Protein Kinase Signaling Networks Driven by Oncogenic Gq/11 in Uveal Melanoma Identified by Phosphoproteomic and Bioinformatic Analyses.

Metastatic uveal melanoma (UM) patients typically survive only 2 to 3 years because effective therapy does not yet exist. Here, to facilitate the discovery of therapeutic targets in UM, we have identified protein kinase signaling mechanisms elicited by the drivers in 90% of UM tumors: mutant constitutively active G protein α-subunits encoded by GNAQ (Gq) or GNA11 (G11). We used the highly specific Gq/11 inhibitor FR900359 (FR) to elucidate signaling networks that drive proliferation, metabolic reprogramming, and dedifferentiation of UM cells. We determined the effects of FR on the proteome and phosphoproteome of UM cells as indicated by bioinformatic analyses with CausalPath and site-specific gene set enrichment analysis. We found that inhibition of oncogenic Gq/11 caused deactivation of PKC, Erk, and the cyclin-dependent kinases CDK1 and CDK2 that drive proliferation. Inhibition of oncogenic Gq/11 in UM cells with low metastatic risk relieved inhibitory phosphorylation of polycomb-repressive complex subunits that regulate melanocytic redifferentiation. Site-specific gene set enrichment analysis, unsupervised analysis, and functional studies indicated that mTORC1 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 drive metabolic reprogramming in UM cells. Together, these results identified protein kinase signaling networks driven by oncogenic Gq/11 that regulate critical aspects of UM cell biology and provide targets for therapeutic investigation.

Oncogenic Gq/11 signaling acutely drives and chronically sustains metabolic reprogramming in uveal melanoma.

Metabolic reprogramming has been shown to occur in uveal melanoma (UM), the most common intraocular tumor in adults. Mechanisms driving metabolic reprogramming in UM are poorly understood. Elucidation of these mechanisms could inform development of new therapeutic strategies for metastatic UM, which has poor prognosis because existing therapies are ineffective. Here, we determined whether metabolic reprogramming is driven by constitutively active mutant α-subunits of the heterotrimeric G proteins Gq or G11 (Gq/11), the oncogenic drivers in ∼90% of UM patients. Using PET-computed tomography imaging, microphysiometry, and GC/MS, we found that inhibition of oncogenic Gq/11 with the small molecule FR900359 (FR) attenuated glucose uptake by UM cells in vivo and in vitro, blunted glycolysis and mitochondrial respiration in UM cell lines and tumor cells isolated from patients, and reduced levels of several glycolytic and tricarboxylic acid cycle intermediates. FR acutely inhibited glycolysis and respiration and chronically attenuated expression of genes in both metabolic processes. UM therefore differs from other melanomas that exhibit a classic Warburg effect. Metabolic reprogramming in UM cell lines and patient samples involved protein kinase C and extracellular signal-regulated protein kinase 1/2 signaling downstream of oncogenic Gq/11. Chronic administration of FR upregulated expression of genes involved in metabolite scavenging and redox homeostasis, potentially as an adaptive mechanism explaining why FR does not efficiently kill UM tumor cells or regress UM tumor xenografts. These results establish that oncogenic Gq/11 signaling is a crucial driver of metabolic reprogramming in UM and lay a foundation for studies aimed at targeting metabolic reprogramming for therapeutic development.

A Building Block Approach for the Total Synthesis of YM-385781.

YM-254890 and FR900359 are potent and selective inhibitors of the Gq/11-signaling pathway. As such, they have been attractive targets for both synthesis and biological studies. Yet in spite of this effort, a versatile synthetic approach to the molecules that allows for the rapid construction of a variety of non-natural and labelled analogs and an increase in the amount of those analogs available remains elusive. We report here a convergent building block approach to the molecules that can solve this challenge.

Targeting primary and metastatic uveal melanoma with a G protein inhibitor.

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.

Proteolytic degradation of regulator of G protein signaling 2 facilitates temporal regulation of Gq/11 signaling and vascular contraction.

Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the Gq/11 class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of Gq/11-evoked Ca2+ signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of Gq/11-coupled receptor signaling in the cardiovascular, immune, and nervous systems.

Regulation of neurite morphogenesis by interaction between R7 regulator of G protein signaling complexes and G protein subunit Gα13.

The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gß5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of Gi/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated Gi/o signaling. Accordingly, we used tandem affinity purification and LC-MS/MS to search for novel proteins that interact with R7-RGS heterotrimers in the mouse brain. Among several proteins detected, we focused on Gα13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα13 in its active or inactive state interacts with R7-RGS heterotrimers containing any R7-RGS isoform. LARG (leukemia-associated Rho guanine nucleotide exchange factor (GEF)), PDZ-RhoGEF, and p115RhoGEF augmented interaction between activated Gα13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα13·R7-RGS complexes. Because Gα13/R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gß5 with or without R7BP. We found that neurite retraction evoked by Gα12/13-dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα12/13 but not Gαi/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα13 to augment signaling pathways that regulate neurite morphogenesis. This mechanism expands the diversity of functions whereby R7-RGS complexes regulate critical aspects of nervous system development and function.

Endothelial cell FGF signaling is required for injury response but not for vascular homeostasis.

Endothelial cells (ECs) express fibroblast growth factor receptors (FGFRs) and are exquisitely sensitive to FGF signals. However, whether the EC or another vascular cell type requires FGF signaling during development, homeostasis, and response to injury is not known. Here, we show that Flk1-Cre or Tie2-Cre mediated deletion of FGFR1 and FGFR2 (Fgfr1/2(Flk1-Cre) or Fgfr1/2(Tie2-Cre) mice), which results in deletion in endothelial and hematopoietic cells, is compatible with normal embryonic development. As adults, Fgfr1/2(Flk1-Cre) mice maintain normal blood pressure and vascular reactivity and integrity under homeostatic conditions. However, neovascularization after skin or eye injury was significantly impaired in both Fgfr1/2(Flk1-Cre) and Fgfr1/2(Tie2-Cre) mice, independent of either hematopoietic cell loss of FGFR1/2 or vascular endothelial growth factor receptor 2 (Vegfr2) haploinsufficiency. Also, impaired neovascularization was associated with delayed cutaneous wound healing. These findings reveal a key requirement for cell-autonomous EC FGFR signaling in injury-induced angiogenesis, but not for vascular homeostasis, identifying the EC FGFR signaling pathway as a target for diseases associated with aberrant vascular proliferation, such as age-related macular degeneration, and for modulating wound healing without the potential toxicity associated with direct manipulation of systemic FGF or VEGF activity.

A mechanism regulating G protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation.

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 Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/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 Gi/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.

GIRK channel modulation by assembly with allosterically regulated RGS proteins.

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/Gß5 complexes under allosteric control of R7 RGS-binding protein (R7BP). Elimination of R7BP occludes the Gß5 subunit that interacts with GIRK channels. R7BP-bound R7-RGS/Gß5 complexes and Gßγ 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 µ-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.

Altered reactivity of resistance vasculature contributes to hypertension in elastin insufficiency.

Elastin (Eln) insufficiency in mice and humans is associated with hypertension and altered structure and mechanical properties of large arteries. However, it is not known to what extent functional or structural changes in resistance arteries contribute to the elevated blood pressure that is characteristic of Eln insufficiency. Here, we investigated how Eln insufficiency affects the structure and function of the resistance vasculature. A functional profile of resistance vasculature in Eln(+/-) mice was generated by assessing small mesenteric artery (MA) contractile and vasodilatory responses to vasoactive agents. We found that Eln haploinsufficiency had a modest effect on phenylephrine-induced vasoconstriction, whereas ANG II-evoked vasoconstriction was markedly increased. Blockade of ANG II type 2 receptors with PD-123319 or modulation of Rho kinase activity with the inhibitor Y-27632 attenuated the augmented vasoconstriction, whereas acute Y-27632 administration normalized blood pressure in Eln(+/-) mice. Sodium nitroprusside- and isoproterenol-induced vasodilatation were normal, whereas ACh-induced vasodilatation was severely impaired in Eln(+/-) MAs. Histologically, the number of smooth muscle layers did not change in Eln(+/-) MAs; however, an additional discontinuous layer of Eln appeared between the smooth muscle layers that was absent in wild-type arteries. We conclude that high blood pressure arising from Eln insufficiency is due partly to permanent changes in vascular tone as a result of increased sensitivity of the resistance vasculature to circulating ANG II and to impaired vasodilatory mechanisms arising from endothelial dysfunction characterized by impaired endothelium-dependent vasodilatation. Eln insufficiency causes augmented ANG II-induced vasoconstriction in part through a novel mechanism that facilitates contraction evoked by ANG II type 2 receptors and altered G protein signaling.

G Protein Activation Occurs via a Largely Universal Mechanism.

Understanding how signaling proteins like G proteins are allosterically activated is a long-standing challenge with significant biological and medical implications. Because it is difficult to directly observe such dynamic processes, much of our understanding is based on inferences from a limited number of static snapshots of relevant protein structures, mutagenesis data, and patterns of sequence conservation. Here, we use computer simulations to directly interrogate allosteric coupling in six G protein α-subunit isoforms covering all four G protein families. To analyze this data, we introduce automated methods for inferring allosteric networks from simulation data and assessing how allostery is conserved or diverged among related protein isoforms. We find that the allosteric networks in these six G protein α subunits are largely conserved and consist of two pathways, which we call pathway-I and pathway-II. This analysis predicts that pathway-I is generally dominant over pathway-II, which we experimentally corroborate by showing that mutations to pathway-I perturb nucleotide exchange more than mutations to pathway-II. In the future, insights into unique elements of each G protein family could inform the design of isoform-specific drugs. More broadly, our tools should also be useful for studying allostery in other proteins and assessing the extent to which this allostery is conserved in related proteins.

Regulator of G protein signaling 2 deficiency causes endothelial dysfunction and impaired endothelium-derived hyperpolarizing factor-mediated relaxation by dysregulating Gi/o signaling.

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for G(q/11)α and G(i/o)α 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)α 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.

Building Chemical Probes Based on the Natural Products YM-254890 and FR900359: Advances toward Scalability.

The biological activity of natural products YM-254890 (YM) and FR900359 (FR) has led to significant interest in both their synthesis and the construction of more simplified analogs. While the simplified analogs lose much of the potency of the natural products, they are of interest in their own right, and their synthesis has revealed synthetic barriers to the family of molecules that need to be addressed if a scalable synthesis of YM and FR analogs is to be constructed. In the work described here, a synthetic route to simplified analogs of YM is examined and strategies for circumventing some of the challenges inherent to constructing the molecules are forwarded.

Gi/o signaling and the palmitoyltransferase DHHC2 regulate palmitate cycling and shuttling of RGS7 family-binding protein.

R7BP (RGS7 family-binding protein) has been proposed to function in neurons as a palmitoylation-regulated protein that shuttles heterodimeric, G(i/o)α-specific GTPase-activating protein (GAP) complexes composed of Gß5 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-Gß5 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.

RGS2 is a primary terminator of ß2-adrenergic receptor-mediated G(i) signaling.

Two major ß-adrenergic receptor (ßAR) subtypes, ß(1)AR and ß(2)AR, are expressed in mammalian heart with ß(1)AR coupling to G(s) and ß(2)AR dually coupling to G(s) and G(i) proteins. In many types of chronic heart failure, myocardial contractile response to both ß(1)AR and ß(2)AR stimulation is severely impaired. The dysfunction of ßAR signaling in failing hearts is largely attributable to an increase in G(i) signaling, because disruption of the G(i) signaling restores myocardial contractile response to ß(1)AR as well as ß(2)AR stimulation. However, the mechanism terminating the ß(2)AR-G(i) signaling remains elusive, while it has been shown activation of the G(i) signaling is dependent on agonist stimulation and subsequent PKA-mediated phosphorylation of the receptor. Here we demonstrate that regulator of G protein signaling 2 (RGS2) is a primary terminator of the ß(2)AR-G(i) signaling. Specifically, prolonged absence of agonist stimulation for 24h impairs the ß(2)AR-G(i) signaling, resulting in enhanced ß(2)AR- but not ß(1)AR-mediated contractile response in cultured adult mouse cardiomyocytes. Increased ß(2)AR contractile response is accompanied by a selective upregulation of RGS2 in the absence of alterations in other major cardiac RGS proteins (RGS3-5) or G(s), G(i) or ßAR subtypes. Administration of a ßAR agonist, isoproterenol (ISO, 1.0 nM), prevents RGS2 upregulation and restores the ß(2)AR-G(i) signaling in cultured cells. Furthermore, RGS2 ablation, similar to ßAR agonist stimulation, sustains the ß(2)AR-G(i) signaling in cultured cells, whereas adenoviral overexpression of RGS2 suppresses agonist-activated ß(2)AR-G(i) signaling in cardiomyocytes and HEK293 cells. These findings not only define RGS2 as a novel negative regulator of the ß(2)AR-G(i) signaling but also provide a potential novel target for the treatment of chronic heart failure.

Regulator of G-protein signaling-2 mediates vascular smooth muscle relaxation and blood pressure.

Nitric oxide (NO) inhibits vascular contraction by activating cGMP-dependent protein kinase I-alpha (PKGI-alpha), which causes dephosphorylation of myosin light chain (MLC) and vascular smooth muscle relaxation. Here we show that PKGI-alpha attenuates signaling by the thrombin receptor protease-activated receptor-1 (PAR-1) through direct activation of regulator of G-protein signaling-2 (RGS-2). NO donors and cGMP cause cGMP-mediated inhibition of PAR-1 and membrane localization of RGS-2. PKGI-alpha binds directly to and phosphorylates RGS-2, which significantly increases GTPase activity of G(q), terminating PAR-1 signaling. Disruption of the RGS-2-PKGI-alpha interaction reverses inhibition of PAR-1 signaling by nitrovasodilators and cGMP. Rgs2-/- mice develop marked hypertension, and their blood vessels show enhanced contraction and decreased cGMP-mediated relaxation. Thus, PKGI-alpha binds to, phosphorylates and activates RGS-2, attenuating receptor-mediated vascular contraction. Our study shows that RGS-2 is required for normal vascular function and blood pressure and is a new drug development target for hypertension.

Uveal melanoma cells use ameboid and mesenchymal mechanisms of cell motility crossing the endothelium.

Uveal melanomas (UMs) are malignant cancers arising from the pigmented layers of the eye. UM cells spread through the bloodstream, and circulating UM cells are detectable in patients before metastases appear. Extravasation of UM cells is necessary for formation of metastases, and transendothelial migration (TEM) is a key step in extravasation. UM cells execute TEM via a stepwise process involving the actin-based processes of ameboid blebbing and mesenchymal lamellipodial protrusion. UM cancers are driven by oncogenic mutations that activate Gαq/11, and this activates TRIO, a guanine nucleotide exchange factor for RhoA and Rac1. We found that pharmacologic inhibition of Gαq/11 in UM cells reduced TEM. Inhibition of the RhoA pathway blocked amoeboid motility but led to enhanced TEM; in contrast, inhibition of the Rac1 pathway decreased mesenchymal motility and reduced TEM. Inhibition of Arp2/3 complex allowed cells to transmigrate without intercalation, a direct mechanism similar to the one often displayed by immune cells. BAP1-deficient (+/-) UM subclones displayed motility behavior and increased levels of TEM, similar to the effects of RhoA inhibitors. We conclude that RhoA and Rac1 signaling pathways, downstream of oncogenic Gαq/11, combine with pathways regulated by BAP1 to control the motility and transmigration of UM cells.

R9AP and R7BP: traffic cops for the RGS7 family in phototransduction and neuronal GPCR signaling.

RGS (regulator of G protein signaling) proteins have emerged as crucial regulators, effectors and integrators in G-protein-coupled receptor (GPCR) signaling networks. Many RGS proteins accelerate GTP hydrolysis by Galpha subunits, thereby regulating G protein activity, whereas certain RGS proteins also transduce Galpha signals to downstream targets. Particularly intriguing are members of the RGS7 (R7) family (RGS6, RGS7, RGS9 and RGS11), which heterodimerize with Gbeta5. In Caenorhabditis elegans, R7-Gbeta5 heterodimers regulate synaptic transmission, anesthetic action and behavior. In vertebrates, they regulate vision, postnatal development, working memory and the action of psychostimulants or morphine. Here we highlight R9AP and R7BP, a related pair of recently identified SNARE-like R7-family binding proteins, which regulate intracellular trafficking, expression and function of R7-Gbeta5 heterodimers in retina and brain. Emerging understanding of R7BP and R9AP promises to provide new insights into neuronal GPCR signaling mechanisms relevant to the causes and treatment of neurological disorders.

Genetic ablation of calcium-independent phospholipase A(2)beta causes hypercontractility and markedly attenuates endothelium-dependent relaxation to acetylcholine.

Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that play prominent roles in regulating vasomotor tone. To identify the role of calcium-independent phospholipase A(2)beta (iPLA(2)beta) in vasomotor function, we measured vascular responses to phenylephrine (PE) and ACh in mesenteric arterioles from wild-type (WT; iPLA(2)beta(+/+)) mice and those lacking the beta-isoform (iPLA(2)beta(-/-)) both ex vivo and in vivo. Vessels isolated from iPLA(2)beta(-/-) mice demonstrated increased constriction to PE, despite lower basal smooth muscle calcium levels, and decreased vasodilation to ACh compared with iPLA(2)beta(+/+) mice. PE constriction resulted in initial intracellular calcium release with subsequent steady-state constriction that depended on extracellular calcium influx. Endothelial denudation had no effect on vessel tone or PE-induced constriction although the dilation to ACh was significantly reduced in iPLA(2)beta(+/+) vessels. In contrast, vessels from iPLA(2)beta(-/-) constricted by 54% after denudation, indicating smooth muscle hypercontractility. In vivo, blood pressure, resting vessel diameter, and constriction of mesenteric vessels to PE were not different in iPLA(2)beta(-/-) vessels compared with WT mouse vessels. However, relaxation after ACh administration in situ was attenuated, indicating an endothelial inability to induce dilation in response to ACh. In cultured endothelial cells, inhibition of iPLA(2)beta with (S)-(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL) decreased endothelial nitric oxide synthase phosphorylation and reduced endothelial agonist-induced intracellular calcium release as well as extracellular calcium influx. We conclude that iPLA(2)beta is an important mediator of vascular relaxation and intracellular calcium homeostasis in both smooth muscle and endothelial cells and that ablation of iPLA(2)beta causes agonist-induced smooth muscle hypercontractility and reduced agonist-induced endothelial dilation.

A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility.

Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.