Visit our Expo - Redox and Inflammation signaling 2012

The use of BRET (Bioluminescence Resonance Energy Transfer) for the study of
tyrosine-kinase receptors
Tarik ISSAD
CNRS UMR8104, INSERM U567, Université René Descartes Paris 5, Institut Cochin,
Department of Cell Biology, 22 rue Méchain, 75014 Paris
e.mail: issad@cochin.inserm.fr
During these last years, we have developed an expertise in the use of BRET technology for
the study of protein-protein interaction, particularly in the field of tyrosine kinase receptors
and their interaction with regulatory partners. We first demonstrated that the BRET
methodology can be used to monitor ligand-induced conformational changes within the
insulin receptor (Boute et al, Mol.Pharm., 2001; Tips, 2002 ; Biochem.Pharm., 2002). We
then used BRET to study, in real time, in living cells, the interaction of the insulin receptor
(IR) with the protein tyrosine phosphatase PTP1B, which is anchored on the cytosolic face of
the endoplasmic reticulum (Boute et al., EMBO Report, 2003). We showed that insulin
rapidly and dose-dependently stimulated the interaction of the IR with PTP1B and we
demonstrated that internalisation of the IR is necessary for this interaction. In addition, we
demonstrated that PTP1B plays an important role in the regulation of the insulin receptor
immature precursor during its biosynthesis in the endoplasmic reticulum (Boute et al., EMBO
Report, 2003 ; Issad et al., Biochimie 2005). We more recently demonstrated that the BRET
methodology can also be used to monitor ligand-induced conformational changes within the
IGF1 Receptor, as well as the interaction of this receptor with PTP1B (Blanquart et al., Mol.
Pharm. 2005).
The receptor-like plasma membrane protein-tyrosine phosphatases PTPalpha and PTPespilon
may also play a role in the regulation of the IR. We have shown that in the basal state, the IR
and these PTPases are pre-associated at the plasma membrane. Using BRET saturation
experiments, we have shown that insulin does not stimulate the recruitment of these PTPases
to the receptor but rather induced conformational changes within these pre-associated
complexes (Lacasa et al., Mol. Pharm. 2005).
Finally, we also provide evidence that the BRET methodology can be use to monitor the
interaction of the insulin receptor with intracellular adaptors positively or negatively involved
in insulin signaling.
Publications
Boute, N., Pernet, K., and Issad, T. (2001) The activation state of the insulin receptor
monitored using the Bioluminescence Resonance Energy Transfer methodology Molecular
Pharmacology, 60: 640-645
Tavaré, J.M. and Issad, T. (2001) Two-dimensional phosphopeptide mapping of receptor
tyrosine kinases Methods in Molecular Biology 124: 67-85 (Humana Press)
Zilberfarb, V., Siquier, K., Strosberg, A.D., and Issad, T. (2001) Effect of dexamethasone on
adipocyte differentiation markers and tumour necrosis factor alpha expression in human
PAZ6 cells Diabetologia, 44 : 377-386
Grosfeld, A.,. Zilberfarb, V., Turban, S., André, J., Guerre-Millo, M., and Issad, T. (2002)
Hypoxia increases leptin expression in human PAZ6 adipose cells Diabetologia 45: 527-530
Lahlou,N., Issad,T., Carel, J-C., Camoin, L., Lebouc,Y., Roger, M., and Girard, J. (2002)
Mutations in the human leptin and leptin receptor genes as models of serum leptin receptor
regulation Diabetes 51: 1980-1985
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