Regulator of G protein signaling 2 inhibits Gαq-dependent uveal melanoma cell growth

Activating mutations in Gα_q/11 are a major driver of uveal melanoma (UM), the most common intraocular cancer in adults. While progress has recently been made in targeting Gα_q/11 for UM therapy, the crucial role for these proteins in normal physiology and their high structural similarity with many other important GTPase proteins renders this approach challenging. The aim of the current study was to validate whether a key regulator of Gq signaling, regulator of G protein signaling 2 (RGS2), can inhibit Gα_q-mediated UM cell growth. We used two UM cell lines, 92.1 and Mel-202, which both contain the most common activating mutation Gα_q^Q209L and developed stable cell lines with doxycycline-inducible RGS2 protein expression. Using cell viability assays, we showed that RGS2 could inhibit cell growth in both of these UM cell lines. We also found that this effect was independent of the canonical GTPase-activating protein activity of RGS2 but was dependent on the association between RGS2 and Gα_q. Furthermore, RGS2 induction resulted in only partial reduction in cell growth as compared to siRNA-mediated Gαq knockdown, perhaps because RGS2 was only able to reduce mitogen-activated protein kinase signaling downstream of phospholipase Cβ, while leaving activation of the Hippo signaling mediators yes-associated protein 1/TAZ, the other major pathway downstream of Gα_q, unaffected. Taken together, our data indicate that RGS2 can inhibit UM cancer cell growth by associating with Gα_q^Q209L as a partial effector antagonist.     

Multiple potassium channel tetramerization domain (KCTD) family members interact with Gβγ, with effects on cAMP signaling

G protein–coupled receptors (GPCRs) initiate an array of intracellular signaling programs by activating heterotrimeric G proteins (Gα and Gβγ subunits). Therefore, G protein modifiers are well positioned to shape GPCR pharmacology. A few members of the potassium channel tetramerization domain (KCTD) protein family have been found to adjust G protein signaling through interaction with Gβγ. However, comprehensive details on the KCTD interaction with Gβγ remain unresolved. Here, we report that nearly all the 25 KCTD proteins interact with Gβγ. In this study, we screened Gβγ interaction capacity across the entire KCTD family using two parallel approaches. In a live cell bioluminescence resonance energy transfer–based assay, we find that roughly half of KCTD proteins interact with Gβγ in an agonist-induced fashion, whereas all KCTD proteins except two were found to interact through coimmunoprecipitation. We observed that the interaction was dependent on an amino acid hot spot in the C terminus of KCTD2, KCTD5, and KCTD17. While KCTD2 and KCTD5 require both the Bric-à-brac, Tramtrack, Broad complex domain and C-terminal regions for Gβγ interaction, we uncovered that the KCTD17 C terminus is sufficient for Gβγ interaction. Finally, we demonstrated the functional consequence of the KCTD–Gβγ interaction by examining sensitization of the adenylyl cyclase–cAMP pathway in live cells. We found that Gβγ-mediated sensitization of adenylyl cyclase 5 was blunted by KCTD. We conclude that the KCTD family broadly engages Gβγ to shape GPCR signal transmission.