Understanding the Role of Gβγ in the Cellular Regulation of Mutationally Activated Gαq/11

Uveal melanoma (UM) is the most common intraocular malignancy in adults. UM metastasizes to the liver in 50% of patients, and there are currently no effective therapies for metastatic UM. Approximately 90% of UM cases harbor mutually exclusive mutations in the GNAQ and GNA11 genes, which code for the heterotrimeric G protein subunits Gα and Gα , respectively. The most common mutations within these genes are within residues Q209 and R183, resulting in Q209L, Q209P, or R183C mutations in UM patients. These mutations prevent GTP hydrolysis and render the proteins constitutively active (CA) to promote oncogenic signaling. The Ras/mitogen-activated protein kinase (MAPK) and Hippo/YAP pathways are commonly stimulated via CA Gα . Although it is generally thought that active, GTP-bound Gα subunits signal independently of Gβγ, we have found that these CA mutants of Gα retain some level of binding to Gβγ and that this interaction is necessary for oncogenic signaling. The main objective of this project is to understand the role of Gβγ in the regulation of CA Gα . We hypothesized that disrupting the interaction between CA Gα and Gβγ can ultimately inhibit oncogenic signaling in UM. To test this hypothesis, we introduced a single point mutation within the N-terminus of the CA Gα mutants, which has been shown previously to disrupt the interaction between wild-type Gα and Gβγ and prevent agonist-dependent signaling of Gα We also overexpressed another Gα subunit, Gα , to bind endogenous Gβγ and thereby prevent Gβγ binding by CA Gα mutants. We found that these methods disrupted binding of CA Gα to Gβγ and further disrupted oncogenic signaling via the Hippo/YAP and MAPK pathways. Interestingly, we found that the Gα Q209L mutant binds more strongly to Gβγ, and signaling by the Gα Q209P and Gα R183C mutants are more sensitive to disruption of Gβγ binding. This was further confirmed within UM cell lines where siRNA knockdown of Gβ and Gβ had a more profound effect on inhibition of signaling in cells with the Gα Q209P mutation compared to cell lines with the Gα Q209L mutation. In conclusion, our study challenges the idea that CA Gα signals independently of Gβγ. Our work also suggests a novel difference in sensitivity between the Gα Q209L, Gα Q209P, and Gα R183C mutants. We propose that disrupting the interaction between CA Gα and Gβγ can be a novel therapeutic target in UM.