Live cell luminescence-based Gαq-PLC-β interaction assay: Development and application at the serotonin 5-HT2A receptor

ABSTRACT

G protein-coupled receptors (GPCRs) remain among the most important drug targets. To characterize their ligands' activity, a range of in vitro assays is available, all with their inherent advantages and limitations. In the Gαq signaling pathway, the interaction between Gαq and phospholipase C (PLC-)β is a receptor-proximal event, and its monitoring does not require modification of the GPCR. With this in mind, we set out to develop a luminescence-based assay gauging this interaction. The resulting assay format encompassed functional complementation of a split nanoluciferase following the interaction of SmBiT, N-terminally fused to PLC-β, with LgBiT, intramolecularly integrated within Gαq. When applied to assess activation of the Gαq-coupled serotonin 2 A receptor (5-HT2AR), an excellent assay performance was demonstrated, as evidenced by an auspicious Z'-factor of 0.75 and by confirming the specificity for both the GPCR and the Gαq signaling pathway. The optimized Gαq-PLC-β assay was successfully applied on a diverse panel of 5-HT2AR ligands and the resulting output was compared with that of a more upstream miniGαq recruitment assay, and more downstream IP1 accumulation and Ca2+ mobilization assays. Overall, the Gαq-PLC-β assay yielded efficacy, potency and relative activity data that correlated very well with those obtained with the IP1 accumulation and miniGαq recruitment assays, although with the former two assays consistently higher potencies and a smaller range of efficacies were observed. Comparison with the Ca2+ mobilization assay did not yield such good correlation, presumably because of kinetic implications. Last, the Gαq-PLC-β interaction assay allowed to detect signals emerging from endogenously expressed GPCRs, indicative of its broad applicability.