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 Gα-, Gβ- and Gγ-subunits, BRET measured very rapid ligand-induced increases in the interaction between receptor and Gαβγ-complexes (t1/2 ∼ 300 ms) followed by a slower (several minutes) decrease, reflecting receptor desensitization. The agonist-promoted increase in GPCR-Gβγ interaction was highly dependent on the identity of the Gα-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.