Enhanced Membrane Binding of Oncogenic G Protein Αqq209l Confers Resistance to Inhibitor YM-254890

Heterotrimeric G proteins couple activated G protein- coupled receptors (GPCRs) to intracellular signaling path-ways. They can also function independently of GPCR activation upon acquiring mutations that prevent GTPase activity and result in constitutive signaling, as occurs with the alpha qQ209L mutation in uveal melanoma. YM-254890 (YM) can inhibit signaling by both GPCR-activated WT alpha q and GPCR-independent alpha qQ209L. Although YM inhibits WT alpha q by binding to alpha q-GDP and preventing GDP/GTP exchange, the mechanism of YM inhibition of cellular alpha qQ209L remains to be fully understood. Here, we show that YM promotes a sub -cellular redistribution of alpha qQ209L from the plasma membrane (PM) to the cytoplasm. To test if this loss of PM localization could contribute to the mechanism of inhibition of alpha qQ209L by YM, we developed and examined N-terminal mutants of alpha qQ209L, termed PM-restricted alpha qQ209L, in which the addi-tion of membrane-binding motifs enhanced PM localization and prevented YM-promoted redistribution. Treatment of cells with YM failed to inhibit signaling by these PM-restricted alpha qQ209L. Additionally, pull-down experiments demonstrated that YM promotes similar conformational changes in both alpha qQ209L and PM-restricted alpha qQ209L, resulting in increased binding to beta gamma and decreased binding to regulator RGS2, and effectors p63RhoGEF-DH/PH and phospholipase C-beta. GPCR-dependent signaling by PM-restricted WT alpha q is strongly inhibited by YM, demonstrating that resistance to YM inhibi-tion by membrane-binding mutants is specific to constitutively active alpha qQ209L. Together, these results indicate that changes in membrane binding impact the ability of YM to inhibit alpha qQ209L and suggest that YM contributes to inhibition of alpha qQ209L by promoting its relocalization.