experiment was repeated three times.
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3.4.9 FAK and YDRREY interactions
Unphosphorylated and autophosphorylated forms of src bound to the YDRREY peptide, but
only the autophosphorylated form bound to the phosphopeptide xDRREx (Figure 3.37). Here
the aim was to determine whether unphosphorylated FAK would bind the xDRREx peptide.
To test this notion, recombinant FAK was allowed to bind to the YDRREY and xDRREx
peptides immobilised on magnetic beads. The beads were washed to remove unbound FAK,
subjected to an in vitro kinase assay, and either left unwashed or washed with PBS. The
presence of autophosphorylated FAK at 125 kDa was analysed by SDS-PAGE (Figure 3.38).
Unphosphorylated FAK bound to both the YDRREY and xDRREx peptides, albeit FAK
bound substantially less well to the xDRREx peptide. Washing of the beads after FAK-
mediated phosphorylation of the YDRREY peptide had little effect on the amount of
phosphorylated FAK detected (compare lanes 1 and 3 with lanes 2 and 4). The results suggest
that FAK can bind to the YDRREY peptide in both its tyrosine phosphorylated and
unphosphorylated forms, albeit binding to the nonphosphorylated from is more substantial.
Unlike src, FAK does not detach from the peptide once the peptide has been phosphorylated.
Figure 3.38 Tyrosine phosphorylation of the YDRREY peptide affects the degree of FAK binding
The peptides YDRREY and xDRREx immobilised on magnetic beads were incubated with unphosphorylated
recombinant FAK, and washed thoroughly to remove unbound FAK. The beads were subjected to an in vitro
kinase assay at 30°C for 30 min, and either left unwashed (lanes 1 and 3) or washed thrice with PBS (lanes 2 and
4). FAK was resolved on a 10% polyacrylamide SDS-gel, and autoradiographed. The position of phosphorylated
FAK at 125 kDa is indicated in the right-hand margin. Molecular weights of marker proteins are indicated in the
left-hand margin in kDa. This experiment was repeated twice.
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3.4.10 FAK and src bind synergistically to the YDRREY peptide
FAK and src both bind to the YDRREY peptide, and are known to bind and phosphorylate
each other (Mitra et al. 2005), raising the hypothesis that they might bind synergistically to
the YDRREY peptide. To test this hypothesis, FAK and src were added alone or together to
the YDRREY peptide immobilised on beads. The beads were thoroughly washed, and
subjected to an in vitro kinase assay. The phosphoproteins were resolved by SDS-PAGE and
autoradiographed (Figure 3.39). The binding of src and FAK to the YDRREY peptide
appeared to be enhanced when they were added together rather than singly. An alternative
explanation to account for the increased intensity of the FAK and src bands when the kinases
are added together is simply that the two kinases have phosphorylated one another. Further
experiments are required to distinguish the two possibilities.
Figure 3.39 Do FAK and src bind synergistically to the YDRREY peptide?
The YDRREY peptide immobilised on magnetic beads was mixed with 0.4 µg of recombinant FAK and src,
either alone or in combination. The beads were washed thoroughly, and subjected to an in vitro kinase assay.
The phosphorylated kinases were resolved by SDS-PAGE and autoradiographed. The positions of the FAK and
src phosphoproteins of 125 and 60 kDa, respectively, are indicated in the right-hand margin. Molecular weights
of marker proteins are indicated in the left-hand margin in kDa. This experiment was repeated twice.
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3.5. The impact of cytoskeletal proteins on the interaction of the YDRREY
peptide with kinases
Here the aim was to determine the impact of cytoskeletal proteins on the interaction of the
YDRREY peptide with FAK and src. Cytoskeletal proteins, including the actin-binding
protein filamin, are phosphorylated by lck (Pal Sharma et al. 2004). Filamin binds to the β1,
β2, and β7 integrin subunit cytoplasmic domains (Sharma et al. 1995; Calderwood et al.
2001). Filamin binds to the β1 and β7 integrin subunits at a region which overlaps the central
conserved NPxY motif. In contrast, filamin binds to the β2 subunit at a site within the
membrane proximal region, overlapping the region where the YDRREY motif resides in the
β7 subunit. Paxillin also binds to a membrane proximal region (Schaller et al. 1995a) in the
β1 subunit, which also overlaps the region containing the YDRREY motif in the β7 subunit.
3.5.1 Filamin disrupts the binding of src to the YDRREY peptide
As several signalling proteins bind to one another and to overlapping regions of the integrin β
subunit cytoplasmic domains, the aim here was to investigate whether the cytoskeletal
proteins filamin and paxillin synergize or antagonize the binding and/or phosphorylation of
the YDRREY peptide by src. The peptides pYDRREY and pFDRREF were immobilised on
magnetic beads and mixed with a combination of recombinant filamin and src, paxillin and
src, or src alone. The beads were washed thoroughly to remove unbound proteins, and
subjected to an in vitro kinase assay to determine whether src bound to the peptides. Bead-
bound protein was resolved by SDS-PAGE to detect the presence of src, which would be
expected to be present in its autophosphorylated form (Figure 3.40). Filamin appeared to
completely disrupt the binding of src to pYDRREY and pFDRREF, as autophosphorylated
src was not detected. In contrast src bound to the pYDRREY and pFDRREF peptides in the
presence of paxillin, albeit binding was slightly decreased. The apparent failure of src to bind
to the pYDRREY and pFDRREF peptides in the presence of filamin was not due to filamin
disruption of src autophosphorylation. Src incubated with filamin or paxillin in solution and
subjected to an in vitro kinase assay still underwent autophosphorylation. There is also the
possibility that the band at approximately 60 kDa represents paxillin, or a combination of
paxillin and src, as both molecules are of similar molecular weight. Distinguishing the various
possibilities will require further investigation.
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Figure 3.40 Filamin disrupts the binding of src to the YDRREY peptide
The peptides pFDRREF and pYDRREY immobilised on magnetic beads were mixed with a combination of 1 μg
of recombinant filamin and 0.4 μg of src, a combination of 1 μg of recombinant paxillin and 0.4 μg of src, or 0.4
μg of src alone at 4°C for 2 hrs. Unbound filamin, paxillin and src were removed by washing thrice with kinase
buffer, and the beads subjected to an in vitro kinase assay at 30°C for 30 min. Src was incubated with filamin
and paxillin in solution and subjected to an in vitro kinase assay to determine whether filamin and paxillin
interfered with autophosphorylation of src (left-hand panel). The presence of autophosphorylated src, and src
incubated with both filamin and paxillin were analysed by SDS-PAGE. Molecular weights of marker proteins
are indicated in the left-hand margin in kDa. This experiment was repeated twice.
3.5.2 Interaction of paxillin with the β7 subunit cytoplasmic domain
As shown above, paxillin did not affect src binding to the YDRREY motif. Paxillin has been
shown to bind to FAK after it is phosphorylated by ERK-2 (Liu et al. 2002). Therefore, the
aim here was to determine whether ERK-2-phosphorylated paxillin and FAK would form a
complex and bind to the YDRREY peptide, or whether paxillin would antagonize the binding
of FAK to the YDRREY peptide. Recombinant ERK-2, paxillin, and FAK were added
together to the pYDRREY peptide or the control peptide (biotin-r9-pptdqsrpvqpflnlttprkpr)
immobilised on magnetic beads. As controls, ERK-2 alone, and ERK-2 in combination with
paxillin, were incubated with the pYDRREY and control peptides immobilised on magnetic
beads. The beads were subjected to an in vitro kinase assay and thoroughly washed. The
bead-bound phosphoproteins were resolved by SDS-PAGE and autoradiographed (Figure
3.41). Autophosphorylated ERK-2 (44 kDa) was not detected in any of the lanes.
Recombinant FAK and paxillin when added in combination with ERK-2 bound to the-
pYDRREY peptide, and were visualized as phosphoproteins of 125 kDa, and 60 kDa,
respectively. Binding of FAK was much weaker when ERK-2 was omitted, suggesting either
that ERK-2 is required to facilitate the binding of paxillin. Thus, the experiment shows that a
trimer of FAK bound to pYDRREY and paxillin bound to FAK is formed.
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Figure 3.41 Erk-phosphorylated paxillin binds to FAK and forms a complex with the YDRREY peptide.
The pYDRREY peptide and the control peptide (biotin-r9-pptdqsrpvqpflnlttprkpr) were immobilised on
magnetic beads and mixed with recombinant ERK-2, paxillin and FAK, either alone or in combination as
indicated. The beads were subjected to an in vitro kinase assay, washed thoroughly, and the bound
phosphoproteins resolved by SDS-PAGE. The SDS-PAGE gel was dried and exposed at -80°C to x-ray film
overnight (left panel) or for 6 hrs (right panel). The positions of FAK (125 kDa) and paxillin (60 kDa) are
indicated in the right-hand margin. Molecular weights of marker proteins are indicated in the left-hand margin in
kDa. This experiment was repeated twice.
3.5.3 FAK, src, and α-actinin bind the YDRREY peptide
The cytoskeletal protein α-actinin has previously been reported to bind the cytoplasmic
domains of the β1 and β2 integrin subunits (Otey et al. 1990; Pavalko et al. 1993; Sampath et
al. 1998). α-actinin binds to src, and is phosphorylated by FAK (Izaguirre et al. 2001), raising
the possibility that the kinases indirectly mediate the interaction of α-actinin with the β7
cytoplasmic domain. In this experiment, α-actinin was subjected to phosphorylation by FAK
and src and tested for binding to the YDRREY peptide or the control peptide (biotin-r9-
pptdqsrpvqpflnlttprkpr) which had been coated on magnetic beads. The beads were washed
thoroughly, and the bound phosphoproteins were resolved by SDS-PAGE (Figure 3.42). α-
actinin was phosphorylated by both FAK and src, as evidenced by the presence of a 100 kDa
α-actinin band when either FAK (125 kDa) or src (60 kDa) were present. The complex of
FAK in combination with α-actinin bound weakly to the YDRREY peptide, compared to the
complex of src and α-actinin. When FAK, src, and actinin were added in combination, it
appeared that the complex that bound the YDRREY peptide was predominantly composed of
src and α-actinin. However, the presence of FAK may have been obscured by the background.
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FAK and α-actinin did not bind to the control peptide. A faint 60 kDa protein bound the
control peptide, which may correspond to peptide-bound src or more likely represents trace
amounts of src from incompletely washed beads. The identity of the band which migrated
with a molecular weight of ~40 kDa is not known. The results suggest that α-actinin can
potentially bind indirectly to the β7 CARD via FAK or src.
Figure 3.42 Binding of src, FAK, and α-actinin to the YDRREY peptide.
Recombinant FAK and src were incubated either alone or together with 1 µg of recombinant α-actinin and the
mix subjected to an in vitro kinase assay. The phosphoproteins were mixed with either the control peptide
(biotin-r9-pptdqsrpvqpflnlttprkpr) or the YDRREY peptide immobilised on magnetic beads, and incubated at
4°C for 2 hrs. The beads were washed thoroughly, and the bound phosphoproteins were resolved by SDS-
PAGE. The positions of FAK (125 kDa), α-actinin (100 kDa), and src (60 kDa) are indicated in the right-hand
margin. The identity of the ~40 kDa band is not known. Molecular weights of marker proteins are indicated in
the left-hand margin in kDa.
3.6. In vivo interactions of β7 integrins with kinases
3.6.1 Co-localisation of a fluoresceinated YDRREY peptide in living cells
After showing that FAK and src interact with the YDRREY motif in an in vitro system, the
next step was to investigate these interactions in living cells. The r9-YDRREY peptide was
fluoresceinated by attachment of a 5-FAM green fluorescent molecule to the 5’-end of the
peptide (FAM-YDRREY). TK-1 cells were incubated with a low concentration of the FAM-
YDRREY peptide (1 µM) in order to prevent disruption by the peptide of α4β7-mediated
binding of TK-1 cells to MAdCAM-1. The cells were washed, activated with Mn 2+ , incubated
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with MAdCAM-1-coated microspheres, fixed with paraformaldehyde and immunostained
with anti-kinase antibodies to observe whether the FAM-YDRREY peptide would localize to
focal adhesions (Figure 3.43). The FAM-YDRREY peptide (green) localised to focal
adhesions at the cell-surface (Figure 3.43A, B). Src (Figure 3.43A) and FAK (Figure 3.43B;
both red), also were concentrated at focal adhesions, and when the images were merged it was
clear that both FAK and src colocalized with the FAM-YDRREY peptide at the focal
adhesions (indicated by the arrows in the merged images). Thus, the YDRREY peptide may
bind FAK and src in vivo.
Figure 3.43 The YDRREY peptide co-localises with FAK and src at focal adhesions in vivo
TK-1 cells were pre-incubated with a fluoresceinated cell-permeable FAM-YDRREY (1 μM) peptide for 30
min, activated with Mn 2+ , and bound to MAdCAM-1-Fc-coated polystyrene microspheres (0.95 μm). The cells
were fixed with paraformaldehyde, and immunostained with rabbit anti-src (A) and rabbit anti-FAK (B)
antibodies (both used at 1:100 dilutions). The primary antibodies were detected using an anti-rabbit-AF594
secondary antibody (1:250), and visualised by confocal microscopy. The green fluorescence indicates the
location of the FAM-YDRREY peptide; the red fluorescence denotes the locations of FAK and src. The merged
photo shows the colocalisation of the peptide with src (A) and FAK (B) as indicated by the yellow coloration.
Focal adhesions are donoted by blue arrows. The bar represents 10 µm.
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3.6.2 Phosphorylation of the integrin β7 subunit in vivo
The above results have shown that the YDRREY motif of the cytoplasmic domain of the
integrin β7 subunit specifically binds to FAK and src in vitro, and possibly in vivo. In
addition, a variety of cytoskeletal proteins were found to influence the binding and
phosphorylation of the YDRREY motif by FAK and src. The NPxY motif within the
cytoplasmic domain of the β3 subunit of αIIbβ3 is phosphorylated during platelet aggregation
(Law et al. 1996; Phillips et al. 2001). The question addressed here is whether the β7
cytoplasmic domain is phosphorylated on tyrosine in vivo.
The YDRREY peptide and a peptide encompassing the full-length β7 cytoplasmic domain
were subjected to an in vitro kinase assay with trace amounts of cell lysate prepared from
activated and non-activated TK-1 cells. Surprisingly the peptides were phosphorylated to the
same extent by the two lysates (data not shown). To partly address the question raised above,
a comparison was made of the tyrosine phosphorylation of non-activated β7 integrins,
activated β7 integrins, and activated ligand-bound β7 integrins. The β7 integrins from
activated and non-activated TK-1 cells were immunoprecipitated and immunoblotted with
anti-phosphotyrosine antibodies. TK-1 cells were initially inactivated by treatment with the
metal ion chelator, EDTA, and the cells were either left unactivated or were activated with
Mn 2+ . The activated TK-1 cells were incubated with MAdCAM-1-coated magnetic beads to
generate ligand-bound β7 integrins. The above cells representing three different states of β7
integrin activation were lysed in the presence of sodium orthovanadate and phosphatase
inhibitors (PhosSTOP, Roche). The soluble fraction was immediately denatured and
immunoprecipitated with anti-β7 antibodies recognising the β7 cytoplasmic domain that had
been crosslinked to Sepharose beads. The immunoprecipitated β7 integrin subunit was
resolved by SDS-PAGE and immunoblotted with an anti-phosphotyrosine antibody (Figure
3.44A, upper panel). β7 immunoprecipitated from unactivated cells was weakly
phosphorylated, giving a single band of approximately 100 kDa (lane 1). In contrast, β7
immunoprecipitated from activated cells, including cells incubated with MAdCAM-1 was
more strongly phosphorylated, giving a major band of 100 kDa, and a more diffuse band of
130 kDa (lanes 2 and 3, respectively).
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The membrane was stripped and re-probed with the anti-β7 cytoplasmic domain antibody that
had been used for the immunoprecipitation. Despite the fact that the antibody had been
covalently cross-linked to Sepharose beads, there is always trace amounts that leak off the
beads when boiled in reducing SDS-loading buffer. Hence the secondary antibody used to
detect the β7 antibody recognised trace amounts of the antibody from the
immunoprecipitation, as evidenced by the reduced antibody bands at 50 and 70 kDa (Figure
3.44A, lower panel). For comparison, an aliquot of the β7 antibody was resolved on SDS-
PAGE, confirming the bands at 50 and 70 kDa represent antibody heavy chains (Figure
3.44B).
The β7 subunit appeared to migrate as two bands of similar intensity of 100 and 130 kDa
irrespective of the activation status of the cells (Figure 3.44A, lanes 1, 2 and 3, respectively).
The presence of two β7 subunit bands is in accord with the fact that the β7 subunit exists as
an immature unglycosylated form (Yuan et al. 1990) and as a larger mature cell-surface
glycosylated form. It is interesting therefore that cells have to be activated in order for
phosphorylation of the mature form to occur. The immature form appears to be constitutively
phosphorylated. There may have been a slight increase in phosphorylation of both forms upon
ligand binding/clustering of α4β7 integrins, but the increase was negligible.
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Figure 3.44 Tyrosine phosphorylation of the β7 subunit in vivo is dependent on the activation status of
cells
TK-1 cells were treated with EDTA to inactivate α4β7and were either left non-activated, or activated with Mn 2+ .
Activated cells were either left unbound or bound to MAdCAM-1 immobilised on magnetic beads. The TK-1
cells were lysed in the presence of phosphatase inhibitors, and the soluble protein fractions were collected and
denatured at 95°C for 5 min. The denatured protein cell lysates from TK-1 cells that were inactivated (lane 1),
activated (lane 2) and activated in the presence of the ligand MAdCAM-1 (lane 3) were immunoprecipitated
with a rabbit antibody against the cytoplasmic domain of the integrin β7 subunit which had been crosslinked to
Sepharose beads. The immunoprecipitates were resolved by SDS-PAGE, and immunoblotted with a mouse antiphosphotyrosine
(1:200) antibody. Antibody reactivity was detected using a goat anti-mouse-HRP secondary
antibody (1:80,000; A, upper panel). Phosphotyrosine bands of approximately 100 and 130 kDa as indicated
are the approximate size of immature and mature forms of the integrin β7 subunit.
The membrane was stripped with NaOH and probed with the rabbit anti-β7 cytoplasmic domain antibody
(1:200), which was detected using a goat anti-rabbit-HRP antibody (1:20,000; A, lower panel). The integrin β7
subunit was resolved as its immature and mature forms of 100 and 130 kDa, respectively, as indicated. Antibody
chains of 50 and 70 kDa, which presumably leached from the antibody-Sepharose matrix were also detected by
the secondary goat anti-rabbit-HRP antibody. The latter confirms that equal amounts of antibody beads were
used in the immunoprecipitations. B) shows β7 antibody alone resolved by SDS-PAGE and Western blotted as
in A (lower panel). Molecular weights of marker proteins are indicated in the left-hand margin in kDa. The
experiments were performed in duplicate.
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3.6.3 Confirmation of FAK and src involvement in β7 integrin signalling
The tyrosine kinases FAK and src bind and phosphorylate the YDRREY peptide in vitro and
possibly in vivo. To unequivocally determine whether FAK and src bind the β7 subunit in
living cells, the β7 subunit was immunoprecipitated from TK-1 cells, and immunoblotted for
the presence of the tyrosine kinases. Thus the soluble fraction from a TK-1 cell lysate was
incubated with the anti-β7 antibody Fib504 (rat-IgG2a) cross-linked to Sepharose beads, and
with a control rat IgG antibody cross-linked to Sepharose beads. The immunoprecipitated
proteins were resolved by SDS-PAGE, transferred onto a PVDF membrane, and
immunoblotted with a rabbit antibody against the β7 subunit cytoplasmic domain (Figure
3.45).
The Fib504 mAb immunoprecipitated the β7 subunit from the soluble fraction of the TK-1
cell lysate (Figure 3.45, lane 5), as evidenced by an intense band of approximately 150 kDa
detected by the rabbit polyclonal antibody against the β7 cytoplasmic domain. Direct
immunoblotting of the soluble fraction of the TK-1 lysate (lane 1) gave bands at ~150 kDa,
which may also represent the β7 subunit. No bands of 150 kDa were detected in control
samples, including rat-IgG-Sepharose alone (lane 2), the immunoprecipitate formed with rat-
IgG-Sepharose (lane 3), Fib504-conjugated Sepharose beads alone (lane 4), non-conjugated
Fib504 antibody (lane 6), non-conjugated rat-IgG antibody (lane 7), Sepharose beads alone
(lane 8), or the insoluble fraction of the TK-1 cell lysate (lane 9). The results indicate that the
β7 integrin was specifically immunoprecipitated by the Fib504 mAb.
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Figure 3.45 The Fib504 mAb specifically immunoprecipitates the β7 subunit from TK-1 cells
The β7 integrin subunit was immunoprecipitated from the soluble fraction of a TK-1 cell lysate (10 7 cells/mL)
with the anti-β7 subunit mAb Fib504 cross-linked to Sepharose. The immunoprecipitate was resolved by SDS-
PAGE and immunoblotted with a rabbit polyclonal antibody against the β7 cytoplasmic domain (1:400). A goat
anti-rabbit-IgG-HRP secondary antibody (1:20,000) was used to detect immunoreactivity. Lane 1, soluble
fraction of the TK-1 lysate; lane 2, control rat-IgG antibody-conjugated Sepharose beads; lane 3, control rat-IgG
antibody-conjugated Sepharose beads incubated with TK-1 lysate; lane 4, Fib504-conjugated Sepharose beads;
lane 5, Fib504-conjugated Sepharose incubated with TK-1 lysate; lane 6, non-conjugated Fib504 antibody; lane
7, non-conjugated control rat-IgG antibody; lane 8, Sepharose beads; lane 9, insoluble fraction of the TK-1
lysate. The band in lane 5 at approximately 150 kDa is the β7 integrin subunit. Molecular weights of marker
proteins are indicated in the left-hand margin in kDa. This experiment was repeated twice.
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3.6.4 Detection of src in β7 integrin immunoprecipitates
After establishing the presence of the β7 integrin in the immunoprecipitate formed with the
Fib504 mAb as shown in Figure 3.45, the membrane was stripped of protein with NaOH, and
immunoblotted with an anti-src antibody (Figure 3.46). A prominent band at 60 kDa was
detected in the β7 immunoprecipitate (Figure 3.46, lane 5). It was also present in the soluble
(lane 1) and insoluble (lane 9) fractions of TK-1 cell lysate, respectively. In contrast, the 60
kDa band was not present in any of the control samples. Additional minor bands appear to
bind non-specifically to antibody as evidenced by their immunoprecipitation with rat-IgG-
Sepharose (lane 3). This result establishes that src binds the β7 integrin expressed in cells.
Figure 3.46 Src is coimmunoprecipitated with the β7 integrin from TK1 cell lysates
The blot in Figure 3.45 was stripped of protein with 0.2 mM NaOH, and probed with a rabbit anti-src antibody
(1:200), followed by a goat anti-rabbit-HRP secondary mAb. The region of the gel between 75 and 50 kDa
where src should migrate was magnified (inset). The lanes are as specified in Figure 3.45. Molecular weights of
marker proteins are indicated in the left-hand margin in kDa. This experiment was repeated twice.
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3.6.5 Detection of FAK in β7 integrin immunoprecipitates
The membrane in Figure 3.45 was re-stripped with NaOH and probed for FAK with an anti-
FAK antibody (Figure 3.47). FAK was detected as a doublet of bands at approximately 100
and 75 kDa (Figure 3.47, lane 5). Bands of similar size were present in the soluble fraction,
but not the insoluble fraction, of a TK-1 cell lysate. A number of lower molecular weight
doublet bands in the soluble fraction of the lysate may signify that FAK is partially degraded.
FAK is known to be cleaved by the protease calpain and caspases into smaller products of
approximately 95 kDa and 77 kDa (Wen et al. 1997; Carragher et al. 2001). This result
establishes that FAK binds the β7 integrin expressed in cells.
Figure 3.47 FAK is coimmunoprecipitated with the β7 integrin from TK1 lysates
The blot in Figure 3.46 was stripped of protein with 0.2 mM NaOH, and probed with a rabbit anti-FAK
antibody (1:200), followed by a goat anti-rabbit-HRP secondary antibody. The region of the gel between 60 and
140 kDa where FAK should migrate was magnified (inset). The lanes are as specified in Figure 3.45. Molecular
weights of marker proteins are indicated in the left-hand margin in kDa. This experiment was repeated twice.
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3.6.6 Localisation of interacting kinases in living cells
FAK and src were detected in immunoprecipitates of the β7 integrin, suggesting that FAK
and src complex with β7 integrins in vivo. The aim here was to colocalize FAK and src with
α4β7 in living TK-1 cells. Activated TK-1 cells allowed to bind to MAdCAM-1-coated
surfaces were polarised and migrated randomly (data not shown). TK-1 cells were activated
with Mn 2+ and bound to glass cover slips coated with MAdCAM-1 at a low concentration to
enable the formation of focal adhesions and cell migration. They were allowed to bind and
spread, then fixed and stained with an antibody against the β7 integrin and antibodies
recognizing either FAK (Figure 3.48), src (Figure 3.49), or lck (Figure 3.50). The β7
integrin was detected with the green fluorophore AF-488, whereas the kinases were detected
with the red fluorophore AF-594. The cells were stained with isotype control antibodies, but
they failed to stain the cells (data not shown).
Figure 3.48 FAK and α4β7 do not strongly colocalize on TK-1 cells
TK-1 cells were activated with Mn 2+ and adhered to coverslips coated with MAdCAM-1 at 2.5 µg/mL. The cells
were permeabilised and co-stained with anti-β7 (1:100) and anti-FAK (1:100) antibodies. The β7 subunit was
visualised with the green fluorophore AF-488 (1:250), while FAK was visualised with the red fluorophore AF-
594 (1:250). The images were merged to detect colocalization (merged). Uropods are indicated by the blue
arrow. Images were captured using epi-fluorescence microscopy. Bar indicates the length of 10 µm. This
experiment was repeated three times.
Both α4β7 and FAK were diffusely expressed throughout cells, but with multiple points of
intense staining giving a punctate pattern of expression (Figure 3.48). α4β7 was strongly
expressed on the uropod while FAK was only very weakly expressed, and largely at pinpoints
of expression. The results suggest that α4β7 does not colocalize with FAK on the uropod, and
does not strongly colocalize with FAK on the cell body
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Src was diffusely expressed throughout the cells, but was also expressed in a punctate
fashion, albeit not as marked as for the β7 integrin. Unlike FAK, src was expressed on the
uropod, where expression largely overlapped that of the α4β7 (Figure 3.49). In contrast,
expression of both molecules in the cell body was mostly punctate, and did not overlap as in
the uropod.
Figure 3.49 Src appears to colocalize with α4β7 in the uropod
TK-1 cells were activated with Mn 2+ and adhered to coverslips coated with MAdCAM-1 at 2.5 µg/mL. The cells
were permeabilised and stained with anti-β7 (1:100) and anti-src (1:100) antibodies. The β7 integrin was
visualised with the green fluorophore AF-488 (1:250), while src was visualised with the red fluorophore AF-594
(1:250). The images were merged to detect colocalization (merged). Uropods are indicated by the blue arrow.
Images were captured using epi-fluorescence microscopy. Bar indicates the length of 10 µm. This experiment
was repeated three times.
Lck was diffusely expressed throughout the cells, and like FAK was poorly expressed on the
uropod in a punctate fashion (Figure 3.50). Lck was also expressed in a fine punctate
expression in the cell body. There was no apparent overlap with α4β7.
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Figure 3.50 Lck and α4β7 do not appear to colocalize on TK-1 cells
TK-1 cells were activated with Mn 2+ and adhered to coverslips coated with MAdCAM-1 at 2.5 µg/mL. The cells
were permeabilised and stained with anti-β7 (1:100) and anti-lck (1:100) antibodies. The β7 integrin was
visualised with the green fluorophore AF-488 (1:250), whereas lck was visualised with the red fluorophore AF-
594 (1:250). The images were merged to detect colocalization (merged). Panel B shows an enlargement of the
uropod of a cell. Uropods are indicated by the blue arrow. Images were captured using epi-fluorescence
microscopy. Bar indicates the length of 20 µm. This experiment was repeated three times.
3.7. Localization of α4β7 with kinases in supra-molecular activation
complexes (SMAC)
SMACs are clusters of receptors and signalling molecules formed on cells when TCR and
integrins on lymphocytes bind their ligands expressed on APCs. Preliminary experiments in
the laboratory had led to the discovery that integrin ligands immobilised on polystyrene
microspheres could cause the clustering of β7 integrins at the cell-surface and the formation
of SMAC-like structures when incubated with activated TK-1 cells (Zhang 1999, unpublished
data). The pseudo-SMAC could be visualised by staining of the β7 integrins with a
fluorescent anti-β7 subunit mAb.
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The aim here was to determine whether src, FAK and lck might co-localise with α4β7 in the
pseudo-SMAC structures. Integrin clustering serves as a useful platform to detect co-
localisation of proteins, as the complexes formed are readily detectable by light microscopy.
For this study, TK-1 cells were activated and incubated with MAdCAM-1-coated magnetic
beads. The SMAC that formed were stained with antibodies against the β7 integrin subunit,
and the tyrosine kinases src, FAK and lck, and analyzed by confocal microscopy.
3.7.1 TK-1 cells bind to MAdCAM-1-coated magnetic beads
Mn 2+ -activated TK-1 cells were incubated with MAdCAM-1-Fc coated magnetic beads and
binding analyzed by light microscopy. The cells were seen to bind to the beads, where some
cells appeared to bind to more than one magnetic bead (Figure 3.51B). Similar results were
achieved with beads ranging in size from 0.95 μm to 5 μm in diameter (data not shown).
Magnetic beads that had not been coated with MAdCAM-1 did not bind to Mn 2+ -activated
TK-1 cells (Figure 3.51A). In addition, non-activated TK-1 cells did not bind to MAdCAM-
1-coated magnetic beads (data not shown).
Figure 3.51 TK-1 cells bind to MAdCAM-1-coated magnetic beads
TK-1 cells were activated with Mn 2+ and incubated with (A) non-coated and (B) MAdCAM-1-Fc-coated (100 ng
MAdCAM-1/μL beads) magnetic beads at RT for 30 min. The cells were fixed in 4% paraformaldehyde, placed
on glass slides and visualised under light microscopy. 40x magnification. This experiment was repeated three
times.
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3.7.2 α4β7 colocalizes with src in SMACs
Mn 2+ -activated TK-1 cells were incubated with 0.9 μm diameter polystyrene microspheres
coated with MAdCAM-1 for molecular colocalization experiments as 0.9 μm diameter
microspheres provide a smaller point of contact for clustering of integrins. In addition,
polystyrene microspheres produce less auto-fluorescence as compared to magnetic beads or
Sepharose beads (data not shown). The cells were fixed and stained with green and red
fluorescent antibodies against the β7 subunit and src, respectively, and analyzed by confocal
microscopy. α4β7 (green) clustered in distinct focal adhesions around the plasma membrane,
whereas src (red) occupied the entire plasma membrane but also clustered in focal adhesions
within the membrane (Figure 3.52A). Merger of the two images clearly illustrated colocalisation
(yellow) of src and β7 integrins in the adhesion clusters. The image of one
representative cell was magnified to better visualize the SMAC (Figure 3.52B).
Several of the β7 clusters appeared to have a circular structure resembling pSMAC-like
structures Figure 3.52B. In contrast, src appeared to occupy both the central SMAC
(cSMAC) and peripherial SMAC (pSMAC).
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Figure 3.52 α4β7 clusters with src in pseudo-SMAC
Polystyrene microspheres (0.95 μm diameter) coated with MAdCAM-1 (100 ng/μL) were incubated with Mn 2+ -
activated TK-1 cells at RT for 30 min. The cells were stained with the rat-anti-β7 mAb Fib504 (1:100) and a
rabbit-anti-src antibody (1:100). The anti-β7 antibody was detected with a secondary anti-rat-AF488 (green)
antibody, and the anti-src antibody was detected with a secondary anti-rabbit-AF594 (red) antibody. Stained
cells were analysed by confocal microscopy. (A) A representative population of TK-1 cells incubated with
MAdCAM-1-coated microspheres and stained with anti-β7 (green) and anti-src (red) antibodies. The two images
were merged to aid visualization of colocalization (merged). (B) A representative single cell was enlarged for
clearer visualisation. Pseudo-SMACs are indicated by blue arrows. Bar indicates the length of 20 µm. This
experiment was repeated three times.
3.7.3 α4β7 colocalizes with FAK in SMACs
TK-1 cells were activated with Mn 2+ , incubated with MAdCAM-1-coated microspheres to
cluster α4β7 integrins, and stained with antibodies against the β7 subunit and FAK.
Fluorescent green and red secondary antibodies were used to detect the primary anti-β7 and
anti-FAK antibodies, respectively, and visualized by confocal microscopy (Figure 3.53).
Both α4β7 (green) and FAK (red) clustered in distinct focal adhesions around the plasma
143

membrane (Figure 3.53A). Colocalization of α4β7 with FAK was obvious even without
having to merge the two images. Nevertheless merger confirmed colocalization.
High magnification of a representative single cell revealed that clusters of α4β7 formed
distinct circular pSMAC-like structures (Figure 3.52B) FAK weakly localised to the
pSMAC-like structures indicating co-localisation with α4β7. It was present at much higher
levels within the cSMAC.
Figure 3.53 α4β7 clusters with FAK in pseudo-SMAC
Polystyrene microspheres (0.95 μm diameter) coated with MAdCAM-1 (100 ng/μL) were incubated with Mn 2+ -
activated TK-1 cells at RT for 30 min. The cells were stained with the rat-anti-β7 mAb Fib504 (1:100) and a
rabbit-anti-FAK antibody (1:100). The anti-β7 antibody was detected with a secondary anti-rat-AF488 (green)
antibody, and the anti-src antibody was detected with a secondary anti-rabbit-AF594 (red) antibody. Stained
cells were analysed by confocal microscopy. (A) A representative population of TK-1 cells incubated with
MAdCAM-1-coated microspheres and stained with anti-beta7 (green) and anti-FAK (red) antibodies. The two
images were merged to aid visualization of colocalization (merged). (B) A representative single cell was
enlarged for clearer visualisation. Pseudo-SMACs are indicated by blue arrows. Bar indicates the length of 20
µm. This experiment was repeated three times.
3.7.4 α4β7 colocalizes with lck in SMACs
TK-1 cells were activated with Mn 2+ , incubated with MAdCAM-1-coated microspheres to
cluster α4β7 integrins, and stained with antibodies against the β7 subunit and lck. Fluorescent
144

green and red secondary antibodies were used to detect the primary anti-β7 and anti-lck
antibodies, respectively, and visualized by confocal microscopy (Figure 3.54). Both α4β7
(green) and lck (red) clustered in distinct focal adhesions around the plasma membrane
(Figure 3.54A).
High magnification of a representative single cell revealed that clusters of α4β7 formed
distinct circular pSMAC-like structures (Figure 3.54B). Lck predominantly localised to the
cSMAC, suggesting it was unlikely to associate with α4β7.
Figure 3.54 Clusters of integrin β7 and lck
Polystyrene microspheres (0.95 μm diameter) coated with MAdCAM-1 (100 ng/μL) were incubated with Mn 2+ -
activated TK-1 cells at RT for 30 min. The cells were stained with the rat-anti-β7 mAb Fib504 (1:100) and a
rabbit-anti-lck antibody (1:100). The anti-β7 antibody was detected with a secondary anti-rat-AF488 (green)
antibody, and the anti-lck antibody was detected with a secondary anti-rabbit-AF594 (red) antibody. Stained
cells were analysed by confocal microscopy. (A) A representative population of TK-1 cells incubated with
MAdCAM-1-coated microspheres and stained with anti-beta7 (green) and anti-lck (red) antibodies. The two
images were merged to aid visualization of colocalization (merged). (B) A representative single cell was
enlarged for clearer visualisation. Pseudo-SMACs are indicated by blue arrows. Bar indicates the length of 20
µm. This experiment was repeated three times.
145

3.8. Mutation of the β7 cytoplasmic domain and effects on cell adhesion
The above results suggested that the β7 subunit CARD associates with the tyrosine kinases
src and FAK both in vitro and in vivo. To further investigate structure-function properties of
the β7 CARD in vivo, the next aim was to examine the effect of mutated tyrosines 736 and
741 in the β7 CARD on α4β7 integrin adhesion. The third downstream tyrosine residue at
position 761 was also mutated for comparison. The tyrosine residues were substituted with
phenylalanines in the full-length β7 sequence as described previously in Section 3.4.1.
Plasmids encoding the mutant β7 subunit were transfected together with a plasmid encoding
the wild-type α4 integrin subunit into HEK-293T cells to analyze α4β7-mediated cell
adhesion to MAdCAM-1.
3.8.1 Cloning of mutated variants of the integrin β7 subunit into mammalian expression
vectors
The initial aim was to clone wild-type integrin β7 cDNA and mutated variants into a
mammalian expression vector. The cloning strategies are summarised in Figure 3.55. The full
length wild-type human β7 (β7wt) cDNA was excised from the pcDM8-huβ7 vector with
HindIII and NotI and ligated it into a pcDNA3.1 H+ vector (Figure 3.54, Step 1). DNAs
encoding variant forms of the integrin β7 cytoplasmic domains carrying the mutations Y736F,
Y741F, Y761F and Y736F+741F had previously been cloned into pGEX-2T vectors by lab
colleagues (refer to Section 3.4.1). They were amplified by PCR (Step 2), and the resultant
products were used in an overlap PCR reaction with a fragment of β7wt cut from the
pcDNA3.1 H+ vector by the restriction enzymes SacI and NotI (Step 3). The overlap PCR
products carrying the mutated tyrosines (Step 4) were ligated in a 3-way ligation with an
empty pcDNA3.1 H+ vector and a cDNA fragment containing the remaining portion of wild-
type β7 cDNA to form a plasmid carrying the complete mutated full-length β7 cDNA (Step
5). All clones were sequenced to confirm the integrity of the cloned sequence (data not
shown).
In addition, the full-length β7 cDNAs were subcloned between the restriction sites NheI and
XhoI of the pIRES2-EGFP vector (Step 6). The pIRES2-EGFP vector expresses the enhanced
green fluorescent protein (EGFP), and also contains an internal ribosomal targeting sequence
to co-express a desired protein, such as the β7 integrin cDNAs. The pIRES2-EGFPcontaining
β7 cDNA vectors were primarily used to control for transfection efficiency.
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Figure 3.55 Cloning strategy for constructing plasmids encoding the full-length β7 subunit with mutated
cytoplasmic tyrosines
Complementary DNA encoding the full-length wild-type human β7 subunit (β7wt) was excised from the clone
pCDM8-β7wt and cloned into the plasmid pcDNA3.1 H+ to form the pcDNA3.1-β7wt vector (Step 1).
Complementary DNA encoding the C-terminal region of the wild-type β7 subunit was amplified from the
pcDNA3.1-β7wt vector with the primers cc24 and cc25. Complementary DNAs encoding the cytoplasmic
domain of the β7 subunit carrying mutations in tyrosines 736, 741, and 761 were PCR-amplified from the
respective pGEX-2T vectors with the primers cc26 and cc27 (Step 2). The PCR products that resulted from the
latter two amplifications were joined together by overlap PCR using the primers cc24 and cc27. The PCR
product, which bore the restriction enzyme site SacI at the 5’-end and NotI at the 3’-end (Step 4), was digested
with SacI and NotI for subcloning. Complementary DNA encoding the N-terminal region of the wild-type fulllength
β7 cDNA was excised from the pcDNA3.1-β7wt vector with the restriction enzymes HindIII and SacI
(Step 3), and ligated together with PCR products encoding the C-terminal region that carried tyrosine mutations.
The ligated fragments were subcloned into the pcDNA3.1 vector to form full-length mutated variants of the β7
subunit (Step 5). The full-length β7 subunit mutants were excised with NheI and XhoI and cloned into the
pIRES2-EGFP vector (Step 6). The boxes show keys to the diagram, indicating the START and STOP codons,
the start of the cytoplasmic domain, and the positions of the tyrosines (Y) that were mutated to phenylalanines
(F). The expression plasmids generated carried the substitutions Y736F, Y741F, Y761F, and Y736+741F in the
cytoplasmic domain.
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3.8.2 Cloning of a wild-type integrin α4 construct
The aim was to clone a full-length cDNA encoding the integrin α4 subunit into the pcDNA6
mammalian expression vector. The cloning strategy is summarised in Figure 3.56. The
available α4 subunit cDNA in the pCDM8 plasmid lacked the first 114 amino acid residues at
the 5’-end of the α4 subunit cDNA (data not shown). mRNA from the human T-lymphocyte
cell line H9 (Step 1) was used as a template to RT-PCR amplify the missing N-terminal
region of the α4 integrin cDNA (Step 2). The PCR product was cloned into pGEM-T and
excised with the restriction enzymes BamHI and EcoRV (Step 3). The integrin α4 cDNA in
the pCDM8 vector was excised with EcoRV and NotI, where the EcoRV site was located 3’
of the missing region (Step 4). The PCR product encoding the missing N-terminus of the α4
subunit was then ligated in a 3-way ligation with the rest of the α4 subunit cDNA into a
pCDNA6-V5his vector (Step 5). The resulting construct was sequenced to confirm its
integrity (data not shown).
Figure 3.56 Schematic diagram of the strategy used to generate a pcDNA6-V5his vector encoding the full-
length α4 subunit
The α4 subunit cDNA provided in a pCDM8 vector lacked an extreme N-terminal sequence (denoted in purple).
RNA was isolated from H9 cells (Step 1) and used as a template for RT-PCR with the primers cc28 and cc29 to
repair the cDNA to obtain a full-length α4 subunit cDNA (Step 2). The resulting PCR product (red) was ligated
into pGEM-T and excised with BamHI and EcoRV (Step 3). The C-terminus of the α4 subunit was excised from
pCDM8 with EcoRV and NotI digestion (4). The latter two α4 subunit fragments were ligated together into the
pcDNA6-V5his vector, producing a construct encoding a full-length wild-type α4 subunit.
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3.8.3 Transfection of HEK-293T cells to express the α4 and β7 subunit plasmids
Confirmation of high transfection efficiency of integrin plasmids
HEK-293T cells were transfected with pcDNA6-V5his and pIRES-2-EGFP plasmids
encoding the α4 and variant β7 subunits, respectively, using the Lipofectamine 2000
transfection reagent. Cells transfected with the α4 expression plasmid were cotransfected
with a pIRES2-EGFP vector to measure transfection efficiency. The transfectants were grown
in normal growth media containing of 5 μg/mL of blasticidin for 48-72 hrs, and the success of
transfection was visualised by viewing GFP fluorescence by using epi-fluorescence
microscopy (Figure 3.57). Transfection efficiencies were similar for all transfections, and
estimated to be approximately 80-90%. GFP was uniformly expressed in cells.
Figure 3.57 Transfection of HEK-293T cells with plasmids encoding the integrin α4 and β7 subunits.
HEK-293T cells were cotransfected with 4 µg of the pcDNA6-V5his vector expressing the native α4 subunit and
4 µg of the pIRES2-EGFP vector (Panel 1). They were also transfected with 4 µg of each of the pIRES-2-EGFP
vectors encoding native β7 (Panel 2), and the β7 subunit with the mutations Y736F (Panel 3), Y741F (Panel 4),
Y761F (Panel 5), and Y736F+741F (Panel 6). Images were captured using epi-fluorescence microscopy. Bar
indicates the length of 20 µm. This experiment was repeated three times.
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Confirmation of integrin expression by immunoblotting
The transfectants were analysed by immunoblotting for expression of the introduced integrin
subunits. Lysates of each of the above transfectants were resolved by SDS-PAGE and
immunoblotted with an antibody recognising the β7 subunit extracellular domain (Figure
3.58). The β7 subunit was expressed at similar levels by each transfectant (lanes 2 to 6). In
contrast, HEK-293T cells transfected with the α4 expression plasmid did not express the β7
subunit. The membrane was probed with an antibody against β-actin to show that equal
amounts of each lysate had been loaded on the gel.
Figure 3.58 Western blot analysis of HEK-293T cells transfected with plasmids encoding α4 and β7
varients
Transfected HEK-293T cells (5 x 10 5 ) were lysed in the presence of NP40, resolved by SDS-PAGE, and
immunoblotted with an antibody against the β7 subunit extracellular domain (sheep anti-huβ7ext-IgG, used at
1:1000), which was detected with an anti-sheep-HRP antibody (1:80,000). The HEK-293T cells were transfected
with plasmids encoding the α4 subunit (lane 1); α4 and β7wt subunits (lane 2); α4 and β7(Y736F) subunits (lane
3); α4 and β7(Y741F) subunits (lane 4); α4 and β7(Y761F) subunits (lane 5); and α4 and β7(Y736+741F)
subunits (lane 6). The membrane was also immunoblotted for β-actin as a loading control. Molecular weights of
marker proteins are indicated in the left-hand margin in kDa. This experiment was repeated three times.
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Staining of transfectants with anti-integrin antibodies to confirm integrin expression
Expression of the α4 and β7 integrins by transfected HEK-293T cells was confirmed by
staining of cells with fluoresceinated anti-integrin antibodies (Figure 3.59). The upper row of
photographs in Figure 3.59 show transfected cells stained with a red fluorescent antibody
against the β7 subunit cytoplasmic domain. Cells transfected with the α4 plasmid did not stain
with the anti-β7 subunit antibody, indicating that HEK-293T cells do not express β7
integrins, as expected. Cells cotransfected with the α4 and β7wt plasmids were brightly
stained, whereas cells cotransfected with the α4 plasmid and one of the mutated β7 plasmids
stained less brightly. The observed difference in staining of β7wt compared to the β7 mutants
may be explained by the fact that the antibody used to detect expression was raised against
the β7 subunit cytoplasmic domain such that mutations of the tyrosine residues to
phenylalanines may cause a decrease in antibody affinity. To examine this possibility,
transfected HEK-293T cells were stained with a green fluorescent Fib504 mAb against the
extracellular domain of the β7 subunit (Figure 3.59, middle row of photographs). As
expected, cells transfected with the α4 plasmid did not stain with the Fib504 mAb. In contrast
to the above results, cells cotransfected with the α4 plasmid and either wild-type or mutant β7
plasmids stained green and showed similar levels of staining.
The transfectants were also stained with a green fluorescent DATK32 antibody against α4β7,
which is complex-specific. Cells transfected with the α4 plasmid did not stain with the
DATK32 mAb. Cells cotransfected with the α4 plasmid and either wild-type or mutant β7
plasmids were stained green and showed similar levels of staining. Not all cells expressed
α4β7, as the transfection efficiency was not 100%.
Taken together, the results show that HEK-293T cells were successfully transfected with the
plasmids encoding the α4 and β7 integrin subunits, leading to detectable levels of cell-surface
expression of α4β7 and mutated forms.
151

Figure 3.59 α4β7 is expressed at the surface of HEK-293T cells transfected with α4 and β7 plasmids
HEK-293T cells were transfected with plasmids encoding the wild-type α4 subunit (pcDNA6-α4wt) alone or together with either wild-type (pcDNA3.1-β7wt) or mutant forms
(pcDNA3.1-β7Y736F, -β7Y741F, -β7Y761F, or -β7Y736+741F) of the integrin β7 subunit. Transfectants were grown for 48 hrs, fixed, and stained with fluorescent antibodies.
The cDNAs used for transfection are indicated at the top of each column of photographs, whereas the antibodies used to detect the encoded integrin subunit are indicated in the
right-hand margin of each row. The top row shows transfectants immunostained with a rabbit polyclonal antibody against the cytoplasmic domain of the β7 subunit, which was
detected using the red fluorescent anti-rabbit-antibody AF594. The middle row shows immunostaining with the Fib504 mAb against the extracellular domain of the β7 subunit,
which was detected with a green fluorescent anti-rat AF488 antibody. The bottom row shows transfectants stained with the DATK32 mAb against the α4β7 complex, which was
detected with a green fluorescent anti-rat AF488 antibody. Cell nuclei were stained blue with DAPI. Images were captured using epi-fluorescence microscopy. Bar indicates a
length of 20 µm. This experiment was repeated three times.
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3.8.4 Testing the ability of transfectants to adhere to integrin ligands
The aim here was to determine whether HEK-293T cells transfected with plasmids encoding
wild-type α4β7 and forms containing mutated cytoplasmic tyrosines (Y736F, Y741F, Y761F,
and Y736+741F) were able to bind to MAdCAM-1 via α4β7. The transfectants were
activated with Mn 2+ , adhered to MAdCAM-1-coated plates, fixed and stained with methylene
blue, and the adherent cells either visualised by light microscopy (Figure 3.60A), or
quantified using a microplate reader (Figure 3.60C). In parallel, Mn 2+ -activated TK-1 cells
were adhered to MAdCAM-1-coated slides for comparison as positive controls (Figure
3.60B and D).
HEK-293T cells transfected with the α4 plasmid that lacked α4β7 expression adhered very
poorly to MAdCAM-1 (Figure 3.60C). In contrast, cells cotransfected with the α4 and β7
plasmids showed a 1.5 fold increase in cell adhesion compared to cells transfected with the
α4 plasmid. Single tyrosine mutations Y736F, Y741F, and Y761F in the β7 cytoplasmic
domain significantly reduced the level of cell binding by 24% to 41% (p = 2.49 × 10 -2 to 3.26
× 10 -2 ), whereas the double mutant Y736+741F significantly reduced cell adhesion by 53%
(p = 1.88 × 10 -3 ), compared to adhesion mediated by wild-type α4β7 (Figure 3.60C).
Visualization of attached cells by light microscopy confirmed the above results (Figure
3.60A).
For comparison, non-activated and Mn 2+ -activated TK-1 cells were bound to MAdCAM-1.
There was a 2-fold increase in binding of activated TK-1 cells compared to non-activated
cells (Figure 3.60D). Under light microscopy, activated TK-1 cells densely covered the
entire surface of MAdCAM-1-coated slides, whereas adherent non-activated TK-1 cells were
more sparsely distributed (Figure 3.60B).
The above results suggest that all three tyrosine residues in the cytoplasmic domain of the β7
subunit are important in mediating the cell adhesion properties of α4β7 integrin.
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Figure 3.60 Testing the ability of HEK 293T transfectants to adhere to MAdCAM-1
HEK-293T cells transfected with plasmids encoding α4, α4β7wt, α4β7(Y736F), α4β7(Y741F), α4β7(Y761F),
and α4β7(Y736+741F) were tested for their ability to bind to MAdCAM-1-coated slides and plates. Slides and
plates were coated with 10 μg/mL of MAdCAM-1 overnight at 4 °C. Cells (5 x 10 5 to 1 x 10 6 per well) were left
to adhere to plates for 30 min at RT, and were then fixed and stained with methylene blue. They were visualised
by light microscopy (A) and quantified using a microplate reader at the wavelength 405 nm (C). For
comparison, non-activated and activated TK-1 cells were adhered to MAdCAM-1-coated slides, visualised by
light microscopy (B), and quantified in a microplate reader (D). Data shown represent the mean and standard
deviation of three wells. * denotes a p-value < 0.05 and ** denotes a p-value of < 0.01. The experiments were
performed twice in triplicate.
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3.9. Identification of binding partners of the β7 cytoplasmic domain
Integrin cytoplasmic domains play a key role in regulating integrin functions. The results of
this thesis so far have shown that a CARD within the β7 cytoplasmic domain binds src and
FAK, and interacts with the cytoskeletal proteins paxillin, filamin and α-actinin. The aim here
was to identify additional binding partners for the β7 cytoplasmic domain, and examine their
involvement in the signalling of β7 integrins.
3.9.1 Pull-down assay
A biotin-labelled synthetic peptide comprising the full-length 52 aa β7 cytoplasmic domain
was bound to streptavidin-coated magnetic beads, and incubated with TK-1 cell lysates. The
beads were washed thoroughly, and the pulled-down proteins resolved by SDS-PAGE, and
stained with Coomassie blue (Figure 3.61, lanes 2 and 4) and by silver staining (data not
shown). A control synthetic peptide (biotin-APTLPPAWQPFLK-OH) was incubated with a
TK-1 cell lysate for comparison (Figure 3.61, lanes 1 and 3). The β7 cytoplasmic domain
peptide specifically precipitated two proteins of approximately 70 kDa when compared to the
control peptide. The 70 kDa proteins were equally precipitated from the lysates of Mn 2+ -
activated and non-activated cells (Figure 3.61, lane 2 compared to lane 4).
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Figure 3.61 SDS-PAGE analysis of proteins precipitated with a synthetic peptide comprising the complete
cytoplasmic domain of the β7 subunit
A synthetic peptide comprising the complete cytoplasmic domain of the β7 subunit was conjugated to magnetic
beads, and incubated with TK-1 cell lysates for 2 hrs at 4°C. The lysates were prepared from non-activated
(lanes 1 and 2) and Mn 2+ -activated (lanes 3 and 4) cells. The pull-down assays were performed with an
unrelated control peptide (biotin-APTLPPAWQPFLK; lanes 1 and 3) and the β7 cytoplasmic domain peptide
(lanes 2 and 4). The affinity-purified proteins were resolved by SDS-PAGE, and stained with Coomassie blue.
Two protein bands of 70 kDa that were specifically precipitated by the β7 cytoplasmic domain peptide
(indicated by arrow in lane 4) were analysed by mass spectrometry. The sizes of molecular weight markers are
indicated in the left-hand margin. This experiment was repeated twice.
3.9.2 Mass spectrometry identifies heat shock proteins as ligands of the β7 subunit
The 70 kDa proteins isolated in the pull-down assay (Figure 3.61, lane 4) by the β7
cytoplasmic domain peptide were sent for mass spectrometry analysis. The whole gel was
sent to the Centre for Genomics and Proteomics at the University of Auckland. The two
distinguishable bands were separately and analysed. The resulting spectra were compared
with a Mus musculus genomic database. Mass spectrometry analysis identified four hsp70
family members as potential ligands of the β7 cytoplasmic domain. They included heat shock
protein 9, heat shock protein 8a, heat shock 70 kDa protein 1-like, and heat shock 70 kDa
protein 1a (Figure 3.62A and B).
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Figure 3.62 Mass spectrometry identifies four heat shock proteins as potential β7 ligands
Mass spectrometry analysis identified four hsp70 family members as potential ligands of the β7 cytoplasmic
domain. They include heat shock protein 9 (A), heat shock protein 8a, heat shock 70 kDa protein 1-like, and
heat shock 70 kDa protein 1a (B). Mass spectrometry analysis was performed by the Centre for Genomics and
Proteomics at the University of Auckland.
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3.9.3 A pull-down assay estabilishes that hsp70 interacts indirectly with the β7 subunit
The synthetic β7 cytoplasmic domain peptide was tested for its ability to precipitate a
recombinant hsp70 protein to determine whether hsp70 interacts directly or indirectly with
the β7 cytoplasmic domain. The β7 cytoplasmic domain peptide and a control peptide
(control peptide 1) were immobilised on magnetic beads and incubated with recombinant
hsp70-1a (ProSpec-Tany TechnoGene Ltd) in the presence and absence of a TK-1 cell lysate.
The beads were washed thoroughly, bound proteins were resolved by SDS-PAGE, and the
gel was silver-stained (Figure 3.63). The control peptide did not precipitate recombinant
hsp70 in the absence or presence of a TK-1 cell lysate (lanes 1 and 2). The β7 cytoplasmic
domain peptide precipitated recombinant hsp70, but only in the presence of a TK-1 cell lysate
(compare lanes 3 and 4). The precipitated doublet of 70 kDa proteins co-migrated with
recombinant hsp70 (lane 5), and were not detectable in the crude TK-1 cell lysate (lane 6).
The results suggest that hsp70 does not bind directly to the β7 cytoplasmic domain, but does
so after facilitation by other proteins.
Figure 3.63 A synthetic β7 cytoplasmic domain peptide precipitates recombinant hsp70 in a pull down
assay
A β7 cytoplasmic domain peptide and a control peptide bound to magnetic beads were incubated with 4 μg of
recombinant human hsp70-1a in the absence or presence of a TK-1 lysate. Lanes 1 and 2, control peptide
incubated with hsp70 in the absence or presence of a TK-1 cell lysate, respectively. Lanes 3 and 4, β7
cytoplasmic domain peptide incubated with hsp70 in the absence or presence of a TK-1 cell lysate, respectively.
Lane 5, 2 μg of recombinant hsp70-1a protein. Lane 6, TK-1 cell lysate (10 4 cells/lane). The arrow indicates the
location of the recombinant hsp70-1a. The left-hand margin indicates the size of the molecular weight markers.
This experiment was repeated three times.
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3.9.4 Coimmunoprecipitation of α4β7 and hsp70
The aim here was to determine whether α4β7 and hsp70 could be coimmunoprecipitated with
each other from a TK-1 cell lysate. The β7 integrin was immunoprecipitated from a TK-1 cell
lysate, the immunoprecipitate resolved by SDS-PAGE, transferred to PVDF membrane, and
immunoblotted with antibodies against hsp70 (Figure 3.64A) and the integrin β7 subunit
(Figure 3.64B). Immunoprecipitates formed with rat-IgG were used as controls. Hsp70 was
immunoprecipitated by antibodies against the β7 subunit, indicating that hsp70 complexes
with the integrin β7 subunit (Figure 3.64A, 70 kDa band indicated by arrow). The specificity
of the immunoprecipitating Fib504 anti-β7 integrin subunit antibody was confirmed by
immunoblotting the membrane with a rabbit polyclonal antibody against the β7 cytoplasmic
domain (Figure 3.64B, 120 kDa band indicated by arrow).
Figure 3.64 Hsp70 is coimmunoprecipitated from a TK-1 cell lysate with the β7 subunit
TK-1 cell lysates were incubated with the rat anti-β7 subunit antibody Fib504, and a control rat IgG. Both
antibodies had been cross-linked to Sepharose beads. The immunoprecipitates were resolved on 10%
polyacrylamide SDS-gels, and immunoblotted with antibodies against hsp70 (A) and the β7 integrin subunit (B).
A band at approximately 70 kDa in (A) indicates the presence of hsp70 in the β7 immunoprecipitate (right lane;
refer to arrow), and a band at approximately 120 kDa in (B) indicates the presence of the β7 subunit (refer to
arrow). No bands of the corresponding sizes were present in the control immunoprecipitates (middle lanes). For
comparison, the unfractionated TK-1 cell lysate was immunoblotted (left lanes). The sizes of the molecular
weight markers are indicated in the left-hand margin. This experiment was repeated twice.
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Hsp70 was immunoprecipitated from a TK-1 cell lysate using anti-hsp70 antibodies, and the
immunoprecipitate immunoblotted with the rabbit anti-β7 subunit antibody to further confirm
that hsp70 forms a complex with α4β7 (Figure 3.65). The TK-1 cell lysate was
immunoprecipitated with the Fib504 mAb against the β7 subunit (lane 3) and an antibody
against hsp70 (lane 7). An immunoprecipitate formed with mouse ascites served as a control
(lane 5). For comparison, the TK-1 cell lysate was loaded into lane 1, and the
immunoprecipitating antibodies in the absence of lysate were included in lanes 2, 4 and 6.
Both the Fib504 mAb and the anti-hsp70 mAb immunoprecipitated the β7 subunit, as
indicated by a protein band at 120 kDa (lanes 3 and 7, respectively). The β7 subunit may
have been partially degraded as indicated by bands of lesser molecular weight. In contrast,
the 120 kDa protein was not immunoprecipitated by the control antibody (lane 5).
Figure 3.65 The β7 subunit is coimmunoprecipitated from a TK-1 cell lysate with hsp70
TK-1 cell lysates were incubated with the anti-β7 mAb Fib504, a mouse ascites to anti-human IgG (control
antibody), and with an anti-hsp70 mAb (mouse ascites). The immunoprecipitates were resolved by SDS-PAGE
and immunoblotted with a rabbit anti-β7 polyclonal antibody. Lane 1, TK-1 cell lysate; lane 2, anti-β7 (Fib504)
antibody alone; lane 3, lysate immunoprecipitated with anti-β7 mAb (Fib504); lane 4, mouse ascites alone; lane
5, lysate immunoprecipitated with mouse ascites; lane 6, anti-hsp70 antibody alone; lane 7, lysate
immunoprecipitated with the anti-hsp70 antibody. Arrow in the right-hand margin indicates the size and location
of the mature β7 subunit. The sizes of the molecular weight markers are indicated in the left-hand margin. This
experiment was repeated twice.
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3.9.5 Localization of hsp70 and hsp90 in TK-1 cells
The results above indicate that hsp70 complexes with the β7 integrin in vivo. The aim here
was to visualise the localisation of endogenous hsp70 and β7 in cells. TK-1 cells were
adhered to MAdCAM-1-coated coverslips, fixed, and co-stained with a red fluorescent
antibody against hsp70 and a green fluorescent antibody against the β7 integrin subunit
Figure 3.66. Staining was visualised by fluorescence microscopy. α4β7 strongly localized to
the uropod (Figure 3.66, refer to arrow), but was also present at what appear to be small focal
points. In contrast, hsp70 was more diffusely expressed throughout the cytoplasm. Merger of
the images failed to reveal co-localisation of α4β7 and hsp70, but it was clear that hsp70 was
not expressed with α4β7 on the uropod.
Figure 3.66 Localisation of hsp70 and α4β7 on TK-1 cells attached and spread on MAdCAM-1
Mn 2+ -activated TK-1 cells were adhered to MAdCAM-1-coated coverslips, fixed with paraformaldehyde, and
stained with rat anti-β7 (Fib504, 1:100) and rabbit anti-hsp70 (1:100) antibodies. The primary antibodies were
detected with anti-rat-AF488 (green, 1:250) and anti-rabbit-AF594 (red, 1:250) antibodies, respectively. Nuclei
were stained with DAPI, and the images were merged to measure co-localisation of hsp70 and β7. The magenta
arrow shows the localisation of the uropod. Images were captured using confocal fluorescence microscopy. Bar
indicates the length of 20 μm. This experiment was repeated three times.
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In addition to localization of hsp70 in T cells, the location of hsp90 was investigated for
comparison. Hsp90 had been included as a control for hsp70 in the above experiment. Hsp90
represents another major family of heat shock proteins involved in chaperone activity
required to protect signalling and adaptor proteins from degradation. TK-1 cells were adhered
to MAdCAM-1-coated coverslips, fixed, and co-stained with a red fluorescent antibody
against hsp90 and a green fluorescent antibody against the β7 integrin subunit (Figure 3.67).
As before, α4β7 was shown to be strongly expressed by the uropod and within small focal
adhesions (Figure 3.67, refer to magnified image B). Hsp90 was diffusely expressed
throughout the cytoplasm (Figure 3.67A), and was weakly expressed by the uropod (Figure
3.67, refer to magnified image B). Merger of the images revealed that α4β7 and hsp90 do not
colocalize within the uropod.
Figure 3.67 Localisation of hsp90 and α4β7 on TK-1 cells attached and spread on MAdCAM-1
Mn 2+ -activated TK-1 cells were adhered to MAdCAM-1-coated coverslips, fixed with paraformaldehyde, and
stained with rat anti-β7 (Fib504, 1:100) and goat anti-hsp90 (1:100) antibodies. The primary antibodies were
detected with anti-rat-AF488 (green, 1:250) and anti-goat-AF594 (red, 1:250) antibodies, respectively (A).
Images were merged to measure co-localisation of hsp90 and β7. The uropod of one cell was magnified (B).
Images were captured using epi-fluorescence microscopy. Bar indicates the length of 20 μm. This experiment
was repeated three times.
162

3.9.6 Hsp70 colocalizes with intracellular β7 ligands following ligand-induced clustering
of α4β7
The aim here was to determine whether hsp70 might colocalize with intracellular ligands for
β7 within SMAC formed by MAdCAM-1-induced clustering of α4β7. A green
fluoresceinated FAM-YDRREY peptide was employed which is expected to interact with
intracellular ligands for α4β7. TK-1 cells were pre-incubated with the FAM-YDRREY
peptide, and then activated with Mn 2+ to induce binding to MAdCAM-1-coated microspheres
and clustering of α4β7 at the cell surface. The cells were fixed and stained with antibodies
against hsp70 to look for co-localisation with the FAM-YDRREY peptide (Figure 3.68). The
FAM-YDRREY peptide localized largely within a small number of clusters which formed
near or at the plasma membrane. In contrast, hsp70 was more diffusely scattered throughout
the cells, but nevertheless it was clearly present within large clusters that appeared to have
similar positions as those containing the FAM-YDRREY peptide (Figure 3.68, refer to blue
arrows). Colocalization of hsp70 and the FAM-YDRREY peptides was confirmed by
merging the images. Interestingly, there were several clusters where hsp70 and the FAM-
YDRREY peptides did not colocalize (Figure 3.68, refer to white arrows)
Figure 3.68 Hsp70 colocalizes with intracellular β7 ligands within SMAC formed with MAdCAM-1coated
microspheres
TK-1 cells were incubated with 1 μM of the green fluorescent peptide FAM-YDRREY and then activated with
Mn 2+ . Clustering of α4β7 was induced by incubating the cells with MAdCAM-1-coated microspheres. The cells
were fixed and stained with a rabbit anti-hsp70 antibody (1:100) and then with a red fluorescent anti-rabbit-
AF594 antibody. The merged images reveal clusters where hsp70 and the FAM-YDRREY peptide co-localize
(blue arrows), and clusters where they do not colocalize (white arrows). Images were captured using epifluorescence
microscopy. Bar indicates the length of 20 μm. This experiment was repeated three times.
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3.9.7 Effect of heat shock on expression of hsp70 and the β7 subunit
Given that hsp70 interacts with α4β7, albeit indirectly, it was decided to determine whether
hsp70 might influence α4β7 expression and/or function. Here, the effect of heat shock on the
expression of hsp70 and the β7 subunit was examined, given that hsp70 is expected to protect
cells against heat shock. TK-1 cells were heat shocked at 42ºC for 45 min and allowed to
recover overnight at 37ºC. Non-heat shocked cells were cultured overnight at 37ºC, and
served as controls. The cells were lysed and the lysates were analysed by SDS-PAGE and
immunoblot analysis. The membrane was separated into three sections according to the
molecular weight ladder, whereby the larger and intermediate sized proteins were
immunoblotted with anti-β7 and anti-hsp70 antibodies, respectively (Figure 3.69). There was
no detectable change in the level of the β7 subunit in the soluble (S) and insoluble (P)
fractions after heat shock treatment. In contrast, the level of hsp70 was increased in both the
soluble and insoluble fractions after heat shock treatment (Figure 3.69). The membrane
containing the smaller proteins was probed an antibody against β-actin to serve as a loading
control.
Figure 3.69 Effect of heat shock on the expression of hsp70 and the β7 subunit by TK-1 cells
164

TK-1 cells were heat shocked at 42ºC for 45 min and allowed to recover overnight at 37ºC. Non-heat shocked
cells were cultured overnight at 37ºC, and served as controls. The soluble (S) and insoluble (P) fractions of
lysates of the latter cells were resolved by SDS-PAGE, and the proteins transferred to PVDF membrane. The
membrane was immunoblotted with antibodies against the integrin β7 subunit (rabbit anti-β7 cytoplasmic
domain), hsp70 (rabbit anti-hsp70) and β-actin (rabbit anti-β-actin). The primary antibodies were detected using
goat anti-rabbit-HRP antibodies (1:20,000). The positions of the β7 subunit, hsp70 and β-actin are indicated by
the arrows in the right-hand margin. The sizes of the molecular weight markers are indicated in the left-hand
margin. This experiment was repeated twice.
3.9.8 Effect of heat shock on activation of α4β7 is time dependent
Here the aim was to determine whether the activation of α4β7 by Mn 2+ might be affected by
heat shock treatment. TK-1 cells were heat shocked at 42˚C for 15, 30 and 45 min, and then
allowed to recover overnight at 37ºC. Non-heat shocked cells were cultured overnight at
37ºC, and served as controls. The cells were either left unactivated or were activated with
Mn 2+ and compared in their ability to adhere to MAdCAM-1-coated slides. As can be seen in
Figure 3.70, α4β7 integrins on resting TK-1 cells are normally inactive and are only able to
marginally support cell binding to MAdCAM-1. In the presence of Mn 2+ , integrins rapidly
switch to an active state. Interestingly, prolonged heat shock treatment for 15 to 45 minutes
activated α4β7 such that cells were able to bind to MAdCAM-1 without prior activation with
Mn 2+ . The degree of cell binding depended on the length of heat shock treatment, where
maximal activation of α4β7 was achieved by heat shocking cells for 45 min. Heat shock
treatment of cells for 45 min produced levels of cell binding that were similar to those
induced by Mn 2+ (Figure 3.70A). While all cells in the experiment were viable as determined
by trypan blue exclusion, heat shock treatment for times greater than 45 min led to increasing
cell death (data not shown). Cells subjected to heat shock for 45 min were also incubated
with antibodies against α4 and β7 prior to cell adhesion (Figure 3.70B). In the presence of
antibodies, there was a decrease in cell adhesion, confirming heat shock activated cell
binding to MAdCAM-1 was mediated by α4β7. The results indicate that heat shock treatment
very gradually induces the activation of α4β7 integrins on TK-1 cells, and that the degree of
activation of α4β7 is dependent on the length of heat shock treatment. The ability of α4β7 to
remain active overnight following heat shock treatment is unusual, as it was previously
reported that Mn 2+ -induced activation of α4β7 is very transient i.e. once activated, α4β7
slowly deactivates over period of to 2 hrs (Zhang et al. 1999).
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Figure 3.70 Effect of heat shock treatment on the binding of TK-1 cells to MAdCAM-1
TK-1 cells were heat shocked at 42˚C for 0, 15, 30 or 45 min, and incubated overnight at 37˚C. Non-heat
shocked cells were cultured overnight at 37ºC, and served as controls. Cells were either left non-activated or
activated with Mn 2+ . Cells were adhered to MAdCAM-1-coated slides at RT for 30 min (A). B) Cells were also
heat shocked at 42˚C for 45 min incubated overnight at 37˚C, and then incubated with antibodies against α4 and
β7 prior to adhesion to MAdCAM-1-coated slides. Bound cells were fixed, stained with 1% methylene blue, and
cell attachment analysed using a microplate reader at a wavelength of 495 nm. Data shown represent the mean
and standard deviation of three wells. The experiment was repeated three times.
3.9.9 Fever-range temperatures can activate α4β7
TK-1 cells were incubated at 37, 39, 42 and 44°C for 45 min to assess whether the degree of
activation of α4β7 was temperature-dependent. In particular would fever-range temperatures
of ~39°C be able to activate α4β7? The cells were allowed to recover overnight at 37°C and
tested for their ability to bind to MAdCAM-1-coated slides (Figure 3.71). TK-1 cells
incubated at 39 and 42°C showed similar levels of binding to MAdCAM-1 i.e. about 1-fold
greater binding than non-heat shocked cells. The level of cell binding was comparable to that
166

of TK-1 cells activated with Mn 2+ . Cells treated for 45 min at 44 °C did not survive (data not
shown).
Figure 3.71 Fever-range temperatures induce TK-1 cell binding to MAdCAM-1
TK-1 cells were incubated at 37, 39, and 42˚C for 45 min. The cells were then incubated overnight at 37˚C.
Non-heat shocked cells were cultured overnight at 37ºC, and served as controls. The Ccells were either left nonactivated
or activated with Mn 2+ and adhered to MAdCAM-1-coated surfaces at RT for 30 min. Bound cells
were fixed, stained with 1% methylene blue, and cell attachment analysed using a microplate reader at a
wavelength of 495 nm. Data shown represent the mean and standard deviation of three wells. The experiment
was repeated three times.
3.9.10 Heat shock leads to prolonged activation of α4β7 even after Mn 2+ -treatment
As mentioned above, it was previously reported that Mn 2+ -induced α4β7 activation is very
transient i.e. once activated, α4β7 slowly deactivates over a period of to 2 hrs (Zhang et al.
1999). However, heat shock treatment appeared to cause the prolonged activation of α4β7,
where α4β7 remained active after overnight culture for at least 16 hrs (Figure 3.70). Here, it
was determined whether treatment with Mn 2+ after heat shocking would render α4β7
activation more labile.
TK-1 cells were heat shocked at 42˚C and allowed to recover overnight at 37˚C. Non-heatshocked
cells were cultured overnight at 37ºC, and served as controls. Cells were harvested
and either left untreated or treated with Mn 2+ for 0, 1 or 2 hrs. They were then allowed to
attach to MAdCAM-1-coated slides (Figure 3.72). As before, heat shocked cells bound to
MAdCAM-1 even without Mn 2+ -induced activation. The cells retained their ability to bind to
MAdCAM-1 even after 2 hrs treatment with Mn 2+ . In contrast, TK-1 cells that had not been
heat shocked were unable to bind to MAdCAM-1 unless they were activated with Mn 2+ .
167

Further, their ability to bind to MAdCAM-1 was transient, where binding was decreased by
54% after 1 hr of Mn 2+ treatment. After a further hour the cells had completely lost their
ability to bind to MAdCAM-1.
Figure 3.72 The prolonged activation of TK-1 cells by heat shocking is not affected by Mn 2+ treatment
TK-1 cells were heat shocked at 42˚C for 45 min, or left untreated, and cultured overnight at 37˚C. Both heat
shocked and untreated cells were activated with Mn 2+ at RT for 0, 1 or 2 hrs before testing their ability to adhere
to MAdCAM-1-coated slides. Cells were left to adhere at RT for 30 min, and then fixed, stained with 0.1%
methylene blue, and attachment analysed using a microplate reader at a wavelength of 495nm. All cells were
confirmed to be viable by trypan blue exclusion. Data shown represent the mean and standard deviation of three
wells. The experiment was repeated three times.
3.9.11 An inhibitor of hsp70 blocks the binding of TK-1 cells to MAdCAM-1
The effects of the hsp70 inhibitor KNK-437 on TK-1 cell binding to MAdCAM-1 was
examined to further study the effects of hsp70 on the function of β7 integrins. KNK-437
inhibits heat shock factor 1 (HSF1) binding to heat shock element (HSE) thereby preventing
expression of inducible hsps (Ohnishi et al. 2004). TK-1 cells were incubated with increasing
concentrations (10, 50 to 100 µM) of KNK-437 at RT for 2 hrs, activated with Mn 2+ , and
tested for binding to MAdCAM-1-coated slides. KNK-437 decreased the binding of TK-1
cells to MAdCAM-1 in a dose dependent manner. It caused significant reductions in cell
binding of 72% (p = 1.53 × 10 -3 ), and 94% (p = 1.48 × 10 -4 ) at 50 and 100 µM, respectively
(Figure 3.73). Treatment of TK-1 cells with DMSO, the dilutent for KNK-437, had no effect
on TK-1 cell adhesion (data not shown). Inhibition of TK-1 cell binding to MAdCAM-1 by
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KNK-437 suggests that hsp70 is involved in the activation and/or signalling pathways of
α4β7.
Figure 3.73 KNK-437 blocks TK-1 cell adhesion to MAdCAM-1
TK-1 cells were incubated in the presence or absence of 0, 10, 50, or 100 µM KNK-437 at 37˚C for 2 hrs and
activated with Mn 2+ . Cells not treated with KNK-437 and left unactivated served as controls. Cells were adhered
to MAdCAM-1-coated slides at RT for 30 min. The bound cells were fixed, stained with 0.1% methylene blue,
and cell attachment analysed using a microplate reader at a wavelength of 495 nm. Data shown represent the
mean and standard deviation of three wells. The experiment was repeated three times. ** denotes a p-value <
0.01 and *** denotes a p-value of < 0.001; compared to the number of cells bound in the absence of peptide.
3.9.12 KNK-437 has no detectable effect on hsp70 and β7 expression.
Here the aim was to determine whether KNK-437 alters the level of expression of hsp70 and
the β7 subunit, which might explain how it blocks cell adhesion. TK-1 cells were incubated
with 0, 25, 50 or 100 μM KNK-437, and cell lysates were subjected to Western blot analysis
with antibodies against the β7 subunit and hsp70 (Figure 3.74). The membrane was also
immunoblotted for β-actin as a loading control. KNK-437 had no detectable effect on the
expression of either hsp70 or the β7 subunit, when levels were compared with those of βactin
169

Figure 3.74 Effect of KNK-437 on the expression of hsp70 and the β7 subunit
TK-1 cells were incubated with 0, 25, 50 and 100 µM KNK-437 at 37˚C overnight. The cells were lysed and the
soluble protein fraction was resolved by SDS-PAGE, and transferred onto PVDF membrane. The membrane
was immunoblotted with antibodies against the β7 subunit, hsp70, and β-actin as a loading control. The
positions of the β7 subunit (120 kDa), hsp70 (70 kDa), and β-actin (40 kDa) are indicated in the right-hand
margin. The sizes of the molecular weight markers are indicated in the left-hand margin. The experiment was
repeated three times.
3.9.13 Serum deprivation activates α4β7
The effect of heat shock on TK-1 cell adhesion to MAdCAM-1 is a stress-related response.
The aim here was to determine whether other stressors might induce the activation of α4β7.
The response of TK-1 cells to serum deprivation which leads to nutrient starvation, and
decreased levels of growth factors was examined. TK-1 cells are normally grown in the
presence of 10% serum. For serum deprivation the serum concentration was decreased to 5
and 2.5%, and the cells were incubated overnight. The cells were either left unactivated or
were activated by Mn 2+ and tested for binding to MAdCAM-1-coated slides. Cells grown in
10% FCS were unable to bind to MAdCAM-1 without activation by Mn 2+ . In contrast, cells
grown in 5% and 2.5% serum bound to MAdCAM-1 without prior activation with Mn 2+ . Cell
binding was increased by 2-fold and 3-fold, respectively (Figure 3.75A). The results show
that activation of α4β7 can be induced by stress caused by low serum levels. Cells were also
grown in 10%, 5% and 2.5% serum and incubated with antibodies against α4 and β7 prior to
cell adhesion (Figure 3.75B). In the presence of antibodies cell adhesion decreased,
confirming that α4β7 mediates cell adhesion in response to serum deprivation.
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Figure 3.75 Effect of serum deprivation on TK-1 cell adhesion to MAdCAM-1
TK-1 cells were cultured in normal conditions of 10% serum, or stressed by culturing in 5% serum and 2.5%
serum overnight (A). For comparison, an aliquot of the cells was heat shocked at 42˚C for 45 min and cultured
overnight in 10% FCS. The cells were either left unactivated or were activated with Mn 2+ , and bound to
MAdCAM-1-coated slides at RT for 30 min. Cells cultured in 10%, 5% and 2.5% serum were also incubated
with antibodies against α4 and β7 for 30 min prior to cell binding assay (B). The bound cells were fixed, stained
with methylene blue, and cell attachment analysed using a microplate reader at a wavelength of 495nm. All cells
were confirmed to be viable by trypan blue exclusion. Data shown represent the mean and standard deviation of
three wells. The experiment was repeated three times.
The level of expression of the β7 subunit and hsp70 by cells deprived of serum was
examined. TK-1 cells were grown overnight in media containing 2.5%, 5%, 7.5% and 10%
serum, and the soluble fraction of the cell lysates was analyzed by SDS-PAGE, and subjected
to Western blot analysis with antibodies against the β7 subunit and hsp70 (Figure 3.76).
Serum deprivation did not lead to any detectable change the levels of the β7 subunit and
hsp70, suggesting that increased binding of TK-1 cells to MAdCAM-1 in response to serum
deprivation was not due to increased expression of α4β7. All cells were confirmed to be
viable by trypan blue exclusion before cell lysis (data not shown).
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Figure 3.76 Expression levels of the β7 subunit and hsp70 remain unchanged after serum depletion
TK-1 cells were cultured overnight in media containing 2.5%, 5%, 7.5%, and 10% serum. The soluble protein
fraction of the cell lysates was resolved by SDS-PAGE, and transferred onto PVDF membrane. The membrane
was cut into two pieces between the 75 and 50 kDa protein markers. The piece containing the higher molecular
weight proteins was immunoblotted with antibodies against the β7 subunit cytoplasmic domain (rabbit anti-β7
cytoplasmic domain, 1:400) and then stripped and probed with an antibody against hsp70 (rabbit anti-hsp70,
1:200). The piece containing the lower molecular proteins was immunoblotted with an antibody against β-actin
(rabbit-anti-β-actin, 1:1000), in order to measure protein loading. The positions of the β7 subunit (120 kDa),
hsp70 (70 kDa), and β-actin (40 kDa) are indicated in the right-hand margin. The sizes of the molecular weight
markers are indicated in the left-hand margin. The multiple bands beneath the major actin band are suggestive of
degradation products, indicating that the proteins analyzed have been partially degraded. The experiment was
repeated three times.
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Chapter 4. Discussion
4.1. Preliminary characterization of α4β7 and αEβ7 expression and
adhesion
Development and characterization of reagents and methods for the study
The initial aim of this thesis was to confirm the expression of β7 integrins on T-cell
lymphoma TK-1 (Ruegg et al. 1992) and T-cell hybridoma MTC-1 (Roberts et al. 1993) cells
which had previously been reported to express the α4β7 and αEβ7 integrins, respectively. The
expression of both integrins on the latter cell lines was detected by immunoblotting and
fluorescence microscopy, confirming previous reports. The principle ligands of α4β7 and
αEβ7 are MAdCAM-1 and E-cadherin respectively, and were required as recombinant forms
in order to serve as substrates to analyze α4β7 and αEβ7-mediated cell adhesion. The cDNAs
of MAdCAM-1 and E-cadherin had previously been fused by lab colleagues, to the CH2/CH3
regions of the human IgG antibody heavy chain Fc domain to produce recombinant
MAdCAM-1-Fc, and E-cadherin-Fc proteins, respectively (Yang et al. 1995; Berg et al.
1999b). The latter reagents were used to produce recombinant forms of MAdCAM-1-Fc and
E-cadherin-Fc, whose purity was confirmed by SDS-PAGE.
In order to assess β7 integrin signalling pathways it was necessary to choose specific integrin
activating agents. Integrins can be activated by a variety of stimuli, including the integrin
pan-activator Mn 2+ (Dransfield et al. 1992), the protein kinase C (PKC) activator phorbol-12myristate
13-acetate (PMA; Rothlein et al. 1986), and the G-protein activator AlF4 - . AlF4 -
mimics the γ phosphate of GTP by promoting the active conformation of heterotrimeric Gproteins
(Coleman et al. 1994) and was found to activate α4β7 on TK-1 cells to bind
MAdCAM-1 (Driessens et al. 1997; Zhang et al. 1999).This thesis confirmed that the binding
of TK-1 cells to MAdCAM-1-coated slides could be induced by activation of α4β7 with
Mn 2+ , PMA and ALF4 - .
While MTC-1 cells were confirmed to express cell-surface αEβ7, the cells did not bind to Ecadherin-coated
slides. However, MTC-1 cells activated with either Mn 2+ , PMA or AlF4 -
bound to E-cadherin in solution via αEβ7, as confirmed by antibody blockade with anti-αE,
anti-β7, and anti-E-cadherin antibodies. This result is unusual as MTC-1 cells have
173

previously been shown to bind to E-cadherin-coated slides (Berg et al. 1999b). Further, it
would be expected that cell binding to E-cadherin-coated slides would involve a multivalent
interaction which would thereby increase the affinity of interaction. A possible explanation
for this is that the recombinant E-cadherin became denatured on the slides, or assumed a
conformation that rendered it inaccessible to αEβ7. In any event, an adhesion assay based on
the binding of E-cadherin in solution to αEβ7 was established, and used in the subsequent
study.
4.2. Analysis of β7 integrin signalling pathways
Integrins are implicated in many different intracellular signalling pathways (Hynes 2002;
Krissansen et al. 2006a). Various chemical inhibitors were tested for their ability to prevent
TK-1 cell adhesion to MAdCAM-1 in order to identify signalling pathways and molecules
responsible for activating the adhesion and/or clustering of α4β7. Genistein, a general
tyrosine kinase inhibitor, was shown to inhibit the G-protein-mediated activation of αLβ2.
Thus, it inhibited AlF4 - -activated, but not PMA or Mn 2+ -activated adhesion of TAM2D2 T-
cell hybridoma cells to ICAM-1 (Driessens et al. 1997). Similarly, α4β7-mediated adhesion
of TK-1 cells to MAdCAM-1 in response to activation with AlF4 - was inhibited by genistein
(Zhang et al. 1999). In contrast to αLβ2, α4β7-mediated adhesion of Mn 2+ -activated TK-1
cells and PMA-differentiated HL60 cells was also inhibited by genistein (Walsh 1996). This
thesis confirmed that genistein inhibits Mn 2+- , PMA- and AlF4 - activation of α4β7-mediated
adhesion. The fact that genistein does not disrupt Mn 2+ and PMA-mediated activation of
αLβ2 is evidence that different integrins utilize different signalling pathways (Driessens et al.
1997).
It has been shown that different integrins within the same family differ in their response to
agonists. For example, it is much more difficult to activate αXβ2 than other β2 integrins (Lu
et al. 2001a), and αIIbβ3 is more resistant to the effects of Mn 2+ than is αVβ3 (Kamata et al.
2005). Several studies have looked into the dose-dependency of activation of integrins by
divalent cations. Conformation studies of the α1 helix of the β1 integrin subunit A-domain
reveal that Mn 2+ is a potent activator of β1 integrins. Mn 2+ binds to the β1 integrin A-domain
MIDAS and promotes a shift in position of the α1 helix (Mould et al. 2002). It can activate
integrins at subnanomolar ranges, with activation being maximal at about 2 mM
concentration. Mg 2+ also binds to the MIDAS and activates integrin ligand-binding, however
at a much lower level. In contrast, Ca 2+ is unable to cause the same conformational changes,
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however it is likely to also bind to the MIDAS site because Ca 2+ can support low affinity
ligand-binding to α5β1, and high affinity binding of activation-independent ligands to α4β1
(Chen et al. 2001). However, the binding of Ca 2+ to sites other than MIDAS appears to favour
an inactive conformation. Increasing doses of Ca 2+ up to 2 mM cause increased reduction in
ligand-binding (Mould et al. 2002). Questions have been raised whether Mn 2+ -induced
activation accurately reflects physiological integrin activation. The shift of the α1 helix by
Mn 2+ parallels the shift of the α1 helix by inside-out signalling of β2 integrins in response to
activating cytoplasmic domain mutations and mAb binding (Lu et al. 2001c). Recently, a
trivalent cation gadolinium (Ga 3+ ) was shown to activate β2 integrins at concentration doses
in the 10 µM range, and promote extended conformation of integrins as does Mn 2+ . However
it did not alter intracellular Ca 2+ levels of neutrophils which is a feature exhibited by Mn 2+
(Zhang et al. 2008b). Mn 2+ stimulation has also been found to activate integrins in a PLC-
dependent fashion (Driessens et al. 1997).
Doses of PMA ranging from 0.1 to 16 nM have been shown to induce integrin-mediated
binding of gastric carcinoma cells to fibronectin. PMA induces the tyrosine phosphorylation
of FA proteins such as FAK on Y397, paxillin on Y118, and c-src on Y416 (Lee et al. 2006).
AlF4 - mimics the γ-phosphate of GTP and activates the heterotrimeric G-proteins (Coleman
et al. 1994). It is thought that G-proteins activate PLCγ through phosphorylation by the
tyrosine kinase syk, which leads to the hydrolysis of PIP2 and formation of IP3 and DAG.
IP3 mobilises intracellular Ca 2+ intracellular stores, and together with DAG activates PKC
and Rap1-GEF (Figure 4.1; Driessens et al. 1997). The activation of Rap1-GTP activates
RIAM and recruits talin to the β integrin cytoplasmic domain leading to integrin activation
(Han et al. 2006). G-proteins can also be activated by agonist G-protein coupled receptors or
tyrosine kinase coupled receptors, which leads to either the activation of PKC through RhoA,
or PKC and DAG by PI3-kinase and PLC. Additionally, PMA also activates DAG, which
leads to the activation of PKC and Rap1-GTP, and so forth (Han et al. 2006). As mentioned
above, DAG together with Ca 2+ activates PKC and Rap1-GEF which leads to Rap1-GTP-
RIAM activation and the recruitment of talin (Figure 4.1).
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Figure 4.1 Pathways which lead to integrin activation.
Depicted are some of the signalling pathways which lead to integrin activation. The Mn 2+ cation can bind
directly to the integrin extracellular domain to activate integrins which mimics inside-out integrin activation.
Alternatively it can activate integrins through a PLC-dependent pathway, which may signal directly through
PKC or through DAG, which recruits Rap1-GEF leading to Rap1-GTP activation. PMA stimulation can directly
activate DAG which signals through PKC or Rap1-GEF leading to Rap1-GTP activation. TCR signalling can
lead to Rap1-GTP activation. AlF4 - activates G-proteins which leads to either PI3-kinase signalling to PKC and
Rap1-GTP activation, or through PLC leading to Rap1-GTP activation. Agonists such as cytokines can activate
G-protein coupled receptors or tyrosine kinase receptors leading to the activation of G-proteins, and the
subsequent activation of RhoA which signals through PKC or through PI3-kinase, or tyrosine kinase syk leading
to the activation of Rap1-GTP. Activated Rap1-GTP interacts with RIAM which recruits talin to the β subunit
cytoplasmic domain and initiates integrin activation. This figure was generated from information gathered from
published data (Driessens et al. 1997; Kinashi 2005; Han et al. 2006)
Stimulation of TK-1 cells via the α4β7 integrin results in the activation of src and MAP-
kinases (Uhlemann et al. 1997). The three MAP kinase cascades are well known signalling
pathways that are activated in response to signals originating from growth factor receptors,
integrins, src and fyn, and G-protein coupled receptors (Ramos 2008). The ERK, JNK and
p38 MAPK pathways have an important role in cell migration, and may be activated via the
Ras-Raf-MEK cascade triggered by integrin engagement (Desban et al. 2006). In addition,
p38 MAPK has been reported to mediate the downregulation of the integrin α4 subunit in
human mammary epithelial cells transformed to overexpress the small G-protein erbB-2,
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which is responsible for remodelling of the cytoskeleton (Woods Ignatoski et al. 2003). This
thesis investigated the involvement of the MAP kinase cascade and src kinases in α4β7
activation, and the consequent adhesion to MAdCAM-1. Specific signalling/kinase inhibitors
were introduced into cells prior to performing the cell adhesion assays. A 3 hour incubation
of TK-1 cells with PD98059 and SB203580, inhibitors of MEK and p38 MAP kinase,
respectively (Cuenda et al. 1995; Dudley et al. 1995), had no observable effect on preventing
Mn 2+ or PMA activated α4β7-mediated cell adhesion (Table 4.1). A brief incubation with the
inhibitors was chosen in order to prevent any changes to gene transcription. This finding is
consistent with previously published data in which treatment of TK-1 cells with SB203580
and PD98059 for 2 hours had no effect on TK-1 cell adhesion to MAdCAM-1. In contrast,
incubation of TK-1 cells with SB203580 for 48 hours decreased cell adhesion to MAdCAM-
1 by approximately 50% (Shafiei 2004). This same study reported that the surface expression
of the β7 integrin subunit was decreased after 48 hours of treatment with SB203580, and in
accord α4 and β7 gene transcription was downregulated.
Table 4.1 Summary of the effects of chemical inhibitors on β7-mediated cell adhesion
Inhibitors Inhibit β7-mediated cell adhesion? Inhibition of other integrins
genistein yes (###) αLβ2 (Driessens et al. 1997).
PD98059 no -
SB203580 no -
JNK-I-1 yes (#) -
JNK-I-2 no -
AG99 no -
PP2 yes (#) -
damnacanthal yes (##) -
radicicol no -
ML-7 yes (##) αLβ2 (Smith et al. 2003)
# denotes inhibition of less then 15% at the highest concentration tested
## denotes inhibition of between 10 and 50% at the highest concentration tested
### denotes inhibition of greater then 50% at the highest concentration tested
This thesis did not examine the role of the JNK pathway in the activation of β7 integrins.
However, recent published findings provide some information on this subject. Jun N-terminal
kinases (JNKs) are a subfamily of the MAPKs that have been implicated in many integrinmediated
signalling events and processes (Dolfi et al. 1998; Oktay et al. 1999; Pankov et al.
2003). The JNK inhibitor, SP600125, was previously found to downregulate β7 gene
transcription after 1 hour of incubation, and decreased the expression of the β7 subunit on the
cell surface of TK-1 cells after 48 hours of treatment (Shafiei 2004). This study showed that
177

one inhibitor of the JNK pathway, JNK inhibitor-1 (JNK-I-1), could significantly decrease
Mn 2+ and PMA-induced β7 integrin activation (Table 4.1). However another inhibitor, JNK
inhibitor-2 (JNK-I-2, SP600125) had no detectable effect. JNK-I-1 is a peptide which blocks
JNK phosphorylation, and thereby blocks the transactivation of c-jun, but has no effect on the
activities of ERK1/2 and p38 (Barr et al. 2002). JNK-I-2 inhibits JNK by competing for ATP,
and thereby inhibits the phosphorylation of c-jun, and blocks the cellular expression of
cytokines such as IL-2, IFN-γ, and TNF-α (Bennett et al. 2001). The reason for the
differences in the abilities of JNK-I-1 and -2 to inhibit β7 integrin activation is not clear.
RTKs which mediate signalling by growth factors and cytokines, are involved in a variety of
biological processes including cell migration (Friedman et al. 2006). There is considerable
evidence demonstrating that integrins co-operate with RTKs. Thus, integrins associate with
RTKs to stimulate downstream signalling pathways, including MAPK that is necessary for
cell migration (Guo et al. 2004). Integrin-dependent adhesion triggers ligand-independent
activation of the RTK EGFR (Cabodi et al. 2004). In the present study, an inhibitor of EGFR
tyrosine kinase, namely AG99, which inhibits the phosphorylation of EGFR and the
activation of ERK1 and ERK2 (Pai et al. 1998), had no effect on α4β7-mediated adhesion of
TK-1 cells (Table 4.1). This result suggests that EGFR, ERK1 and ERK2 are not involved in
the activation/clustering of α4β7 induced by Mn 2+ or PMA. It does not rule out the possibility
that EGFR, ERK1 and ERK2 are involved in another function of α4β7 signalling, such as cell
migration, spreading, etc.
The src family of tyrosine kinases (SFK) are intrinsically involved in many signalling
transduction pathways, including integrin signalling (Playford et al. 2004; Mitra et al. 2006).
The SFK inhibitor PP2 (Hanke et al. 1996) and the lck inhibitor damnacanthal (Faltynek et al.
1995) only slightly inhibited α4β7-mediated cell adhesion by PMA-activated TK-1 cells. In
contrast, the src inhibitor radicicol (Kwon et al. 1992) did not appear to prevent α4β7mediated
adhesion of TK-1 cells to MAdCAM-1 (Table 4.1). The only minor inhibition of
α4β7-mediated cell adhesion by PP2 (PMA activation) and damnacanthal (Mn 2+ activation)
suggests that SFKs are not responsible for integrin activation in response to the agents used in
this study. Alternatively, SFKs may show redundancy in their ability to mediate integrin
activation, such that it may be necessary to inactivate several SFKs in order to demonstrate
the importance of SFKs for α4β7 activation. For example, combinational targeting of SFKs
by gene deletion leads to an increased frequency of perinatal lethality (Stein et al. 1994).
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Myosin light chain kinase (MLCK) phosphorylates and regulates myosin binding to actin
filaments which is required for actin contraction (Bresnick 1999). MLCK was found to
contribute to αLβ2-mediated attachment and migration of T cells on ICAM-1 (Smith et al.
2003). In the current study, inhibition of MLCK by ML-7 (Saitoh et al. 1987) significantly
decreased PMA and Mn 2+ mediated activation of TK-1 cell adhesion to MAdCAM-1 (Table
4.1). This result suggests that MLCK is required for the activation/clustering of α4β7 and
attachment of TK-1 cells to MAdCAM-1. Inhibition of cell adhesion by ML-7 was not
complete indicating that other components are involved.
Blockade of α4β7-mediated TK-1 cell adhesion to MAdCAM-1 with chemical inhibitors has
identified several signalling molecule candidates that participate in α4β7 signalling.
Nevertheless, the above approach is confounded by two factors. Integrin signalling can
involve multiple redundant pathways, and hence when one pathway or molecule is inhibited,
another pathway or molecule may compensate. Hence, it cannot be assumed that the failure
of an inhibitor to inhibit α4β7-mediated TK-1 cell adhesion necessarily means that the
pathway it inhibits does not play a role in α4β7 activation/clustering. Another factor concerns
the specificity of the chemical inhibitors. Chemical inhibitors are not specific, and in
particular can inhibit other molecules when used at high concentrations. Hence it cannot be
assumed that the ability of an inhibitor to inhibit α4β7-mediated TK-1 cell adhesion
necessarily means that the nominated target molecule is responsible. The above results can
only give clues to potential candidates, and further work is required using other types of
inhibitor e.g. using RNAi- knockdown, cell-permeable inhibitor peptide technology etc.
In this study, the effectiveness or viability of the inhibitors was not tested in parallel, but
relied on the manufacturers’ product certification. The inhibitors that showed no activity
should be tested in parallel in assays where they are known to exert activity.
The length of incubation with the inhibitors needs further consideration. A 3 hour incubation
of cells with the inhibitor was employed, according to a previously published thesis from the
host laboratory (Walsh 1996). Shorter or longer incubations with the inhibitors may produce
different results depending on the pathways involved and how the inhibitors interact with
them. Therefore a time-course monitoring of inhibitor activity may be useful.
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4.3. The YDRREY motif
The YDRREY motif within the β7 subunit cytoplasmic domain was previously found to be a
cell adhesion regulatory domain (CARD; Krissansen et al. 2006b). Cell-permeable peptides
containing the YDRREY motif prevented T-cells from binding to MAdCAM-1 and VCAM-1
by preventing α4β7 activation and clustering. The tyrosine residues flanking the motif were
deemed to be critical for the peptide’s activity, as substitution of the tyrosines with
phenylalanines abrogated the ability of the peptide to prevent cell adhesion. This thesis
confirmed that a cell-permeable form of the YDRREY peptide could prevent activated TK-1
cells from binding to MAdCAM-1. In addition, the peptide was found to prevent αEβ7-
mediated adhesion of H9 T-cells from binding to E-cadherin. Thus, the YDRREY motif is a
CARD for both α4β7 and αEβ7.
The YDRREY motif is unique to the β7 integrins. The β1 and β5 subunits contain the core
DRRE sequence but lack the flanking tyrosines. The β2 subunit, with its partial motif
SDLREY, is the only other integrin β subunit that contains a flanking tyrosine. A key
question was whether the YDRREY peptide acted as a kinase “sink” i.e. whether it competed
with the natural kinase substrates by being a scaffold for phosphorylation. The YDRREY
peptide was synthesized as an already phosphorylated form with phosphorylated tyrosines.
The YDRREY phosphopeptide showed a similar level of activity in preventing α4β7-
mediated adhesion of TK-1 cells to MAdCAM-1, suggesting that the YDRREY peptide does
not serve as a kinase “sink”. Interestingly, the proximity of the tyrosines also had no effect on
the activity of the peptide. Thus, the YDRGGGGREY peptide containing a spacer of four
glycine residues was as active as the native peptide. A peptide in which the internal DRRE
sequence was modified to either DGGE or EEEE still retained some activity, albeit the
concentration of peptide required to achieve similar levels of inhibition was much greater
(approximately 2 fold compared to the YDRREY peptide). A 4-mer multimer of the
YDRREY peptide, YDRREYGYDRREYGYDRREYGYDRREY, prevented α4β7-mediated
adhesion of TK-1 cells to MAdCAM-1 as effectively as the native peptide. It was anticipated
that the multimer by acting in a multivalent fashion would be substantially more active than
the native peptide, but this was not the case. Surprisingly, the 4-mer
FDRREFGFDRREFGFDRREFGFDRREF in which the tyrosines had been substituted with
phenylalanines, retained activity which was surprising given that the flanking tyrosines had
been found to be essential for peptide activity. Taking all of the above data into account, the
180

esults suggest that the tyrosine residues are important for the activity of the YDRREY
peptide, but the proximity of the tyrosines and their state of phosphorylation is not critical.
The core region DRRE also contributes to the activity of the CARD where the acidic
residues, but not the internal basic residues, are essential. A multimer of FDRREF appears to
provide a sequence of amino acids that can compensate for the lack of tyrosines.
Interestingly, substitution of the SDLREY motif in the β2 subunit for SDRREY induces
clustering of LFA-1 in K562 cells (Bleijs et al. 2001).
4.4. Phylogenetic study of the sequence of the YDRREY motif in the β7
subunit
Further insights into the structure-function relationships of the YDRREY motif can be gained
by studying the motif present in the β7 subunits of different species. The Ensembl website
(www.ensembl.org) was searched for nucleotide sequences encoding all orthologues of the
β7 subunit. Mammalian orthologues were identified for chimpanzee, gorilla, orangutan,
megabat, horse, hyrax, guinea pig, dolphin, mouse, mouse lemur, macaque, elephant,
microbat, and armadillo. Non-mammaliam orthologues were identified for frog, anole lizard,
zebrafish, and hagfish. The deduced protein sequences of the cytoplasmic domains for each
orthologue are listed in Table 4.2. The β7 integrin cytoplasmic domain is well conserved in
mammals, with protein identity ranging from 99% for human, chimpanzee, gorilla, and
orangutan sequences, to 53% and 48% for cats and armadillo, respectively, in comparison
with the human sequence (data not shown).
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Species
Human (Homo sapiens)
Chimpanzee (Pan troglodytes)
Gorilla (Gorilla gorilla)
Orangutan (Pongo pygmaeus)
Megabat (Pteropus vampyrus)
Horse (Equus caballus)
Cow (Bos taurus)
Hyrax (Procavia capensis)
Rat (Rattus norvegicus)
Guinea Pig (Cavia porcellus)
Dolphin (Tursiops truncatus)
Mouse (Mus musculus)
Mouse Lemur (Microcebus murinus)
Macaque (Macaca mulatta)
Elephant (Loxodonta africana)
Kangaroo rat (Dipodomys ordii)
Pika (Ochotona princeps)
Rabbit (Oryctolagus cuniculus)
Microbat (Myotis lucifugus)
Dog (Canis familiaris)
Cat (Felis catus)
Armadillo (Dasypus novemcinctus)
Frog (Xenopus tropicalis)
Anole Lizard (Anolis carolinensis)
Zebrafish (Danio rerio)
Hagfish (Eptatretus burgeri)
182
Cytoplasmic domain sequence (1-20)
RLSVEIYDRR EYSRFEKEQQ
RLSVEIYDRR EYSRFEKEQQ
RLSVEIYDRR EYSRFEKEQQ
RLSVEIYDRR EYSRFEKEQQ
RLSVEIYDRR EYNRFEKERQ
RLSVEIYDRR EYRRFEKERQ
RLSVEIYDRR EFHRFEKERQ
RISVEIYDRR EYSRFEKEQK
RLSVEVYDRL EYSRFEKERQ
RLLVEIYDRR EYSRFEKEQL
RLSVEIYDRR EYSRFEKEQQ
RLSVEIYDRR EYRRFEKEQQ
RLSVEIYDRR EYRRFEKEQQ
RLSVEIYDRR EYSRFEKEQQ
RISVEIYDRR EYSRFEKEQQ
RLLVEIYDRK EYIRFEKEQQ
RVLVEIYDRR EFSRFEKERQ
RLLVEIYDRR EFNRFEKERQ
RLSVEIYDRR EYKRFEKERQ
RLSVEIYDRR EFSRFEKEQK
RLSVEIYDRQ EYNRFEKERQ
RLSVEIYDRR EYNRFEKEQR
SITVEIYDRQ EYNRFQKERS
RVVVDVYDRR EFNRFEKECQ
RLLLELYDYR EYQSFVKMQN
KLLTTLYDRR EYXKFEMERS
Table 4.2 Alignment of the first 20 amino acid residues of the β7 subunit cytoplasmic domain of various
animal species
Comparison of protein sequences comprising the first 20 amino acid residues of the β7 subunit cytoplasmic
domains of different animal species. The conserved YDRREY motif is highlighted in green, with differences
between humans and other mammals highlighted in red. The table was generated using the Ensembl website
(www.ensembl.org), which is a joint project between European Bioinformatics Institute (EBI), an outstation of
the European Molecular Biology Laboratory (EMBL), and the Wellcome Trust Sanger Institute (WTSI).
The core region of the YDRREY motif in rat (YDRLEY), kangaroo rat (YDRKEY), and cat
(YDRQEY) has a substitution of the second arginine residue for either L, K, or Q,
respectively. The terminal tyrosine residue in cow, pika, rabbit, and dog is substituted with
phenylalanine giving the YDRREF sequence (Table 4.2). There is no example where both

tyrosines are substituted, where an acidic residue is substituted, or where both arginines are
substituted. Thus, in some species subtle changes in the YDRREY motif are permitted,
presumably because structure-function relationships are slightly different, for example the
way in which the YDRREY motif engages its target signalling molecules which are
presumably orthologues of those in humans and will have slightly different structures. The
YDRREY motif is completely conserved in some non-mammalian species including the
hagfish (Eptatretus burgeri). The motif exists as YDRQEY in the frog (Xenopus tropicalis)
and as YDYREY in zebrafish (Danio rerio). In the anole lizard (Anolis carolinensis) the Cterminal
tyrosine is substituted with phenylalanine (YDRREF).
4.5. The NPLY motif
A cell-permeable peptide representing the NPLY motif also prevented α4β7 from binding to
MAdCAM-1. The NPLY motif is found in several integrin β subunits including the β3, β5,
β6 and β7 subunits. The β1 subunit has an NPIY motif at the corresponding region, whereas
the β2 subunit has an NPLF motif, being the only integrin β subunit lacking the terminal
tyrosine. Thus, the NPLY motif, unlike the YDRREY motif, is not specific to the β7 subunit.
The membrane proximal NPxY motif is known to bind several phosphotyrosine binding
(PTB) proteins including talin and DOK1. The NPLY motif may bind to talin and thereby
prevent talin from binding to β integrin subunits to initiate integrin activation. The NPLY
motif is completely conserved in the β7 subunits of all species examined (Table 4.3) with the
exception of the anole lizard (Anolis carolinensis) and zebrafish (Danio rerio) for which the
terminal tyrosine is substituted with a phenylalanine residue. The armadillo sequence
terminates before the NPLY motif, however this sequence may be incomplete. An NPRF
motif (Table 4.3) which was not examined in this thesis is conserved in all species except
guinea pig, mouse lemur, kangaroo rat, pika, dog, frog, anole lizard, zebrafish and hagfish.
183

Species
Human (Homo sapiens)
Chimpanzee (Pan troglodytes)
Gorilla (Gorilla gorilla)
Orangutan (Pongo pygmaeus)
Megabat (Pteropus vampyrus)
Horse (Equus caballus)
Cow (Bos taurus)
Hyrax (Procavia capensis)
Rat (Rattus norvegicus)
Guinea Pig (Cavia porcellus)
Dolphin (Tursiops truncatus)
Mouse (Mus musculus)
Mouse Lemur (Microcebus murinus)
Macaque (Macaca mulatta)
Elephant (Loxodonta africana)
Kangaroo rat (Dipodomys ordii)
Pika (Ochotona princeps)
Rabbit (Oryctolagus cuniculus)
Microbat (Myotis lucifugus)
Dog (Canis familiaris)
Cat (Felis catus)
Armadillo (Dasypus novemcinctus)
Frog (Xenopus tropicalis)
Anole Lizard (Anolis carolinensis)
Zebrafish (Danio rerio)
Hagfish (Eptatretus burgeri)
Cytoplasmic domain sequence (21-end)
QLNWKQDSNP LYKSAITTTI NPRFQEADSP TL
QLNWKQDSNP LYKSAITTTI NPRFQEADSP TL
QLNWKQDSNP LYKSAITTTI NPRFQEADSP TL
QLNWKQDSNP LYKSAITTTI NPRFQEADSP TL
QLNWKQDSNP LYKSAITTTV NPRFQETESP LL
QLNWKQDSNP LYKSAITTTV NPRFQEADSP PL
HLNWKQDHNP LYQSAITTTV NPRFQEADSP VL
QLKWKQDNNP LYKSAITTTI NPRFQTESPS L
QLNWKQDSNP LYKSAVTTTV NPRFQGGNKQ SLSLPLTQEA D
QLNWKQDSNP LYKSAITTTI NPQFQRAESP VL
QLHWKQENNP LYKSAITTTV NPRFQEAEGA PL
QLNWKQDNNP LYKSAITTTV NPRFQGTNGR SPSLSLTREA D
QLNWKQDNNP LYKSAITTTI NPCFQDADRP AL
QLNWKQDSNP LYKSAITTTI NPRFQEADSP IL
QLNWNQDNNP LYKSAITTTI NPRFQGRESP LL
QLNWKQDNNP LYQSAITTTV NPNFQGTDGS SL
QLNWNKDNNP LYQSAITTTV NPFYQEAEKT PLR
QLNWNQDNNP LYRSAVTTTI NPRFQEPERP LL
QPNWKQDRNP LYKSAVTTII NPRFQAPESP L
HLNWKQENNP LYRSAITTTV NPQFQETGSL LS
QLNWKQDSNP LYRSAITTTV NPRFQEARSP FL
HLNWKQ
NAQWNELNNP LYKSATTTVK NPHFIEYVRP NV
SAKWNEMSNP LFRSATTTIM NPRY
QTQWKEAQNP LFKGATTTVM NPLHMQNEA
QAKWNKDDNP LYKSATTTVV NPKFDEN
Table 4.3 Alignment of the protein sequences from the distal region of the β7 subunit cytoplasmic of
various animal species
Comparison of protein sequences comprising the distal region (21 st amino acid residue to last residue) of the β7
subunit cytoplasmic domains of different animal species. Conserved regions are highlighted, the NPLY motif in
blue, and the NPRF motif in yellow. Differences in the conserved regions between humans and other mammals
are highlighted red. The table was generated using the Ensembl website (www.ensembl.org), which is joint
project between European Bioinformatics Institute (EBI), an outstation of the European Molecular Biology
Laboratory (EMBL), and the Wellcome Trust Sanger Institute (WTSI).
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4.6. Interaction of the YDRREY motif with tyrosine kinases
Tyrosine residues in the YDRREY motif are important for the function of the β7 CARD as
evidenced by the fact that substitution of the tyrosine residues with phenylalanines destroys
the ability of peptide to disrupt α4β7-mediated cell adhesion. Notably, the activity of the
peptide was not dependent on its state of tyrosine phosphorylation, as both synthetic
phosphorylated and unphosphorylated forms of the peptide were active. Nevertheless, it is
conceivable that the peptide has to be phosphorylated, and that the unphosphorylated cell-
permeable peptide becomes phosphorylated by cellular kinases once it enters the cell. Indeed,
TK-1 cell lysates were shown to phosphorylate the YDRREY peptide. In this thesis, the study
of the tyrosines phosphorylated in the β7 cytoplasmic domain was extended to include all 3
cytoplasmic tyrosines at positions 736, 741, 761. The aim was to identify which of the
tyrosine residues is phosphorylatable, and to identify the kinase(s) responsible. A GST-fusion
protein of the β7 cytoplasmic domain was phosphorylatable using a TK-1 cell lysate. The
identity of the tyrosine residues that were phosphorylatable was determined by creating GST-
fusion proteins of the β7 cytoplasmic domain carrying tyrosine to phenylalanine substitutions
at positions 736, 741, 761 and 736+741 combined. Substitution of each tyrosine with
phenylalanine reduced the degree of phosphorylation of the cytoplasmic domain, as
evidenced by reduced incorporation of 32 P. The data clearly showed that all three tyrosines in
the β7 cytoplasmic domain are potentially phosphorylatable.
Immunoprecipitation of α4β7 from activated and non-activated TK-1 cells revealed
differences in the phosphorylation state of the β7 subunit as detected with an antiphosphotyrosine
antibody. The β7 subunit isolated from activated cells was more highly
phosphorylated compared to that isolated from non-activated cells. There was no detectable
difference in the degree of tyrosine phosphorylation of the β7 subunit isolated from activated
TK-1 cells as compared to activated TK-1 cells containing bound ligand. The results suggest
that the tyrosine phosphorylation state of the β7 subunit is increased upon cell activation,
involving either the YDRREY or NPLY motif or both.
Phosphorylation of conserved motifs within the cytoplasmic domains of integrins can
modulate the binding of intracellular proteins. Phosphorylation of the β-integrin cytoplasmic
domain on threonine residues 783, 783, 785, or β2 residue 758, potentially mediated by
protein kinase C (PKC), inhibits the binding of filamin, but not talin. Phosphorylation of the
β2 subunit cytoplasmic domain on threonine residue 758 enables a 14-3-3 protein to bind
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(Fagerholm et al. 2005; Kiema et al. 2006; Takala et al. 2008). 14-3-3 proteins have the
ability to bind a multitude of functionally diverse signalling proteins (Morrison 2009).
Tyrosine phosphorylation of the conserved NP(I/L)Y motif, possibly mediated by src,
inhibits talin binding to the β3 subunit but enhances the binding of other PTB-domain-
containing proteins (Fagerholm et al. 2004; Kiema et al. 2006; Oxley et al. 2008). In this
thesis, the tyrosine kinases FAK, src and lck were shown to phosphorylate the YDRREY
motif. Src may not phosphorylate the initial tyrosine residue as the peptide YDRRE missing
the C-terminal tyrosine was not phosphorylated by src. FAK and src were found to bind
directly to the YDRREY multimer. Surprisingly, substitution of the internal arginines with
glycines or glutamic acid residues had a little effect on FAK and src binding, suggesting that
the internal arginines may serve largely as a spacer. FAK was previously reported to bind to
the integrin β1 (KLLMIIHDRREFA), β2 (KALIHLSDLREYR) and β3 (KLLITIHDRKEFA)
cytoplasmic domains at regions corresponding to the YDRREY motif. Replacement of the
aspartic and glutamic acid residues in the β1 subunit DRRE motif with alanines diminished
FAK-binding (Schaller et al. 1995a). Src and fyn were reported to bind to the extreme C-
terminal region 760-RGT-763 of the integrin β3 cytoplasmic domain via their SH3 domains
(Arias-Salgado et al. 2003). Recently fyn was reported to bind to the membrane proximal
region 721-IHDRK-725 of the β3 subunit, a region that overlaps the YDRREY motif (Reddy
et al. 2008). In the present study, src also bound to the FDRREFDRREFDRREFDRREF
peptide, a multimeric form of the FDRREF peptide, suggesting that src can bind to both the
DRRE core and to an YDXXEY peptide. It is possible that both motifs work together to
increase the affinity of binding. As regards the YDXXEY motif, it is interesting that synthetic
polymers of tyrosine and glutamic acid residues serve as substrates for PTK (Braun et al.
1984).
The present study found that autophosphorylated and unphosphorylated src binds to the nontyrosine
phosphorylated form of the YDRREY peptide. In contrast, only autophosphorylated
forms of src can bind to the phosphorylated YDRREY peptide. This was expected as
autophosphorylation of src activates the src kinase freeing the SH2 and SH3 domains. The
SH2 domains are known to bind preferentially to the phosphorylated pY-E-E-I motif due to
the polar and electrostatic interactions of the glutamic acid residues at pY +1 and +2 positions
(Songyang et al. 1993). As other residues can be accommodated in these positions, it has
been predicted that src would bind to the phosphorylated form of the YDRREY motif. In
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contrast, FAK bound equally to both the tyrosine phosphorylated and non-phosphorylated
forms.
FAK and src appeared to bind synergistically to the YDRREY motif. FAK and src can transphosphorylate
each other to enable their activation (Mitra et al. 2005; Mitra et al. 2006).
Whether the YDRREY motif preferentially binds src over FAK or vice versa would be
interesting to determine. Taken together, FAK and src show differences in their abilities to
phosphorylate and bind to the YDRREY motif, yet can cooperate to increase binding.
4.7. Probing the function of β7 subunit cytoplasmic domain tyrosine
phosphorylation sites by mutational analysis
The role of the three tyrosine phosphorylation sites in the β7 subunit cytoplasmic domain in
regulating α4β7-mediated cell adhesion was studied by transfection of plasmids expressing
mutant forms of the β7 subunit in which the tyrosines had been substituted with
phenylalanines. The cDNA of the wild-type β7 subunit, and mutant forms bearing the
substitutions Y736F, Y741F, Y761F, and Y736+41F were successfully cloned into a
mammalian expression vector and transfected into HEK-293T cells engineered to express the
integrin α4 subunit. The transfectants expressed good levels of expression of α4β7, despite
the fact that HEK-293T cells don’t normally express the leukocyte-restricted α4β7 receptor.
HEK-293T cells were used because of their ease of transfectability, whereas leukocytes are
known to be notoriously difficult to transfect at high efficiency. Another study also utilized
HEK-293T cells to successfully express α4β7 (Arthos et al. 2008). A concern in using HEK-
293T cells is that they are derived from kidney cells, whereas the β7 integrins are restricted in
their expression to leukocytes. It was not known whether HEK-293T cells would express all
the signalling molecules required to activate ectopically-expressed α4β7. Fortunately, Mn 2+ -
activation of HEK-293T cells induced the adhesiveness of α4β7, and its binding to
MAdCAM-1. Individual substitution of the three tyrosines with phenylalanines decreased the
binding of the α4β7 integrin to MAdCAM-1 in each case. Not surprisingly therefore,
combined substitution of both tyrosines 736 and 741 caused the greatest decrease in α4β7mediated
cell binding. These results suggest that each tyrosine is potentially involved in
signalling by β7 integrins, and confirm that the β7 CARD is critical for β7 integrin activation.
While the precise mechanism for the decrease in α4β7-mediated cell adhesion was not
identified in this study, several possibilities can be gleaned from the literature. Talin is known
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to bind to the NPxY motif. Tyrosine to alanine substitution of this motif in the β1A (Y788A),
β3 (Y747A), and β7 (Y761A) subunits disrupts talin and other PTB proteins from binding to
the integrin subunits (Calderwood et al. 2003). In accord, in the current thesis, substitution of
Y761 with phenylalanine decreased α4β7-mediated cell adhesion possibly due to disruption
of the binding of talin and other PTB proteins. Similarly, mutations of Y736F and Y741F
may disrupt the binding of either talin, src, FAK or other signalling proteins such as WAIT-1
which control β7 integrin activation.
4.8. Interaction of the YDRREY motif with cytoskeletal proteins
It has been established that the binding of talin to the β subunit cytoplasmic domain of
integrins is critical for integrin activation (Tadokoro et al. 2003). In this interaction, the
phosphotyrosine binding domain of talin binds to the membrane proximal region of the β
subunit cytoplasmic domain (Wegener et al. 2007). This thesis examined the interaction
between the β7 cytoplasmic domain motif YDRREY and adaptor proteins such as paxillin,
filamin and α-actinin.
Paxillin is an essential scaffolding protein which is recruited early to integrin adhesion
plaques (Deakin et al. 2008). Paxillin binds to the β1 and β3 subunits at a membrane-
proximal site (β3, 742-KLLITIHDRKE-752) that overlaps the YDRREY motif (Schaller et
al. 1995a; Chen et al. 2000). Paxillin also binds to the α4 subunit cytoplasmic domain (Liu et
al. 1999). The results of this thesis demonstrated that paxillin does not affect src binding to
the YDRREY peptide, suggesting either that paxillin does not bind directly to the YDRREY
motif or it binds with lesser affinity than src. Paxillin binds to FAK (Hayashi et al. 2002), and
is phosphorylated by ERK-2 (Liu et al. 2002). In the present study, a complex of paxillin,
FAK, and ERK-2 was able to bind to the YDRREY peptide (Figure 4.2). Paxillin can recruit
FAK to focal adhesions (Hildebrand et al. 1995; Tachibana et al. 1995). This provides the
possibility for paxillin to bind to the α4 subunit, and recruit FAK to the cytoplasmic interface,
where it might be in a position to bind and/or phosphorylate the β7 subunit.
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Figure 4.2 Proteins which may interact with the β7 cytoplasmic domain
Depicted is the sequence of the β7 cytoplasmic domain, and underlined in black are the regions where
cytoskeletal proteins or kinases may interact directly with the β7 cytoplasmic domain or the cytoplasmic
domains of other integrins. The green arrow indicates the interaction of FAK and src with the YDRREY motif,
as reported in this thesis. The blue lines indicate complexes formed between cytoskeletal elements and kinases
which bind to the YDRREY motif. The red line denotes the antagonistic effect filamin has on src binding to
YDRREY. This figure was constructed from information in the published literature and the results reported in
this thesis.
Filamin is an actin binding protein. Tight binding of filamin to the β1 and β7 subunit tails
decreased cell migration (Calderwood et al. 2001). The present study found that filamin
inhibited src binding to the YDRREY motif, but the mechanism for this inhibition was not
determined. Filamin is known to bind to the region 35-AITTTI-40 (refer to Table 4.3, and
Figure 4.2) between the NPLY and NPRF motifs in the β7 subunits, and to corresponding
regions in the β1 and β2 subunits (Calderwood et al. 2001). It has also been reported to bind
to a membrane-proximal region corresponding to where the YDRREY motif would reside in
the β1 integrin subunit (Schaller et al. 1995a). Therefore it is possible that filamin prevents
src binding to the YDRREY motif by binding to the YDRREY sequence itself. The SFK
member lck phosphorylates filamin which modulates actin filament cross-linking (Pal
Sharma et al. 2004). An alternative possibility is that filamin binds to src and thereby
prevents it from binding to the YDRREY peptide.
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α-actinin is a cytoskeletal protein which has previously been found to bind the cytoplasmic
domains of the β1 and β2 integrin subunits at a site immediately downstream of the region
corresponding to the YDRREY motif (Otey et al. 1990; Pavalko et al. 1993; Sampath et al.
1998). α-actinin binds to src and is phosphorylated by FAK (Izaguirre et al. 2001). The
present study showed that complexes of α-actinin with src or FAK can bind to the YDRREY
peptide. This result suggests that src or FAK could recruit α-actinin to the β7 subunit
cytoplasmic domain, and thereby provide a link to the cytoskeletal network. It is unlikely that
α-actinin binds directly to the YDRREY motif, as the binding sites for α-actinin within the β1
and β2 integrin subunits were downstream of the YDRREY motif as mentioned above. It is
possible, however, that α-actinin binds to a site downstream of the YDRREY motif in the β7
subunit cytoplasmic domain which could lead to the recruitment of kinases such as src and
FAK (shown diagrammatically in Figure 4.2). In this fashion, multiple assemblies of kinases
could accumulate at the cytoplasmic domain, potentially enhancing signalling.
This thesis did not investigate the roles of other β subunit interacting proteins such as talin
and kindlin, which have recently been reported to activate integrins (Shi et al. 2007;
Montanez et al. 2008; Tadokoro et al. 2003; Tanentzapf et al. 2006). As shown in the diagram
in Figure 4.2, talin binds to the central NPLY motif and to the region around the terminal
tyrosine of the YDRREY motif of the β3 integrin (Wegener et al. 2007; Rodius et al. 2008).
Pull-down assays with the YDRREY peptide to isolate interacting proteins from TK-1 cell
lysates did not show talin binding (data not shown). However, it cannot be excluded that talin
binds to part of the YDRREY motif. Therefore it would be interesting to investigate which
flanking residues might be required to allow talin to interact with the YDRREY motif.
Kindlin binds to the membrane distal region of the cytoplasmic domains of the β1 and β3
subunits (Shi et al. 2007; Montanez et al. 2008), therefore it is unlikely to interact with the
YDRREY motif, however it may still influence the interactions of YDRREY-binding
proteins.
In summary, this thesis has investigated potential interactions between the YDRREY motif,
cytoplasmic adaptor proteins, and the kinases src and FAK. It has not determined the exact
sequence of interaction, but has provided an insight into potential molecular interactions that
may impact upon intracellular signalling by β7 integrins. In addition, these experiments were
conducted with an isolated YDRREY peptide. Answers to whether the identified interactions
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hold true in vivo, and for which states of integrin activation, binding, cell migration and so
forth, will only come from undertaking considerable further research.
4.9. In vivo interactions of the YDRREY peptide with cellular kinases
A fluorescently labelled cell-permeable YDRREY peptide was introduced into TK-1 cells to
determine where the peptide would localize once inside the cells. The peptide was found to
cluster near the plasma membrane, possibly in FAs. FAK and src also co-localised within
these regions, suggesting that these regions may be sites of interaction between FAK and src
and the YDRREY peptide.
The above discussion has considered the interaction of FAK and src with the YDRREY
peptide, which is a contrived interaction. The following discussion will examine the evidence
that FAK and src kinase associate with the β7 subunit in vivo. FAK and src were both present
in α4β7 immunoprecipitates, indicating that they associate either directly or indirectly with
the α4 and/or β7 subunits.
Integrin expression on migrating leukocytes can be crudely separated into three different
zones, namely the lamellipodium/lamella at the leading edge, the focal zone at the midsection,
and the uropod at the trailing edge (Figure 4.3; Evans et al. 2009). Adhesive
junctions maintained as migrating cells move over each other, for example immune cells
moving over antigen presenting cells, are termed kinapses [combining roots indicating
movement (kin- from kinesis) and fastening (-apse from haptein)] (Dustin 2008a; Dustin
2008b). Kinapses are asymmetric and can also be separated into three different zones where
SMAC form. The lamellipodium is analogous to the dSMAC, the focal zone to the pSMAC,
and the uropod to cSMAC (cSMAC; Dustin 2008a; Evans et al. 2009). Focal adhesions form
at the pSMAC, travel to and mature within the dSMAC, then to the cSMAC. TCR
microclusters can form at the leading lamellipodium. TCR microclusters and integrin clusters
intermix within the integrin-rich lamella, and adhesion and TCR structures disengage at the
trailing uropod as the T cell moves. In this thesis, immunofluorescence studies were used to
localize FAK and src with respect to α4β7 on the surface of TK-1 cells bound to MAdCAM-
1-coated slides. The results suggested that FAK and src, but not lck, colocalize with α4β7.
MAdCAM-1 was coated on the slides at low concentrations to facilitate cell spreading and
migration. Fluorescence visualisation revealed that the uropod of migrating cells highly
expressed the β7 subunit, as previously reported for LFA-1 (reviewed in Evans et al. 2009).
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Src was also strongly expressed by the uropod, whereas FAK was weakly expressed, and lck
was undetectable. The β7 subunit and the kinases were also diffusely expressed throughout
the cytoplasm. FAK is thought to perform a variety of roles within a migrating cell from
participating in leading edge formation, FA turnover, and trailing edge retraction (Tomar et
al. 2009). Therefore FAK is expected to have a homogenous distribution as described in this
thesis. The strong presence of both β7 integrin and src in the uropod gives the potential for
molecular interactions between the two molecules. The uropod functions to tether LFA-1-
expressing T cells when they are confronted by chemokine-expressing APC. This attachment
at the rear allows the cells to scan their immediate surroundings at the leading edge. In
addition, during leukocyte transmigration the trailing edge can maintain a grip on the surface
as the leading edge penetrates through the endothelial junction (Evans et al. 2009). It is
possible that kinapses play a similar role in α4β7-expressing T-cells. A summary of the
expression profiles of α4β7 and the signalling molecules src, FAK, and lck on migrating TK-
1 cells is represented schematically in Figure 4.3.
Figure 4.3 The location of zones on migrating TK-1 cells.
A schematic of a TK-1 cell migrating on MAdCAM-1 showing the different kinapse zones and the expression
levels of α4β7, src, FAK, and lck. The leading edge (lamellipodium) expresses low levels of α4β7 and src, and
the mid-zone (focal zone) expresses higher levels of α4β7 and src. The trailing edge (uropod) expresses the
highest levels of α4β7 and src. FAK is expressed evenly throughout the different zones, whereas lck is
expressed evenly through the lamellipodium and focal zones, but weakly in the uropod. This figure was adapted
from (Evans et al. 2009) based on the results from this thesis.
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4.10. α4β7 clusters within SMACs
α4β7 can be induced to cluster on the surface of TK-1 cells by the binding of cells to
MAdCAM-1-coated microspheres (Zhang et al. 1999). TK-1 cells bound to both MAdCAM-
1-coated microspheres and Sepharose/magnetic beads. Confocal microscopy of α4β7 clusters
revealed they were part of multifocal immunological SMACs. This study found that α4β7
localized within a ring-like structure similar to the pSMAC. A parallel can be drawn with the
localization of α4β1 to the pSMAC of mature immunological synapses (Freiberg et al. 2002).
Confocal microscopy revealed that src co-localised with α4β7 within the pSMAC-like
regions, but was also located in the cSMAC, as well as around the entire plasma membrane.
FAK strongly localized within the cSMAC and weakly in the pSMAC with α4β7. Lck has
been reported to colocalize with the TCR within cSMAC (Freiberg et al. 2002). In accord, lck
was predominantly located within the cSMAC in the present study.
Increased filamin binding to integrins restricts integrin-dependent cell migration by inhibiting
transient memebrane protusions and cell polarization (Calderwood et al. 2001). Therefore,
during various stages of cell adhesion, migration and spreading, a dynamic balance must exist
between the binding of integrin cytoplasmic domains to cytoplasmic proteins which inhibit
and promote integrin activation. Taken together, the results reported in this study indicate that
α4β7 forms part of the structure of SMACs, as does α4β1. It may help to recruit src, but not
FAK which was predominantly located in the cSMAC. Whether these results are
representative of naturally occurring phenomena will need further analysis.
4.11. The affect of stress on the function of β7 integrins
Pull-down assays using a synthetic full-length β7 cytoplasmic domain peptide identified four
proteins which belong to the hsp70 family as potential intracellular ligands for the β7
cytoplasmic domain. Confirmation of an interaction between the β7 cytoplasmic domain and
hsp70 was obtained by demonstrating an interaction between recombinant hsp70-1a and the
synthetic full-length β7 cytoplasmic domain peptide. However, the interaction between hsp70
and the β7 cytoplasmic domain peptide was not direct, but rather was facilitated by the
presence of an unknown factor in a TK-1 cell lysate. This result suggests that the interaction
of hsp70 and the β7 cytoplasmic domain requires an adaptor protein, or some other factor to
be present.
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Hsp70 is a chaperone which assists in the folding of misfolded proteins (Daugaard et al.
2007), therefore it was possible that the binding of hsp70 to the β7 peptide might be
nonspecific i.e. hsp70 was just fulfilling its role as a chaperone. However this was not the
case, as the interaction of hsp70 and β7 integrin was confirmed to take place in vivo, as
evidenced by immunoprecipitation analysis of TK-1 cell lysates. Thus, hsp70 was present in
immune-complexes formed with an anti-β7 antibody, and vice versa, the β7 subunit was
present in immune-complexes formed with an anti-hsp70 antibody. Hsp70 did not co-localise
with β7 integrin in the uropod, but was detected at sites where fluoresceinated YDRREY
clustered in TK-1 cells bound to MAdCAM-1-coated microspheres.
This thesis addressed potential functional relationships between hsp70 and α4β7. Firstly,
hsp70 and α4β7 expression was examined in resting cells and in cells heat-shocked at 42°C.
Whilst α4β7 expression was not changed by heat-shocking, hsp70 expression was increased.
It was discovered that heat-shock treatment of TK-1 cells induced the activation of α4β7
adhesiveness, and delayed its deactivation. The dependence of activation on the temperature
of the heat shock treatment was examined. Physiological fever-range temperatures of 39°C
found at inflammatory sites (Chen et al. 2006b) produced similar levels of integrin activation
as induced by heat shocking at 42°C. Fever range hyperthermia has been found to stimulate
α4β7 integrin-dependent lymphocyte adhesion to high endothelial venules in the Peyer’s
patch and mesenteric lymph node.(Evans et al. 2000). In addition, cultured endothelial cells
cultured in fever range hyperthermia conditions augmented actin polymerisation and
enhanced endothelial-derived factors to transactivate α4β7 (Shah et al. 2002). This suggests
that febrile temperatures associated with infection may promote lymphocyte targeting to
those sites. Myocardial stress associated with heat shock, was reported to cause an increased
interaction between integrins and FAK, an increase of paxillin in the membrane fraction of
cell lysates, and protection against lethal ischemic injury (Wei et al. 2003). The same group
reported that heat stress led to the activation of AKT via FAK mediated pathways in rat
myocytes (Wei et al. 2008).
The hsp70 inhibitor, KNK437, causes a decrease in the adhesion of multiple myeloma cells to
stromal cells and fibronectin (Nimmanapalli et al. 2008). Similarly, this study showed that
KNK437 prevented the binding of TK-1 cells to MAdCAM-1, apparently due to a decrease in
hsp70 expression. Thus, hsp70 is critical for the α4β7-mediated adhesion of TK-1 cells to
MAdCAM-1, where it may potentiate α4β7 activation and/or clustering.
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Given that heat shocking activated α4β7-mediated adhesion, the possibility that other
stressors might do the same was explored. Cells were stressed by culture in low serum media,
which led to increased activation of α4β7-mediated adhesion. Whether hsp70 was involved
was not explored.
4.12. Summary
Cell adhesion studies of the binding of TK-1 cells to MAdCAM-1-Fc using specific
signalling inhibitors revealed potential intracellular pathways that may control α4β7
activation and clustering. This study suggests the involvement of JNK, SFK, lck and MLCK.
Intracellular signalling pathways are complex, involving many molecules, which are often
further complicated due to crosstalk between the pathways. Therefore inhibition of one
molecule in one pathway may not be enough to identify the signalling pathways involved
unless it is one which is critical or one in which many pathways converge.
Studies of the YDRREY CARD motif, and its importance to TK-1 cell adhesion to
MAdCAM-1-Fc, revealed that the flanking tyrosines residues, the core DRRE, and the acidic
residues were all functionally important, depending on the overall composition of the motif.
Changes to the central DRRE core such as substitution of the internal arginines was
allowable, and even the tyrosines were redundant in the case of the FDRREF multimer,
which retained the ability to block TK-1 cell adhesion. The proximity of the flanking
tyrosines was found not to be a crucial factor. A cell-permeable peptide based on the NPLY
motif was also able to prevent TK-1 cell adhesion to MAdCAM-1, suggesting multiple
interactions between intracellular signalling molecules and the β7 cytoplasmic domain
control α4β7-mediated cell adhesion. Phylogenetic comparison of the sequence of the β7
cytoplasmic domain revealed that the YDRREY motif was well conserved between species.
Only single amino acid substitutions in the motif, in either the core or flanking tyrosines,
were evident in some species.
The tyrosine residues in the motif were phosphorylatable by tyrosines kinases such as FAK
and src. FAK and src were also able to bind to the YDRREY motif. FAK could bind both
phosphorylated and unphosphorylated forms, whereas src was found to bind to only the
unphosphorylated form. Cytoskeletal adaptor proteins affected the binding of FAK and src to
the YDRREY motif. Filamin prevented src from binding to the YDRREY motif, whereas
paxillin had no effect.
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Tyrosine residues within the β7 integrin cytoplasmic domain were found to be important for
activation of α4β7 and its adhesive function. Substitution of the tyrosines with phenylalanines
resulted in decreased α4β7-mediated adhesion of HEK293T cells to MAdCAM-1.
Immunoprecipitation and immunofluorescence studies confirmed that α4β7 interacted with
FAK and src. Thus, FAK and src were present in immunoprecipitates formed with an anti-β7
subunit antibody. FAK and src both co-localised with the α4β7 at the cell-surface following
ligand-induced clustering. SMAC-like formations developed following clustering of α4β7
with ligand-coated microspheres, providing a useful tool to identify proteins present in focal
adhesions in future studies.
Hsp70 was unexpectedly identified as a potential intracellular binding partner of the β7
subunit. It was found to indirectly associate with the β7 subunit. Blocking hsp70 activity
decreased α4β7-mediated cell adhesion to MAdCAM-1. This finding then led to the
discovery that certain stressors could induce α4β7-mediated cell adhesion. Both heat shock
and serum-depletion stress induced the activation of α4β7, which was prolonged compared to
that induced by Mn 2+ -activation. This discovery provides a potential link between the heat
generated during inflammation and the activation of T cells, where heat may lower the
threshold of activation.
The YDRREY motif which in part controls β7 integrin-mediated cell adhesion could
potentially be a useful target for therapeutic intervention in the treatment of chronic
inflammatory diseases, including IBD, and multiple sclerosis.
4.13. Future directions
This thesis has generated several interesting findings that could be further explored. Outlined
below are a number of experiments which could help advance the aims of the thesis and our
understanding of the signalling pathways of β7 integrins.
Unravelling β7 integrin-mediated intracellular signalling pathways
The chemical inhibition of TK-1 cell adhesion to MAdCAM-1 was not as fruitful as had been
expected, however potential signalling pathways were identified. As briefly mentioned
above, use of a combination of inhibitors might be more productive, however the choice of
inhibitors would need to be carefully considered. To further these experiments it might be
useful to look into the effect of chemical inhibitors on cell migration or cell spreading, which
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involve downstream signalling pathways after integrin activation, including the MAPK or
JNK pathways. The experiments could be carried out using time lapse microscopy to observe
the effects of chemical inhibitors on cell function in real time for a prolonged period. The
findings could be validated by use of siRNAs targeted against key components of signaling
pathways, given the potential non-specificity of chemical inhibition.
Identification of kinases that regulate β7 integrins and investigation of their roles in
signaling by β7 integrins
The roles of the kinases src and FAK, which bind and phosphorylate the YDRREY motif in
the β7 subunit cytoplasmic domain need to be further explored. Are src and FAK involved
independently, or do they have synergistic or antagonistic effects? The results in this thesis
suggests synergy, however further experiments need to be done to determine the
spatiotemporal binding and phosphorylation of the YDRREY motif by each kinase. Recent
advances in integrin signalling have provided a clearer picture of integrin activation, such as
the ability of the talin head domain to bind and activate integrins. The results of this thesis
raise several questions regarding the activation of β7 integrins. How do src and FAK fit into
the pathways which lead to β7 integrin activation? Do the kinases act before talin binding or
after? What are the roles of cytoskeletal or adaptor proteins in signaling by β7 integrins, and
how do they interact with the kinases. Addressing these questions would lead to a more
complete picture of the signalling pathways which regulate theactivation of β7 integrins.
Fluorescently-tagged integrins and kinases coupled with live cell confocal microscopy could
be used to visualise the interactions between the kinases and integrins in real-time during cell
activation, adhesion, cell spreading, cell migration and integrin clustering. Such studies could
include the β7 cytoplasmic domain mutants described in this study.
Small interference RNA (siRNA) would be a useful tool for studying the roles src and FAK
play in regulating the functions of β7 integrins. It would be interesting to observe the effect of
knockdown of src and FAK on β7 integrin-mediated cell adhesion, migration and spreading.
Potential problems to be surmounted would be the difficulty of transfecting siRNA into
leukocytes and the fact that src and FAK have complex roles in other signalling pathways
thereby making it difficult to observe specific integrin-related effects. The former could be
overcome by using viral expression of antisense constructs against src and FAK.
197

It would be useful to screen a library of recombinant kinases with the YDRREY peptide as a
substrate to identify those that are capable of phosphorylating the CARD. Are src and Fak
just two of several kinases that can bind and phosphorylate the YDRREY motif?
SMACs
The study of β7 integrins in SMACs would be useful to elucidate the roles of β7 integrins in
antigen presentation or other forms of cell communication. T cells expressing β7 integrins
could be bound to ligand-coated surfaces (plates, microspheres) or to the ligand-expressing
endothelial cells. The integrins would be fluorescently tagged and visualized using real-time
confocal microscopy. Identifying changes in integrin localisation in lamellipodia, the focal
zone and uropod during migration could be worthwhile.
Heat shock and stress
As reported here, heat shock and serum depletion caused integrin activation, however the
mechanism which led to integrin activation remains to be determined. Pathways involved in
integrin activation could be elucidated by immunoprecipitation of β7 complexes and
identification of proteins in the complexes by mass spectrometry. siRNA and chemical
inhibitors targeted against components of intracellular signalling pathways could be used to
inhibit activation in response to heat shock, and serum starvation, with Mn 2+ cation activation
as a control. This study only investigated the involvement of hsp70 in β7 integrin activation.
Whether hsp70 regulates the activation of other integrins in response to heat shock warrants
investigation.
Therapeutic agents based on the YDRREY motif
The activity of the β7 CARD in its peptide form in preventing β7 integrin activation has
therapeutic potential as β7 integrins contribute to the development and/or pathogenesis of
several inflammatory diseases. Peptidomimetic modification of the YDRREY sequence could
be used to create a more powerful small molecular inhibitor that is specific for β7 integrins.
Modifications would include those to increase stability, and overall pharmacodynamics.
Modified peptide sequences would initially be tested for their ability to block β7 integrinmediated
adhesion in an in vitro cell adhesion model. Peptides with improved properties
would then be tested for their ability to prevent the development and/or attenuate the severity
of selected inflammatory diseases (e.g. multiple sclersosis, inflammatory bowel disease),
198

using known animal models. In addition, recent studies have found that the HIV protein
gp120 binds to α4β7, similarly to the way in which α4β7 binds MAdCAM-1, causing rapid
activation of LFA-1 (αLβ2) and cell-to-cell spreading of HIV. Therefore it would be
interesting to see if the YDRREY peptide can prevent the binding of gp120 to α4β7, and
either reduce cell infection by HIV or reduce cell-to-cell spread of the virus.
199

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