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 alpha subunits with unique properties. Of four G alpha 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 G alpha 12 in living cells. In this kinetic assay, agonist-induced release of Venus-tagged G beta gamma subunits from G alpha 12 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 G alpha 12 in addition to G alpha o, and G alpha q, but not to G alpha s. 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 G alpha subunits. We further showed the utility of this assay to other GPCRs by demonstrating G alpha 12 coupling of cholecystokinin A receptor (CCKAR). Introduction of the G alpha 12-coupling BRET assay is expected to accelerate characterization of GPCR actions on this understudied G protein.