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Live cell optical assay for precise characterization of receptors coupling to Gα12

Heterotrimeric G proteins are essential mediators of G protein‐coupled receptors (GPCRs) signalling to intracellular effectors. There is a considerable diversity of G protein subunits that channel signals initiated by GPCRs into specific outcome. In particular, mammalian genomes contain 16 conserved genes encoding G protein α subunits with unique properties. Of four Gα subfamilies (Gi/o, Gq, Gs and G12/13), members of the G12/13 group have received considerable attention for their roles in carcinogenesis. However, our ability to study activation of G12/13 by GPCRs with the power to distinguish between the two subunits is limited. Here, we present an adaptation of the bioluminescence resonance energy transfer (BRET)‐based assay to specifically monitor activity of Gα12 in living cells. In this kinetic assay, agonist‐induced release of Venus‐tagged Gβγ subunits from Gα12 is followed in real time using nano‐luciferase (Nluc)–tagged BRET donor. Using this assay, we characterized bradykinin B2 receptor (BDKRB2) and found that the receptor couples to Gα12 in addition to Gαo, and Gαq, but not to Gαs. We demonstrated the utility of this assay to quantify rates of G protein activation and inactivation as well as performing dose‐response studies while rank ordering signalling via individual Gα subunits. We further showed the utility of this assay to other GPCRs by demonstrating Gα12 coupling of cholecystokinin A receptor (CCKAR). Introduction of the Gα12‐coupling BRET assay is expected to accelerate characterization of GPCR actions on this understudied G protein.