Membrane Targeting of Constitutively Active Gαq Regulates Inhibition by YM‐254980

Abstract

Uveal melanoma, the most common cancer of the eye in adults, is driven by 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). This mutation is found in more than 90% of cases and up to 50% of patients experience untreatable metastases. YM‐254980 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, suggesting a requirement of Gαq/11 translocating off the membrane to be inhibited. 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 abolish 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. To further define the mechanism of YM insensitivity of membrane targeting mutants of GαqQL, we are currently examining other CA Gα subunits, specifically Gα16 (3 sites of palmitoylation) and Gα13 (2 sites of palmitoylation), that have been mutated to introduce a YM binding site. We have generated myristoylated mutants of these Gα subunits, and are testing if they, like myristoylated GαqQL, are resistant to inhibition by YM. These experiments will highlight the requirements of membrane association for signaling by GαqQL and reveal insights into the mechanism of YM inhibition.