title: Real-time monitoring of receptor and G-protein interactions in living cells.
abstract: G protein-coupled receptors (GPCRs) represent the largest family of proteins involved in signal transduction. Here we present a bioluminescence resonance energy transfer (BRET) assay that directly monitors in real time the early interactions between human GPCRs and their cognate G-protein subunits in living human cells. In addition to detecting basal precoupling of the receptors to Galpha-, Gbeta- and Ggamma-subunits, BRET measured very rapid ligand-induced increases in the interaction between receptor and Galphabetagamma-complexes (t(1/2) approximately 300 ms) followed by a slower (several minutes) decrease, reflecting receptor desensitization. The agonist-promoted increase in GPCR-Gbetagamma interaction was highly dependent on the identity of the Galpha-subunit present in the complex. Therefore, this G protein-activity biosensor provides a novel tool to directly probe the dynamics and selectivity of receptor-mediated, G-protein activation-deactivation cycles that could be advantageously used to identify ligands for orphan GPCRs.
authors.affiliation: Department of Biochemistry, Université de Montréal, P.O. Box 6128 Down-town station, Montréal, H3C 3J7, Canada.
authors.forename: Céline
authors.initials: C
authors.lastname: Galés
authors.forename: R Victor
authors.initials: RV
authors.lastname: Rebois
authors.forename: Mireille
authors.initials: M
authors.lastname: Hogue
authors.forename: Phan
authors.initials: P
authors.lastname: Trieu
authors.forename: Andreas
authors.initials: A
authors.lastname: Breit
authors.forename: Terence E
authors.initials: TE
authors.lastname: Hébert
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
mesh_terms: D002478:Cells, Cultured; D003199:Computer Systems; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins; D025941:Protein Interaction Mapping / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; D019204:GTP-Binding Proteins