Constitutively active G protein α subunits cause cancer, cholera, Sturge-Weber syndrome, and other disorders. Therapeutic intervention by targeted inhibition of constitutively active Gα subunits in these disorders has yet to be achieved. We found that constitutively active Gα_q in uveal melanoma (UM) cells was inhibited by the cyclic depsipeptide FR900359 (FR). FR allosterically inhibited guanosine diphosphate–for–guanosine triphosphate (GDP/GTP) exchange to trap constitutively active Gα_q in inactive, GDP-bound Gαβγ heterotrimers. Allosteric inhibition of other Gα subunits was achieved by the introduction of an FR-binding site. In UM cells driven by constitutively active Gα_q, FR inhibited second messenger signaling, arrested cell proliferation, reinstated melanocytic differentiation, and stimulated apoptosis. In contrast, FR had no effect on BRAF-driven UM cells. FR promoted UM cell differentiation by reactivating polycomb repressive complex 2 (PRC2)–mediated gene silencing, a heretofore unrecognized effector system of constitutively active Gα_q in UM. Constitutively active Gα_q and PRC2 therefore provide therapeutic targets for UM. The development of FR analogs specific for other Gα subunit subtypes may provide novel therapeutic approaches for diseases driven by constitutively active Gα subunits or multiple G protein–coupled receptors (GPCRs) where targeting a single receptor is ineffective.

G-protein-activated inward-rectifying K+ (GIRK) channels hyperpolarize neurons to inhibit synaptic transmission throughout the nervous system. By accelerating G-protein deactivation kinetics, the regulator of G-protein signaling (RGS) protein family modulates the timing of GIRK activity. Despite many investigations, whether RGS proteins modulate GIRK activity in neurons by mechanisms involving kinetic coupling, collision coupling, or macromolecular complex formation has remained unknown. Here we show that GIRK modulation occurs by channel assembly with R7-RGS/G beta 5 complexes under allosteric control of R7 RGS-binding protein (R7BP). Elimination of R7BP occludes the G beta 5 subunit that interacts with GIRK channels. R7BP-bound R7-RGS/G beta 5 complexes and G beta gamma dimers interact noncompetitively with the intracellular domain of GIRK channels to facilitate rapid activation and deactivation of GIRK currents. By disrupting this allosterically regulated assembly mechanism, R7BP ablation augments GIRK activity. This enhanced GIRK activity increases the drug effects of agonists acting at G-protein-coupled receptors that signal via GIRK channels, as indicated by greater antinociceptive effects of GABA(B) or mu-opioid receptor agonists. These findings show that GIRK current modulation in vivo requires channel assembly with allosterically regulated RGS protein complexes, which provide a target for modulating GIRK activity in neurological disorders in which these channels have crucial roles, including pain, epilepsy, Parkinson's disease and Down syndrome.

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for G(q/11)alpha and G(i/o)alpha subunits. RGS2 deficiency is linked to hypertension in mice and humans, although causative mechanisms are not understood. Because endothelial dysfunction and increased peripheral resistance are hallmarks of hypertension, determining whether RGS2 regulates microvascular reactivity may reveal mechanisms relevant to cardiovascular disease. Here we have determined the effects of systemic versus endothelium-or vascular smooth muscle-specific deletion of RGS2 on microvascular contraction and relaxation. Contraction and relaxation of mesenteric resistance arteries were analyzed in response to phenylephrine, sodium nitroprusside, or acetylcholine with or without inhibitors of nitric oxide (NO) synthase or K+ channels that mediate endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation. The results showed that deleting RGS2 in vascular smooth muscle had minor effects. Systemic or endothelium-specific deletion of RGS2 strikingly inhibited acetylcholine-evoked relaxation. Endothelium-specific deletion of RGS2 had little effect on NO-dependent relaxation but markedly impaired EDHF-dependent relaxation. Acute, inducible deletion of RGS2 in endothelium did not affect blood pressure significantly. Impaired EDHF-mediated vasodilatation was rescued by blocking G(i/o)alpha activation with pertussis toxin. These findings indicated that systemic or endothelium-specific RGS2 deficiency causes endothelial dysfunction resulting in impaired EDHF-dependent vasodilatation. RGS2 deficiency enables endothelial G(i/o) activity to inhibit EDHF-dependent relaxation, whereas RGS2 sufficiency facilitates EDHF-evoked relaxation by squelching endothelial G(i/o) activity. Mutation or down-regulation of RGS2 in hypertension patients therefore may contribute to endothelial dysfunction and defective EDHF-dependent relaxation. Blunting G(i/o) signaling might improve endothelial function in such patients.

Uveal melanoma (UM) is the most common intraocular tumor in adults. Nearly half of UM patients develop metastatic disease and often succumb within months because effective therapy is lacking. A novel therapeutic approach has been suggested by the discovery that UM cell lines driven by mutant constitutively active Gq or G11 can be targeted by FR900359 (FR) or YM-254890, which are bioavailable, selective inhibitors of the Gq/11/14 subfamily of heterotrimeric G proteins. Here, we have addressed the therapeutic potential of FR for UM. We found that FR inhibited all oncogenic Gq/11 mutants reported in UM. FR arrested growth of all Gq/11-driven UM cell lines tested, but induced apoptosis only in a few. Similarly, FR inhibited growth of, but did not efficiently kill, UM tumor cells from biopsies of primary or metastatic tumors. FR evoked melanocytic redifferentiation of UM tumor cells with low (class 1), but not high (class 2), metastatic potential. FR administered systemically below its LD50 strongly inhibited growth of PDX-derived class 1 and class 2 UM tumors in mouse xenograft models, and reduced blood pressure transiently. FR did not regress xenografted UM tumors, or significantly affect heart rate, liver function, hematopoiesis, or behavior. These results indicated the existence of a therapeutic window in which FR can be explored for treating UM, and potentially other diseases caused by constitutively active Gq/11.

Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of G_i/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from G_i/o-gated G protein-regulated inwardly rectifying K⁺ (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating G_i/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.

R7BP (RGS7 family-binding protein) has been proposed to function in neurons as a palmitoylation-regulated protein that shuttles heterodimeric, G_i/oα-specific GTPase-activating protein (GAP) complexes composed of Gβ5 and RGS7 (R7) isoforms between the plasma membrane and nucleus. To test this hypothesis we studied R7BP palmitoylation and localization in neuronal cells. We report that R7BP undergoes dynamic, signal-regulated palmitate turnover; the palmitoyltransferase DHHC2 mediates de novo and turnover palmitoylation of R7BP; DHHC2 silencing redistributes R7BP from the plasma membrane to the nucleus; and G_i/o signaling inhibits R7BP depalmitoylation whereas G_i/o inactivation induces nuclear accumulation of R7BP. In concert with previous evidence, our findings suggest that agonist-induced changes in palmitoylation state facilitate GAP action by (i) promoting Giα depalmitoylation to create optimal GAP substrates, and (ii) inhibiting R7BP depalmitoylation to stabilize membrane association of R7-Gβ5 GAP complexes. Regulated palmitate turnover may also enable R7BP-bound GAPs to shuttle between sites of low and high G_i/o activity or the plasma membrane and nucleus, potentially providing spatio-temporal control of signaling by G_i/o-coupled receptors.