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Nat Methods. 2005 Mar;2(3):177-84. Epub 2005 Feb 17.

Real-time monitoring of receptor and G-protein interactions in living cells.

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Department of Biochemistry, Université de Montréal, P.O. Box 6128 Down-town station, Montréal, H3C 3J7, Canada.


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.

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