The guanine nucleotide exchange factor Ric-8A regulates the sensitivity of constitutively active Gαq to the inhibitor...

Heterotrimeric G proteins are stimulated under normal circumstances by G protein-coupled receptors to promote downstream intracellular signaling. Mutations can occur in αq at glutamine 209 (Q209) that cause constitutive, G protein-coupled receptor independent signaling due to disruption of GTPase activity. Specifically, Q209L/P mutations are oncogenic drivers of uveal melanoma. YM-254890 (YM) has been shown to selectively inhibit both WT and constitutively active (CA) αqQ209L/P by preventing the release of GDP and exchange for GTP, thereby halting downstream signaling. Because αqQL/P are thought to be primarily GTP-bound and GTPase deficient, the current mechanistic understanding of YM inhibition needs further investigation to clarify how a GDP-dissociation inhibitor could potently inhibit these oncogenic mutants. Here, we expand on the current knowledge of CA αq cellular regulation by demonstrating a direct role for the αq chaperone and guanine nucleotide exchange factor Ric-8A in YM sensitivity. Through signaling assays in RIC-8A KO cells, we found that myristoylated αqQL/P mutants (αqAG-QL/P), previously demonstrated to be YM-resistant, became YM-sensitive, and this was reversed by reintroduction of Ric-8A. Additionally, αqQL demonstrated increased YM sensitivity in the absence of Ric-8A, which was directly altered by the reintroduction of Ric-8A. Pull-down and BRET assays with the RGS-homology domain of GRK2, which can only bind activated αq, further demonstrated that Ric-8A expression enhances activation of αq, its ability to bind effectors, and therefore its ability to signal. With the understanding of YM acting as a GDP-dissociation inhibitor, we propose that Ric-8A hinders YM inhibitory effects by promoting GTP-bound, activated αqQL/P.