GPCRs and Ric‐8A Both Regulate the RGS14:Gαi1 Complex

Like other Regulators of G protein Signaling (RGS) proteins, RGS14 acts as a GTPase accelerating protein (GAP) to terminate Gαi/o signaling. However, unlike other RGS proteins, RGS14 also contains a GPR/GoLoco domain that binds Gαi1/3‐GDP in cells and in vitro . The non‐receptor guanine nucleotide exchange factor (GEF) Ric‐8A is then able to recognize and act on this RGS14:Gαi1 complex to play a role in unconventional G protein signaling independent of G protein‐coupled receptors (GPCRs). Here we provide evidence that RGS14 may form Gαi‐dependent complexes with GPCRs, and that these complexes are regulated by Ric‐8A. Using live cell bioluminescence resonance energy transfer (BRET), we show that RGS14‐Luc exhibits specific and saturable BRET with the α 2A ‐adrenergic receptor‐Venus (α 2A ‐AR‐Venus) in a Gαi1‐dependent manner. A reduction in RGS14‐Luc:α 2A ‐AR‐Venus BRET is observed upon receptor stimulation by the agonist UK14304. Ric‐8A enhances the agonist‐induced increase in the dissociation of the RGS14:α 2A ‐AR complex in the presence of Gαi1. Together, these findings indicate that RGS14:Gαi1 complexes may associate with and be regulated by GPCRs, and that these complexes may be substrates for other signaling partners.