Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.
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.
Maurel
Damien
D
Centre National de la Recherche Scientifique (CNRS), UMR 5203, Institut de Génomique Fonctionnelle, 141 Rue de la Cardonille, Montpellier F-34000, France.
Comps-Agrar
Laëtitia
L
Brock
Carsten
C
Rives
Marie-Laure
ML
Bourrier
Emmanuel
E
Ayoub
Mohammed Akli
MA
Bazin
Hervé
H
Tinel
Norbert
N
Durroux
Thierry
T
Prézeau
Laurent
L
Trinquet
Eric
E
Pin
Jean-Philippe
JP
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
D043562:Receptors, G-Protein-Coupled; D018080:Receptors, GABA-B; D005680:gamma-Aminobutyric Acid