FR AB - Science Reference

S4
A crack in the egg: protein-protein interactions in failing hearts.
A.R. Marks; Columbia Univ., 630 West 168th Street, New York, NY 10032
Calcium (Ca 2�) ions are second messengers in numerous signaling pathways
in all cell types. In the heart Ca 2� regulates muscle contraction, electrical signals
that determine the cardiac rhythm, and probably plays a role in controlling
cell growth. In the past decade elucidation of the molecular structure
of the intracellular Ca 2� release channels on the sarcoplasmic and endoplasmic
reticulum (SR/ER) has lead to an understanding of how these molecules
regulate Ca 2� homeostasis in the heart. Consequently the role of
these channels (ryanodine receptors, RyR, and inositol 1,4,5-trisphosphate
receptors, IP3R) in cardiac pathophysiology is beginning to be understood.
Intracellular Ca 2� release channels form a unique class of ion channels distinguished
on the basis of structure size, and function. RyRs and IP3Rs have
large cytoplasmic domains that are involved in the regulation of the channel
pore located in the carboxy terminal 10% of the channel sequence. These
enormous cytoplasmic domains serve as scaffolds for targeting proteins that
bind kinases and phosphatases to the channels. Protein kinase A (PKA)
phosphorylation of RyR2 dissociates the channel regulatory protein FKBP12.6
and regulates the channel open probability (Po). We have defined a macromolecular
complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases
PP1 and PP2A, and an anchoring protein mAKAP. In failing human
hearts RyR2 is PKA hyperphosphorylated resulting in defective channel function
due to increased sensitivity to Ca 2�-induced activation resulting in
impaired excitation-contraction (EC) coupling in failing hearts.
S6
VLiPs: a technique to study ligand binding to GPCRs in their
natural environment.
T.H. Jessen, N. Hunt; EVOTEC BioSystems AG, Hamburg, Schnackenburgallee
114, Hamburg, Hamburg 22525, Germany
Virus Like Particles (VLiPs) are generated by insect cells or mammalian cells
through an engineering process of the retroviral export mechanism and the
respective G-protein coupled receptor (GPCR). The retroviral gag protein
interacts non-covalently but tightly and specifically with the C-terminus of the
receptor of interest. The interaction leads to an enrichment of the receptor
in its natural environment, the cell membrane, before budding of the VLiPs
into the supernatant occurs.
VLiPs carry up to 100 receptor molecules and turn out to be ideally suited
for screening due to their robustness, long lived stability and physiological
binding properties. Data obtained in binding and competition experiments
are identical to data from literature. Currently, functional receptor screens are
under investigation. The method allows to explore a number of other protein
classes as well.
SPEAKER ABSTRACTS
ABRF 2001 ABSTRACTS
S5
Distinguishing agonist and antagonist binding to GPCRs using
coupled plasmon-waveguide resonance analysis.
G. Tollin, Z. Salamon, S. Cowell, V. Hruby; Univ. of Arizona,
Biosciences West, Tucson, AZ 85721
Structural changes accompanying the binding of ligands to the cloned human
�-opioid receptor immobilized in a solid-supported lipid bilayer have been
investigated using coupled plasmon-waveguide resonance spectroscopy.
This highly sensitive new technique directly monitors mass density, conformation,
and molecular orientation changes occurring within a single bilayer,
and also allows direct determination of binding constants without structural
modification of materials. Although both agonist binding and antagonist
binding to the receptor cause increases in molecular ordering within the proteolipid
membrane, only agonist binding induces an increase in thickness
and molecular packing density of the membrane. This is a consequence of
mass movements perpendicular to the plane of the bilayer occurring within
the lipid and receptor components. These results are consistent with models
of receptor function that involve changes in the orientation of transmembrane
helices.
S7
A knockout for every gene and a chip for every purpose.
M. Sussman; Univ. of Wisconsin, 425 Henry Mall, Madison, WI 53706
We will report on progress in the development of two genomic technologiessaturation
reverse genetics using a collection of insertionally mutagenized
‘knockout’ Arabidopsis plants, and a maskless array synthesizer (MAS) for
producing high density DNA oligonucleotide arrays ‘on the fly’. Our reverse
genetic approach involves the rapid screening of several hundred thousand
insertionally mutagenized Arabidopsis lines, for the isolation of knockout
plants for any gene of interest. The bias of T-DNA for insertion at particular
regions of the genome will be described and compared to published
accounts of transposon bias. We have previously reported on a mathematical
treatment required to isolate a knockout in every gene (Krysan et al.,
1996), and we extend this analysis by taking into account our observations
on the small but significant bias for the distributiion of T-DNA sites. Progress
towards the establishment of a computer database for T-DNA insertions,
using TAIL PCR to generate flanking sequences, will also be described. As a
general tool for genotyping and for discerning the effects of a particular
mutation on global genome expression, we have been testing existing and
new technologies based on high density oligonucleotide arrays. Recent
experiments utilizing a maskless array synthesizer (Singh-Gasson et al., 1999)
which is capable of generating a half million different oligonucleotides on a
2 cm squared glass surface, will be described. The MAS uses a digital
micromirror device developed by Texas Instruments, to generate virtual
masks for use in photolithography and is a powerful tool for bringing combinatorial
chemistry to the benchtop in research laboratories. Krysan, P.J.,
Young, J.C., Tax, F. and Sussman, M.R. 1996 Identification of T-DNA insertions
within Arabidopsis genes involved in signal transduction and ion transport.
Proc. Natl. Acad. Sci. 93:8145–8150. Singh-Gasson, S., Green, R.D,,
Yue, Y., Nelson, C., Blattner, F., Sussman, M.R. and Cerrina, F. 1999. Maskless
fabrication of light-directed oligonucleotide microarrays usig a digital
micromirror array. Nature Biotechnology 17:974–978.
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 227