Ric‐8A binds to and regulates the activation state of the RGS14:Gai1‐GDP signaling complex

We have shown RGS14 to be a scaffolding protein in brain that binds Gai, H‐Ras, and Raf kinases to regulate MAPkinase signaling. Like other RGS proteins, RGS14 acts as a GTPase activating protein (GAP) to terminate Gai/o signaling. Unlike other RGS proteins, RGS14 contains a GPR/GoLoco (GL) domain that binds inactive Gai1/3‐GDP in cells and in vitro . Here we provide evidence that RGS14 acts as a GL protein in unconventional G protein signaling, independent of G protein‐coupled receptors (GPCRs). Consistent with this idea, we find in cells that RGS14 binds Ric‐8A, a non‐GPCR guanine nucleotide exchange factor (GEF) that regulates other GL:Gai‐GDP complexes. When co‐expressed with Gai1‐GDP in cells, both RGS14 and Ric‐8A are recruited from the cytosol to the plasma membrane, suggesting formation of a protein complex. Ric‐8A induces dissociation of the RGS14:Gai1‐GDP complex in cells and in vitro . This dissociation allows Ric‐8A to act as a GEF towards Gai1; however, RGS14 inhibits the stimulatory effects of Ric‐8A on the GTP binding and steady‐state GTPase activity of Gai1. Native RGS14 and Ric‐8A co‐localize to the same hippocampal neurons, further supporting a functional link between these proteins. These findings indicate that Ric‐8A is a novel regulator of the RGS14:Gai1 signaling complex. Studies are underway to determine how MAPkinase signaling modulates Ric‐8A effects on RGS14/Gai1 signaling in brain.