Re-evaluating Gα protein-response element specificity in GPCR signaling

Reporter gene assays are widely employed to investigate activation of G-protein-coupled receptors (GPCRs). However, the increasing complexity of GPCR signaling-particularly the capability of many receptors to couple with multiple Gα subunits-has created a growing need for reliable methods to dissect individual Gα-mediated pathways. Recent insights into the crosstalk and interdependence among Gα subfamilies have further complicated our understanding of the overarching GPCR signaling landscape. While previous studies have linked specific Gα-proteins to distinct transcriptional response elements, the precise specificity of these associations remains unclear. Here, we systematically identified the relationship between Gα proteins and four commonly used GPCR-regulated response elements-CRE, SRE, NFAT-RE, and SRF-RE-using a panel of Gα knockout (KO) cell lines. Contrary to the traditional model of near-exclusive Gα-response element pairings, our results reveal that each reporter is modulated by multiple Gα subfamilies. Each response element exhibited varying degrees of activation by different Gα proteins, with CRE being predominantly regulated by Gα_s/olf, and SRE, NFAT-RE, and SRF-RE primarily regulated by Gα_q/11. Based on these results, we provide an updated framework that redefines the subfamily-specific Gα regulation of downstream transcriptional responses and underscores the need for caution when interpreting reporter assays to assess Gα-protein activity.