Understanding YM-254890 Inhibition of Constitutively Active Gαq

Abstract ID 94446 Poster Board 167 In more than 90% of uveal melanoma cases, the oncogenic driver is an activating glutamine to leucine or proline mutation at residue 209 of the α subunit of G proteins Gq or G11 (Gαq/11QL or Gαq/11QP). Up to 50% of patients experience untreatable metastases. YM-254890 is a promising inhibitor of constitutively active (CA) Gαq/11, though its mechanism of action is not well understood. Evidence supports that although Gαq/11 has 2 sites of palmitoylation that allow for association with the plasma membrane (PM), these proteins are still found in subcellular locations. We have shown that more strongly targeting GαqQL to membranes by adding an N-terminal myristoylation site renders it insensitive to YM. To further understand how PM-restricted GαqQL loses sensitivity to YM inhibition of signaling, we have generated additional membrane targeting mutants of GαqQL. Although palmitoylation at cysteines 9 and 10 of GαqQL is essential for signaling, preventing palmitoylation by mutating both cysteines 9 and 10 to serines in the context of myristoylated GαqQL does not reduce signaling, as assayed by TEAD- and SRE-dependent luciferase reporter assays in HEK293 q/11 knockout cells. Moreover, signaling by this myristoylated, palmitoylation-deficient GαqQL mutant remains insensitive to YM, indicating that introduction of a single site for myristoylation, in the absence of any palmitoylation, is sufficient to render GαqQL resistant to YM. In addition to the importance of membrane localization for YM inhibition, we are investigating the role of other effector proteins, such as Gαq chaperone Ric8A, in facilitating YM inhibition or resistance. Increasing amounts of Ric8A decrease YM-sensitivity of an otherwise sensitive αqQL. Interestingly, we have also shown that loss of Ric8A confers YM-sensitivity to an otherwise resistant myristoylated GαqQL without abolishing signaling. TEAD and SRE luciferase assays, in addition to effector pulldowns, have revealed that these effects are not due to loss of expression of αq. These experiments reveal insights into the mechanism of YM inhibition, by considering both the impact of αqQL cellular localization and novel aspects of αq cellular regulation by effectors.