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Nat Methods. 2008 Jun;5(6):561-7. doi: 10.1038/nmeth.1213. Epub 2008 May 18.

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

Author information

1
Centre National de la Recherche Scientifique (CNRS), UMR 5203, Institut de Génomique Fonctionnelle, 141 Rue de la Cardonille, Montpellier F-34000, France.

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.

PMID:
18488035
PMCID:
PMC2642604
DOI:
10.1038/nmeth.1213
[Indexed for MEDLINE]
Free PMC Article

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