Real-time monitoring of receptor and G-protein interactions in living cells.
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.
Galés
Céline
C
Department of Biochemistry, Université de Montréal, P.O. Box 6128 Down-town station, Montréal, H3C 3J7, Canada.
Rebois
R Victor
RV
Hogue
Mireille
M
Trieu
Phan
P
Breit
Andreas
A
Hébert
Terence E
TE
Bouvier
Michel
M
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*
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; D019204:GTP-Binding Proteins