title: Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.
abstract: 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 gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) 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.
authors.affiliation: Centre National de la Recherche Scientifique (CNRS), UMR 5203, Institut de Génomique Fonctionnelle, 141 Rue de la Cardonille, Montpellier F-34000, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Maurel
authors.forename: Laëtitia
authors.initials: L
authors.lastname: Comps-Agrar
authors.forename: Carsten
authors.initials: C
authors.lastname: Brock
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Rives
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Bourrier
authors.forename: Mohammed Akli
authors.initials: MA
authors.lastname: Ayoub
authors.forename: Hervé
authors.initials: H
authors.lastname: Bazin
authors.forename: Norbert
authors.initials: N
authors.lastname: Tinel
authors.forename: Thierry
authors.initials: T
authors.lastname: Durroux
authors.forename: Laurent
authors.initials: L
authors.lastname: Prézeau
authors.forename: Eric
authors.initials: E
authors.lastname: Trinquet
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pin
mesh_terms: D000818:Animals; D001703:Biophysics / Q000379:methods*; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008954:Models, Biological; D025941:Protein Interaction Mapping / Q000379:methods*; D020836:Protein Structure, Quaternary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018080:Receptors, GABA-B / Q000737:chemistry; D013499:Surface Properties; D005680:gamma-Aminobutyric Acid
chemical_list: D043562:Receptors, G-Protein-Coupled; D018080:Receptors, GABA-B; D005680:gamma-Aminobutyric Acid