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Nature Methods 5, 561–567 (1 June 2008) | doi:10.1038/nmeth.1213

Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization

Damien Maurel , La|[euml]|titia Comps-Agrar , Carsten Brock , Marie-Laure Rives , Emmanuel Bourrier , Mohammed Akli Ayoub , Herv|[eacute]| Bazin , Norbert Tinel , Thierry Durroux , Laurent Pr|[eacute]|zeau , Eric Trinquet & Jean-Philippe Pin

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput–compatible format. Using this approach, we examined whether G protein–coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers—a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and γ-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABAB receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.