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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.

SUBMITTER: Maurel D 

PROVIDER: S-EPMC2642604 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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

Maurel Damien D   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  

Nature methods 20080518 6


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 a  ...[more]

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