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Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gβγ subunits liberated following activation of Gα i/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gβγ released from Gα s and Gα q Activation of the G s -coupled adenosine 2B receptor and the G q -coupled muscarinic acetylcholine M1 receptor inhibited the activity of TRPM3 heterologously expressed in HEK293 cells. This inhibition was prevented when the Gβγ sink βARK1-ct (C terminus of β-adrenergic receptor kinase-1) was coexpressed with TRPM3. In neurons isolated from mouse dorsal root ganglion (DRG), native TRPM3 channels were inhibited by activating G s -coupled prostaglandin-EP2 and G q -coupled bradykinin B2 (BK2) receptors. The G i/o inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gα i/o or through the major protein kinases activated downstream of G-protein-coupled receptor (GPCR) activation. When DRG neurons were dialyzed with GRK2i, which sequesters free Gβγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in WT mice and did not develop in Trpm3 -/- mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gβγ protein in heterologous expression systems, primary neurons and in vivo , and suggest a critical role for this ion channel in inflammatory heat hypersensitivity. SIGNIFICANCE STATEMENT The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gα i/o -coupled GPCRs inhibit TRPM3 through a direct interaction between Gβγ subunits and TRPM3. Since Gβγ proteins can be liberated from other Gα subunits than Gα i/o , we examined whether activation of G s - and G q -coupled receptors also influence TRPM3 via Gβγ. Our results demonstrate that activation of G s - and G q -coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gβγ liberation. We also demonstrated that G s - and G q -coupled receptors inhibit TRPM3 in vivo , thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gβγ inhibition of TRPM3 as an effector mechanism shared by the major Gα subunits., (Copyright © 2019 the authors.)

Proteases sustain hyperexcitability and pain by cleaving protease-activated receptor-2 (PAR 2 ) on nociceptors through distinct mechanisms. Whereas trypsin induces PAR 2 coupling to Gα q , Gα s , and β-arrestins, cathepsin-S (CS) and neutrophil elastase (NE) cleave PAR 2 at distinct sites and activate it by biased mechanisms that induce coupling to Gα s , but not to Gα q or β-arrestins. Because proteases activate PAR 2 by irreversible cleavage, and activated PAR 2 is degraded in lysosomes, sustained extracellular protease-mediated signaling requires mobilization of intact PAR 2 from the Golgi apparatus or de novo synthesis of new receptors by incompletely understood mechanisms. We found here that trypsin, CS, and NE stimulate PAR 2 -dependent activation of protein kinase D (PKD) in the Golgi of HEK293 cells, in which PKD regulates protein trafficking. The proteases stimulated translocation of the PKD activator Gβγ to the Golgi, coinciding with PAR 2 mobilization from the Golgi. Proteases also induced translocation of a photoconverted PAR 2 -Kaede fusion protein from the Golgi to the plasma membrane of KNRK cells. After incubation of HEK293 cells and dorsal root ganglia neurons with CS, NE, or trypsin, PAR 2 responsiveness initially declined, consistent with PAR 2 cleavage and desensitization, and then gradually recovered. Inhibitors of PKD, Gβγ, and protein translation inhibited recovery of PAR 2 responsiveness. PKD and Gβγ inhibitors also attenuated protease-evoked mechanical allodynia in mice. We conclude that proteases that activate PAR 2 by canonical and biased mechanisms stimulate PKD in the Golgi; PAR 2 mobilization and de novo synthesis repopulate the cell surface with intact receptors and sustain nociceptive signaling by extracellular proteases., (© 2019 Zhao et al.)

G protein βγ subunit (Gβγ) is a major signal transducer and controls processes ranging from cell migration to gene transcription. Despite having significant subtype heterogeneity and exhibiting diverse cell- and tissue-specific expression levels, Gβγ is often considered a unified signaling entity with a defined functionality. However, the molecular and mechanistic basis of Gβγ's signaling specificity is unknown. Here, we demonstrate that Gγ subunits, bearing the sole plasma membrane (PM)-anchoring motif, control the PM affinity of Gβγ and thereby differentially modulate Gβγ effector signaling in a Gγ-specific manner. Both Gβγ signaling activity and the migration rate of macrophages are strongly dependent on the PM affinity of Gγ. We also found that the type of C-terminal prenylation and five to six pre-Caa X motif residues at the PM-interacting region of Gγ control the PM affinity of Gβγ. We further show that the overall PM affinity of the Gβγ pool of a cell type is a strong predictor of its Gβγ signaling-activation efficacy. A kinetic model encompassing multiple Gγ types and parameterized for empirical Gβγ behaviors not only recapitulated experimentally observed signaling of Gβγ, but also suggested a Gγ-dependent, active-inactive conformational switch for the PM-bound Gβγ, regulating effector signaling. Overall, our results unveil crucial aspects of signaling and cell migration regulation by Gγ type-specific PM affinities of Gβγ., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Transient receptor potential (TRP) ion channels in peripheral sensory neurons are functionally regulated by hydrolysis of the phosphoinositide PI(4,5)P 2 and changes in the level of protein kinase mediated phosphorylation following activation of various G protein coupled receptors. We now show that the activity of TRPM3 expressed in mouse dorsal root ganglion (DRG) neurons is inhibited by agonists of the G i -coupled µ opioid, GABA-B and NPY receptors. These agonist effects are mediated by direct inhibition of TRPM3 by Gβγ subunits, rather than by a canonical cAMP mediated mechanism. The activity of TRPM3 in DRG neurons is also negatively modulated by tonic, constitutive GPCR activity as TRPM3 responses can be potentiated by GPCR inverse agonists. GPCR regulation of TRPM3 is also seen in vivo where G i/o GPCRs agonists inhibited and inverse agonists potentiated TRPM3 mediated nociceptive behavioural responses.

Signaling mediated by G protein-coupled receptors (GPCRs) is essential for the migration of cells toward chemoattractants. The recruitment of neutrophils to injured tissues in zebrafish larvae is a useful model for studying neutrophil migration and trafficking in vivo. Indeed, the study of this process led to the discovery that PI3Kγ is required for the polarity and motility of neutrophils, features that are necessary for the directed migration of these cells to wounds. However, the mechanism by which PI3Kγ is activated remains to be determined. Here we show that signaling by specifically the heterotrimeric G protein subunit Gβ1 is critical for neutrophil migration in response to wounding. In embryos treated with small-molecule inhibitors of Gβγ signaling, neutrophils failed to migrate to wound sites. Although both the Gβ1 and Gβ4 isoforms are expressed in migrating neutrophils, only deficiency for the former (morpholino-based knockdown) interfered with the directed migration of neutrophils towards wounds. The Gβ1 deficiency also impaired the ability of cells to change cell shape and reduced their general motility, defects that are similar to those in neutrophils deficient for PI3Kγ. Transplantation assays showed that the requirement for Gβ1 in neutrophil migration is cell autonomous. Finally, live imaging revealed that Gβ1 is required for polarized activation of PI3K, and for the actin dynamics that enable neutrophil migration. Collectively, our data indicate that Gβ1 signaling controls proper neutrophil migration by activating PI3K and modulating actin dynamics. Moreover, they illustrate a role for a specific Gβ isoform in chemotaxis in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on βγ signaling at the Golgi, where βγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit βγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit βγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by βγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by βγ inhibition, demonstrating that βγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate βγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for βγ regulation of TGN to PM transport and a novel role for βγ in mediating Golgi fragmentation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

A defining feature of the brain cancer glioblastoma is its highly invasive nature. When glioblastoma cells are isolated from patients using serum free conditions, they accurately recapitulate this invasive behaviour in animal models. The Rac subclass of Rho GTPases has been shown to promote invasive behaviour in glioblastoma cells isolated in this manner. However the guanine nucleotide exchange factors responsible for activating Rac in this context have not been characterized previously. PREX1 is a Rac guanine nucleotide exchange factor that is synergistically activated by binding of G protein αγ subunits and the phosphoinositide 3-kinase pathway second messenger phosphatidylinositol 3,4,5 trisphosphate. This makes it of particular interest in glioblastoma, as the phosphoinositide 3-kinase pathway is aberrantly activated by mutation in almost all cases. We show that PREX1 is expressed in glioblastoma cells isolated under serum-free conditions and in patient biopsies. PREX1 promotes the motility and invasion of glioblastoma cells, promoting Rac-mediated activation of p21-associated kinases and atypical PKC, which have established roles in cell motility. Glioblastoma cell motility was inhibited by either inhibition of phosphoinositide 3-kinase or inhibition of G protein βγ subunits. Motility was also inhibited by the generic dopamine receptor inhibitor haloperidol or a combination of the selective dopamine receptor D2 and D4 inhibitors L-741,626 and L-745,870. This establishes a role for dopamine receptor signaling via G protein βγ subunits in glioblastoma invasion and shows that phosphoinositide 3-kinase mutations in glioblastoma require a context of basal G protein-coupled receptor activity in order to promote this invasion.

The acute intoxicating effects of ethanol in the central nervous system result from the modulation of several molecular targets. It is widely accepted that ethanol enhances the activity of the glycine receptor (GlyR), thus enhancing inhibitory neurotransmission, leading to motor effects, sedation, and respiratory depression. We previously reported that small peptides interfered with the binding of Gβγ to the GlyR and consequently inhibited the ethanol-induced potentiation of the receptor. Now, using virtual screening, we identified a subset of small molecules capable of interacting with the binding site of Gβγ. One of these compounds, M554, inhibited the ethanol potentiation of the GlyR in both evoked currents and synaptic transmission in vitro When this compound was tested in vivo in mice treated with ethanol (1-3.5 g/kg), it was found to induce a faster recovery of motor incoordination in rotarod experiments and a shorter sedative effect in loss of righting reflex assays. This study describes a novel molecule that might be relevant for the design of useful therapeutic compounds in the treatment of acute alcohol intoxication., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: Despite considerable advances in the understanding of systemic lupus erythematosus (SLE), there is still an urgent need for new and more targeted treatment approaches. We previously demonstrated that small-molecule blockade of G protein βγ subunit (Gβγ) signaling inhibits acute inflammation through inhibition of chemokine receptor signal transduction. We undertook this study to determine whether inhibition of Gβγ signaling ameliorates disease in a mouse model of SLE., Methods: Lupus-prone (NZB × NZW)F1 female mice were prophylactically or therapeutically treated with the small-molecule Gβγ inhibitor gallein. Tissue samples were analyzed by flow cytometry and immunohistochemistry. The development and extent of nephritis were assessed by monitoring proteinuria and by immunohistochemical analysis. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay, and total IgG and anti-double-stranded DNA (anti-dsDNA) antibody-secreting cells were measured by enzyme-linked immunospot assay., Results: Gallein inhibited accumulation of T cells and germinal center (GC) B cells in the spleen. Both prophylactic and therapeutic treatment reduced GC size, decreased antibody-secreting cell production in the spleen, and markedly decreased accumulation of autoreactive anti-dsDNA antibody-secreting cells in kidneys. Gallein also reduced immune complex deposition in kidneys. Finally, gallein treatment dramatically inhibited kidney inflammation, prevented glomerular damage, and decreased proteinuria. Mechanistically, gallein inhibited immune cell migration and signaling in response to chemokines in vitro, which suggests that its mechanisms of action in vivo are inhibition of migration of immune cells to sites of inflammation and inhibition of immune cell maturation., Conclusion: Overall, these data demonstrate the potential use of gallein or novel inhibitors of Gβγ signaling in SLE treatment., (© 2016, American College of Rheumatology.)

Agonist-evoked endocytosis of G protein-coupled receptors has been extensively studied. The mechanisms by which agonists stimulate mobilization and plasma membrane translocation of G protein-coupled receptors from intracellular stores are unexplored. Protease-activated receptor-2 (PAR2) traffics to lysosomes, and sustained protease signaling requires mobilization and plasma membrane trafficking of PAR2 from Golgi stores. We evaluated the contribution of protein kinase D (PKD) and Gβγ to this process. In HEK293 and KNRK cells, the PAR2 agonists trypsin and 2-furoyl-LIGRLO-NH2 activated PKD in the Golgi apparatus, where PKD regulates protein trafficking. PAR2 activation induced translocation of Gβγ, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between Gγ-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and Gβγ (gallein) prevented PAR2-stimulated activation of PKD. CRT0066101, PKD1 siRNA, and gallein all inhibited recovery of PAR2-evoked Ca(2+) signaling. PAR2 with a photoconvertible Kaede tag was expressed in KNRK cells to examine receptor translocation from the Golgi apparatus to the plasma membrane. Irradiation of the Golgi region (405 nm) induced green-red photo-conversion of PAR2-Kaede. Trypsin depleted PAR2-Kaede from the Golgi apparatus and repleted PAR2-Kaede at the plasma membrane. CRT0066101 inhibited PAR2-Kaede translocation to the plasma membrane. CRT0066101 also inhibited sustained protease signaling to colonocytes and nociceptive neurons that naturally express PAR2 and mediate protease-evoked inflammation and nociception. Our results reveal a major role for PKD and Gβγ in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Gαq inhibitor UBO-QIC (FR900359) is becoming an important pharmacological tool, but its selectivity against other G proteins and their subunits, especially βγ, has not been well characterized. We examined UBO-QIC's effect on diverse signaling pathways mediated via various G protein-coupled receptors (GPCRs) and G protein subunits by comparison with known Gαi inhibitor pertussis toxin. As expected, UBO-QIC inhibited Gαq signaling in all assay systems examined. However, other non-Gαq-events, e.g. Gβγ-mediated intracellular calcium release and inositol phosphate production, following activation of Gi-coupled A1 adenosine and M2 muscarinic acetylcholine receptors, were also blocked by low concentrations of UBO-QIC, indicating that its effect is not limited to Gαq. Thus, UBO-QIC also inhibits Gβγ-mediated signaling similarly to pertussis toxin, although UBO-QIC does not affect Gαi-mediated inhibition or Gαs-mediated stimulation of adenylyl cyclase activity. However, the blockade by UBO-QIC of GPCR signaling, such as carbachol- or adenosine-mediated calcium or inositol phosphate increases, does not always indicate inhibition of Gαq-mediated events, as the βγ subunits released from Gi proteins following the activation of Gi-coupled receptors, e.g. M2 and A1Rs, may produce similar signaling events. Furthermore, UBO-QIC completely inhibited Akt signaling, but only partially blocked ERK1/2 activity stimulated by the Gq-coupled P2Y1R. Thus, we have revealed new aspects of the pharmacological interactions of UBO-QIC., (Published by Elsevier Inc.)

G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein βγ (Gβγ) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of Gβγ, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. Gβ1 and Gβ2 mRNA accounted for >99% of Gβ mRNA, and small interfering RNA (siRNA)-mediated silencing of Gβ1 but not Gβ2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking Gβγ enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking Gβγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous Gβγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gβγ in CD4+ T helper cells could have applications for autoimmune diseases.

The dopamine D2 receptor (DRD2) is a G protein-coupled receptor (GPCR) that is generally considered to be a primary target in the treatment of schizophrenia. First generation antipsychotic drugs (e.g. haloperidol) are antagonists of the DRD2, while second generation antipsychotic drugs (e.g. olanzapine) antagonize DRD2 and 5HT2A receptors. Notably, both these classes of drugs may cause side effects associated with D2 receptor antagonism (e.g. hyperprolactemia and extrapyramidal symptoms). The novel, "third generation" antipsychotic drug, aripiprazole is also used to treat schizophrenia, with the remarkable advantage that its tendency to cause extrapyramidal symptoms is minimal. Aripiprazole is considered a partial agonist of the DRD2, but it also has partial agonist/antagonist activity for other GPCRs. Further, aripiprazole has been reported to have a unique activity profile in functional assays with the DRD2. In the present study the molecular pharmacology of aripiprazole was further examined in HEK cell models stably expressing the DRD2 and specific isoforms of adenylyl cyclase to assess functional responses of Gα and Gβγ subunits. Additional studies examined the activity of aripiprazole in DRD2-mediated heterologous sensitization of adenylyl cyclase and cell-based dynamic mass redistribution (DMR). Aripiprazole displayed a unique functional profile for modulation of G proteins, being a partial agonist for Gαi/o and a robust antagonist for Gβγ signaling. Additionally, aripiprazole was a weak partial agonist for both heterologous sensitization and dynamic mass redistribution., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Heterotrimeric G-proteins play a crucial role in the control of renal epithelial cell function during homeostasis and in response to injury. In this report, G-protein βγ subunit (Gβγ) dimer activity was evaluated during the process of tubular repair after renal ischemia-reperfusion injury (IRI) in male Sprague Dawley rats. Rats were treated with a small molecule inhibitor of Gβγ activity, gallein (30 or 100 mg/kg), 1 hour after reperfusion and every 24 hours for 3 additional days. After IRI, renal dysfunction was prolonged after the high-dose gallein treatment in comparison with vehicle treatment during the 7-day recovery period. Renal tubular repair in the outer medulla 7 days after IRI was significantly (P < 0.001) attenuated after treatment with high-dose gallein (100 mg/kg) in comparison with low-dose gallein (30 mg/kg), or the vehicle and fluorescein control groups. Gallein treatment significantly reduced (P < 0.05) the number of proliferating cell nuclear antigen-positive tubular epithelial cells at 24 hours after the ischemia-reperfusion phase in vivo. In vitro application of gallein on normal rat kidney (NRK-52E) proximal tubule cells significantly reduced (P < 0.05) S-phase cell cycle entry compared with vehicle-treated cells as determined by 5'-bromo-2'-deoxyuridine incorporation. Taken together, these data suggest that Gβγ signaling contributes to the maintenance and repair of renal tubular epithelium and may be a novel therapeutic target for the development of drugs to treat acute kidney injury., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Our laboratory has identified a number of small molecules that bind to G protein βγ subunits (Gβγ) by competing for peptide binding to the Gβγ "hot spot." M119/Gallein were identified as inhibitors of Gβγ subunit signaling. Here we examine the activity of another molecule identified in this screen, 12155, which we show that in contrast to M119/Gallein had no effect on Gβγ-mediated phospholipase C or phosphoinositide 3-kinase (PI3K) γ activation in vitro. Also in direct contrast to M119/Gallein, 12155 caused receptor-independent Ca(2+) release, and activated other downstream targets of Gβγ including extracellular signal regulated kinase (ERK), protein kinase B (Akt) in HL60 cells differentiated to neutrophils. We show that 12155 releases Gβγ in vitro from Gαi1β1γ2 heterotrimers by causing its dissociation from GαGDP without inducing nucleotide exchange in the Gα subunit. We used this novel probe to examine the hypothesis that Gβγ release is sufficient to direct chemotaxis of neutrophils in the absence of receptor or G protein α subunit activation. 12155 directed chemotaxis of HL60 cells and primary neutrophils in a transwell migration assay with responses similar to those seen for the natural chemotactic peptide n-formyl-Met-Leu-Phe. These data indicate that release of free Gβγ is sufficient to drive directional chemotaxis in a G protein-coupled receptor signaling-independent manner., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Inhibition of Gβγ-subunit signaling to phospholipase C β3 has been shown to potentiate morphine-mediated antinociception while attenuating the development of tolerance and dependence in mice. The objective of this study was to determine the effect of Gβγ-subunit inhibition on antinociception and other pharmacological effects, such as respiratory depression, constipation, and hyperlocomotion, mediated by the μ-opioid receptor. The Gβγ-subunit inhibitor, gallein, was administered to C57BL/6J mice by intraperitoneal injection before morphine, and data were compared with mice treated with vehicle, morphine, or gallein alone. Morphine-induced antinociception was measured using the 55°C warm-water tail-withdrawal test. Pretreatment with gallein produced a dose-dependent potentiation of morphine-mediated antinociception, producing up to a 10-fold leftward shift in the morphine dose-response curve and extending the duration of antinociception induced by a single dose of morphine. Gallein pretreatment also prevented acute antinociceptive tolerance induced by morphine. In contrast, the dose-dependent respiratory depression and hyperlocomotion induced by morphine were not potentiated by gallein pretreatment. Similarly, gallein pretreatment did not potentiate morphine-conditioned place preference responses or morphine-induced constipation, as measured as a reduction in excreta. These results suggest that selectively inhibiting Gβγ-mediated signaling may selectively increase μ-opioid receptor-mediated antinociception without matching increases in adverse physiological effects.

Background: Intimal hyperplasia remains the principal lesion in the development of restenosis after vessel wall injury. G-protein coupled receptors are involved in smooth muscle cell proliferation but the role of Gβγ in arterial intimal hyperplasia has not been well defined. The aim of this study is to characterize the expression of Gβγ G-proteins in the developing intimal hyperplasia in a murine model and the impact of disruption of Gβγ signaling on intimal hyperplasia development., Methods: The murine femoral wire injury model was employed. Specimens were perfusion-fixed and sections were stained with H&E and Movat's stains such that morphometry could be performed using an Image-Pro system. Additional specimens of femoral artery were also harvested and snap frozen for Western blotting for the Gβγ expression and for Western blotting and zymography to allow for the study of gelatinase and plasminogen activator expression and activation. Contralateral vessels were used as controls. Additional vessels were immersed in pluronic gel containing an adenovirus with the Gβγ inhibitor βARK(CT)., Results: The injured femoral arteries developed intimal hyperplasia, while sham vessels did not produce such a response. Cell proliferation peaked at 3-5 d and cell migration at 7 d after injury. There was a marked time-dependent increase in Gβγ over the 28 d following injury. Inhibition of Gβγ with βARK(CT) inhibited cell proliferation, cell migration and the development of intimal hyperplasia. Inhibition of Gβγ decreased peak uPA activity and expression without increasing early PAI-1 activity and expression. Inhibition of Gβγ reduced peak MMP-2 activity at d 1 but not at d 7 and also reduced peak MMP-9 activity at d 3. Protein expression for both MMP-2 and MMP-9 was also transiently decreased. There were no changes in TIMP-1 and TIMP-2 expression and activity., Conclusions: These data demonstrate a time-dependent increase in Gβγ G-protein expression following wire injury in the mouse. Inhibition of Gβγ alters cell proliferation and migration with associated changes in MMP-2, MMP-9, and uPA expression and activity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Heart failure (HF) is devastating disease with poor prognosis. Elevated sympathetic nervous system activity and outflow, leading to pathologic attenuation and desensitization of β-adrenergic receptors (β-ARs) signaling and responsiveness, are salient characteristic of HF progression. These pathologic effects on β-AR signaling and HF progression occur in part due to Gβγ-mediated signaling, including recruitment of receptor desensitizing kinases such as G-protein coupled receptor (GPCR) kinase 2 (GRK2) and phosphoinositide 3-kinase (PI3K), which subsequently phosphorylate agonist occupied GPCRs. Additionally, chronic GPCR signaling signals chronically dissociated Gβγ subunits to interact with multiple effector molecules that activate various signaling cascades involved in HF pathophysiology. Importantly, targeting Gβγ signaling with large peptide inhibitors has proven a promising therapeutic paradigm in the treatment of HF. We recently described an approach to identify small molecule Gβγ inhibitors that selectively block particular Gβγ functions by specifically targeting a Gβγ protein-protein interaction "hot spot." Here we describe their effects on Gβγ downstream signaling pathways, including their role in HF pathophysiology. We suggest a promising therapeutic role for small molecule inhibition of pathologic Gβγ signaling in the treatment of HF. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure.", (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The extracellular-regulated kinase (ERK1/2) is a key conduit for transduction of signals from growth factor receptors to the nucleus. Previous work has shown that ERK1/2 activation in response to IGF-1 may require the participation of G proteins, but the role of the receptor tyrosine kinase in this process has not been clearly resolved. This investigation of IGF-1 receptor function was therefore designed to examine the contribution of the receptor tyrosine kinase to ERK1/2 activation. Phosphorylation of ERK1/2 in smooth muscle cells following treatment with IGF-1 was not blocked by pretreatment with AG1024 or picropodophylin, inhibitors of the IGF-1 receptor tyrosine kinase. Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor. ERK1/2 activation was unaffected by the phosphatidylinositol 3-kinase inhibitor LY-294002, but was sensitive to inhibitors of Src kinase, phospholipase C and Gβγ subunit signalling. Treatment with αIR-3, a neutralizing monoclonal antibody, also stimulated ERK1/2 phosphorylation without concomitant activation of the receptor tyrosine kinase. Phosphoprotein mapping of IGF-1 and αIR-3 treated cells confirmed that antibody-induced ERK1/2 phosphorylation occurred in the absence of tyrosine kinase phosphorylation, and enabled extension of these findings to p38 MAPK. These results suggest that stimulation of ERK1/2 phosphorylation by IGF-1 does not require activation of the receptor tyrosine kinase., (Copyright © 2010 Elsevier Inc. All rights reserved.)

G protein-coupled receptors transduce signals through heterotrimeric G protein Gα and Gβγ subunits, both of which interact with downstream effectors to regulate cell function. Gβγ signaling has been implicated in the pathophysiology of several diseases, suggesting that Gβγ could be an important pharmaceutical target. Previously, we used a combination of virtual and manual screening to find small molecules that bind to a protein-protein interaction "hot spot" on Gβγ and block regulation of physiological effectors. One of the most potent and effective compounds from this screen was selenocystamine. In this study, we investigated the mechanism of action of selenocystamine and found that selenocysteamine forms a covalent complex with Gβγ by a reversible redox mechanism. Mass spectrometry and site-directed mutagenesis suggest that selenocysteamine preferentially modifies GβCys204, but also a second undefined site. The high potency of selenocystamine in Gβγ inhibition seems to arise from both high reactivity of the diselenide group and binding to a specific site on Gβ. Using structural information about the "hot spot," we developed a strategy to selectively target redox reversible compounds to a specific site on Gβγ using peptide carriers such as SIGCAFKILGY(-cysteamine) [SIGC(-cysteamine)]. Mass spectrometry and site-directed mutagenesis indicate that SIGC(-cysteamine) specifically and efficiently leads to cysteamine (half-cystamine) modification of a single site on Gβ, likely GβCys204, and inhibits Gβγ more than a hundred times more potently than cystamine. These data support the concept that covalent modifiers can be specifically targeted to the Gβγ "hot spot" through rational incorporation into molecules that noncovalently bind to Gβγ.

Rationale: Excess signaling through cardiac Gbetagamma subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G protein-coupled receptor kinase (GRK)2 to agonist-stimulated beta-adrenergic receptors (beta-ARs) in HF, leading to chronic beta-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting Gbetagamma signaling and its interaction with GRK2 could be of therapeutic value in HF., Objective: We sought to investigate small molecule Gbetagamma inhibition in HF., Methods and Results: We recently described novel small molecule Gbetagamma inhibitors that selectively block Gbetagamma-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of Gbetagamma and GRK2 in vitro and in HL60 cells. Here, we show they reduced beta-AR-mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to beta-AR agonist. To evaluate their cardiac-specific effects in vivo, we initially used an acute pharmacological HF model (30 mg/kg per day isoproterenol, 7 days). Concurrent daily injections prevented HF and partially normalized cardiac morphology and GRK2 expression in this acute HF model. To investigate possible efficacy in halting progression of preexisting HF, calsequestrin cardiac transgenic mice (CSQ) with extant HF received daily injections for 28 days. The compound alone halted HF progression and partially normalized heart size, morphology, and cardiac expression of HF marker genes (GRK2, atrial natriuretic factor, and beta-myosin heavy chain)., Conclusions: These data suggest a promising therapeutic role for small molecule inhibition of pathological Gbetagamma signaling in the treatment of HF.

Direct interaction with the beta subunit of the heterotrimeric G protein complex causes voltage-dependent inhibition of N-type calcium channels. To further characterize the molecular determinants of this interaction, we performed scanning mutagenesis of residues 372-387 and 410-428 of the N-type channel alpha1 subunit, in which individual residues were replaced by either alanine or cysteine. We coexpressed wild type Gbeta1gamma2 subunits with either wild type or point mutant N-type calcium channels, and voltage-dependent, G protein-mediated inhibition of the channels (VDI) was assessed using patch clamp recordings. The resulting data indicate that Arg376 and Val416 of the alpha1 subunit, residues which are surface-exposed in the presence of the calcium channel beta subunit, contribute significantly to the functional inhibition by Gbeta1. To further characterize the roles of Arg376 and Val416 in this interaction, we performed secondary mutagenesis of these residues, coexpressing the resulting mutants with wild type Gbeta1gamma2 subunits and with several isoforms of the auxiliary beta subunit of the N-type channel, again assessing VDI using patch clamp recordings. The results confirm the importance of Arg376 for G protein-mediated inhibition and show that a single amino acid substitution to phenylalanine drastically alters the abilities of auxiliary calcium channel subunits to regulate G protein inhibition of the channel.

The effects of norepinephrine (NE), an inhibitor of insulin secretion, were examined on membrane potential and the ATP-sensitive K+ channel (K ATP) in INS 832/13 cells. Membrane potential was monitored under the whole cell current clamp mode. NE hyperpolarized the cell membrane, an effect that was abolished by tolbutamide. The effect of NE on K ATP channels was investigated in parallel using outside-out single channel recording. This revealed that NE enhanced the open activities of the K ATP channels approximately 2-fold without changing the single channel conductance, demonstrating that NE-induced hyperpolarization was mediated by activation of the K ATP channels. The NE effect was abolished in cells preincubated with pertussis toxin, indicating coupling to heterotrimeric G i/G o proteins. To identify the G proteins involved, antisera raised against alpha and beta subunits (anti-G alpha common, anti-G beta, anti-G alpha i1/2/3, and anti-G alpha o) were used. Anti-G alpha common totally blocked the effects of NE on membrane potential and K ATP channels. Individually, anti-G alpha i1/2/3 and anti-G alpha o only partially inhibited the action of NE on K ATP channels. However, the combination of both completely eliminated the action. Antibodies against G beta had no effects. To confirm these results and to further identify the G protein subunits involved, the blocking effects of peptides containing the sequence of 11 amino acids at the C termini of the alpha subunits were used. The data obtained were similar to those derived from the antibody work with the additional information that G alpha i3 and G alpha o1 were not involved. In conclusion, both G i and G o proteins are required for the full effect of norepinephrine to activate the K ATP channel.

G protein betagamma subunit-dependent signaling is important for chemoattractant-dependent leukocyte chemotaxis. Selective small molecule targeting of phosphoinositide 3-kinase (PI3-kinase) gamma catalytic activity is a target of interest for anti-inflammatory pharmaceutical development. In this study, we examined whether small-molecule inhibition of Gbetagamma-dependent signaling, including Gbetagamma-dependent activation of PI3-kinase gamma and Rac1, could inhibit chemoattractant-dependent neutrophil migration in vitro and inflammation in vivo. Small-molecule Gbetagamma inhibitors suppressed fMLP-stimulated Rac activation, superoxide production, and PI3-kinase activation in differentiated HL60 cells. These compounds also blocked fMLP-dependent chemotaxis in HL60 cells and primary human neutrophils. Systemic administration inhibited paw edema and neutrophil infiltration in a mouse carrageenan-induced paw edema model. Overall, the data demonstrate that targeting Gbetagamma-regulation may be an effective anti-inflammation strategy.

The influence of alpha2-autoreceptors on the facilitation of [3H]-noradrenaline release mediated by angiotensin II was studied in prostatic portions of rat vas deferens preincubated with [3H]-noradrenaline. Angiotensin II enhanced tritium overflow evoked by trains of 100 pulses at 8 Hz, an effect that was attenuated by the AT1-receptor antagonist losartan (0.3-1 microM), at concentrations suggesting the involvement of the AT1B subtype. The effect of angiotensin II was also attenuated by inhibition of phospholipase C (PLC) and protein kinase C (PKC) indicating that prejunctional AT1-receptors are coupled to the PLC-PKC pathway. Angiotensin II (0.3-100 nM) enhanced tritium overflow more markedly, up to 64%, under conditions that favor alpha2-autoinhibition, observed when stimulation consisted of 100 pulses at 8 Hz, than under poor alpha2-autoinhibition conditions, only up to 14%, observed when alpha2-adrenoceptors were blocked with yohimbine (1 microM) or when stimulation consisted of 20 pulses at 50 Hz. Activation of PKC with 12-myristate 13-acetate (PMA, 0.1-3 microM) also enhanced tritium overflow more markedly under strong alpha2-autoinhibition conditions. Inhibition of Gi/o-proteins with pertussis toxin (8 microg/ml) or blockade of Gbetagamma subunits with the anti-betagamma peptide MPS-Phos (30 microM) attenuated the effects of angiotensin II and PMA. The results indicate that activation of AT1-receptors coupled to the PLC-PKC pathway enhances noradrenaline release, an effect that is markedly favoured by an ongoing activation of alpha2-autoreceptors. Interaction between alpha2-adrenoceptors and AT1-receptors seems to involve the betagamma subunits released from the Gi/o-proteins coupled to alpha2-adrenoceptors and protein kinase C activated by AT1-receptors.

Signal transduction involving heterotrimeric G proteins is universal among fungi, animals, and plants. In plants and fungi, the best understood function for the G protein complex is its modulation of cell proliferation and one of several important signals that are known to modulate the rate at which these cells proliferate is D-glucose. Arabidopsis thaliana seedlings lacking the beta subunit (AGB1) of the G protein complex have altered cell division in the hypocotyl and are D-glucose hypersensitive. With the aim to discover new elements in G protein signaling, we screened for gain-of-function suppressors of altered cell proliferation during early development in the agb1-2 mutant background. One agb1-2-dependent suppressor, designated sgb1-1(D) for suppressor of G protein beta1 (agb1-2), restored to wild type the altered cell division in the hypocotyl and sugar hypersensitivity of the agb1-2 mutant. Consistent with AGB1 localization, SGB1 is found at the highest steady-state level in tissues with active cell division, and this level increases in hypocotyls when grown on D-glucose and sucrose. SGB1 is shown here to be a Golgi-localized hexose transporter and acts genetically with AGB1 in early seedling development.

A large number of hormones and local agonists activating guanine-binding protein-coupled receptors (GPCR) play a major role in cancer progression. Here, we characterize the new imidazo-pyrazine derivative BIM-46174, which acts as a selective inhibitor of heterotrimeric G-protein complex. BIM-46174 prevents the heterotrimeric G-protein signaling linked to several GPCRs mediating (a) cyclic AMP generation (Galphas), (b) calcium release (Galphaq), and (c) cancer cell invasion by Wnt-2 frizzled receptors and high-affinity neurotensin receptors (Galphao/i and Galphaq). BIM-46174 inhibits the growth of a large panel of human cancer cell lines, including anticancer drug-resistant cells. Exposure of cancer cells to BIM-46174 leads to caspase-3-dependent apoptosis and poly(ADP-ribose) polymerase cleavage. National Cancer Institute COMPARE analysis for BIM-46174 supports its novel pharmacologic profile compared with 12,000 anticancer agents. The growth rate of human tumor xenografts in athymic mice is significantly reduced after administration of BIM-46174 combined with either cisplatin, farnesyltransferase inhibitor, or topoisomerase inhibitors. Our data validate the feasibility of targeting heterotrimeric G-protein functions downstream the GPCRs to improve anticancer chemotherapy.

Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. Interestingly, recent results suggest that G proteins also interact with microtubules and participate in cell division and differentiation. It has been shown earlier that both alpha and betagamma subunits of G proteins modulate microtubule assembly in vitro. Since G protein activation and subsequent dissociation of alpha and betagamma subunits are necessary for G proteins to participate in signaling processes, here we asked if similar activation is required for modulation of microtubule assembly by G proteins. We reconstituted Galphabetagamma heterotrimer from myristoylated-Galpha and prenylated-Gbetagamma, and found that the heterotrimer blocks Gi1alpha activation of tubulin GTPase and inhibits the ability of Gbeta1gamma2 to promote in vitro microtubule assembly. Results suggest that G protein activation is required for functional coupling between Galpha/Gbetagamma and tubulin/microtubules, and supports the notion that regulation of microtubules is an integral component of G protein mediated signaling.

Most drugs target a receptor for a hormone or neurotransmitter. A newer strategy for drug development is to target a downstream signaling element, such as the G protein associated with a receptor. Suramin is considered a lead compound targeting this moiety. It inhibits binding of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) to G proteins and reduces agonist binding to G protein-coupled receptors. Suramin is thought to uncouple the G protein from its associated receptor, although there is no direct evidence for this mechanism. We have now examined the effect of suramin on G protein signaling for the vasoactive intestinal peptide (VIP) receptor in lung. The primary experimental strategy was a two-step cross-linking reaction that covalently captures the VIP-receptor-G protein ternary complex. Such cross-linking provided the first direct evidence that suramin physically disrupts receptor-G protein coupling. We investigated how this uncoupling relates to the inhibition of GTPgammaS binding. Suramin indiscriminately hindered the dissociation of various guanine nucleotides from the G protein, implying that its action is not allosteric. Further cross-linking studies suggested that suramin does not obstruct the receptor docking site directly but appears to block the interface between G protein alpha and betagamma subunits. Observations with a purified system of recombinant G protein subunits without a receptor yielded direct evidence that suramin suppresses the association between these subunits. This action can explain how it both disrupts receptor-G protein coupling and inhibits guanine nucleotide release. The improved understanding of suramin's action advances the development of selective inhibitors of G protein signaling.

The present study is designed to explore the role of G protein-coupled receptors (GPCRs) in the protection afforded by ischemic preconditioning (PC). We used TG mice with cardiac-specific overexpression of a Gbetagamma-sequestering peptide, betaARKct (TG betaARKct mice), to test whether the protection of PC is Gbetagamma-dependent. To test the role of G(i) protein, we used wild-type mice pretreated with the G(i) inhibitor pertussis toxin. Recovery of left ventricular developed pressure and infarct size were measured as indices of protection. PC induced protection in wild-type mice, but this protection was blocked by pertussis toxin treatment and was also blocked in TG betaARKct mice. To determine the mechanism of Gbetagamma-induced protection in PC, we investigated one of the downstream targets of Gbetagamma, the PI3K/p70S6K pathway. PC-induced phosphorylation of p70S6K was not blocked in TG betaARKct hearts; therefore, we investigated other targets of Gbetagamma. Recent studies suggest a role for Gbetagamma in GPCR internalization. We found that betaARKct, a specific PI3K inhibitor wortmannin, and bafilomycin A1, which all block receptor recycling, all blocked the protective effect of PC. To additionally test whether PI3K is involved in PC-activated receptor internalization and endosomal signaling, we used TG mice with cardiac-specific overexpression of a catalytically inactive mutant PI3Kgamma, which disrupts the recruitment of functional PI3K to agonist-activated GPCRs in vivo. We found that the catalytically inactive mutant of PI3Kgamma blocks the protection of PC. In summary, these data suggest the novel finding that the cardioprotective effect of PC requires receptor internalization.

N-type voltage-dependent Ca(2+) channels (N-VDCCs) play important roles in neurotransmitter release and certain postsynaptic phenomena. These channels are modulated by a number of intracellular factors, notably by Gbetagamma subunits of G proteins, which inhibit N-VDCCs in a voltage-dependent (VD) manner. Here we show that an increase in intracellular Na(+) concentration inhibits N-VDCCs in hippocampal pyramidal neurones and in Xenopus oocytes. In acutely dissociated hippocampal neurones, Ba(2+) current via N-VDCCs was inhibited by Na(+) influx caused by the activation of NMDA receptor channels. In Xenopus oocytes expressing N-VDCCs, Ba(2+) currents were inhibited by Na(+) influx and enhanced by depletion of Na(+), after incubation in a Na(+)-free extracellular solution. The Na(+)-induced inhibition was accompanied by the development of VD facilitation, a hallmark of a Gbetagamma-dependent process. Na(+)-induced regulation of N-VDCCs is Gbetagamma dependent, as suggested by the blocking of Na(+) effects by Gbetagamma scavengers and by excess Gbetagamma, and may be mediated by the Na(+)-induced dissociation of Galphabetagamma heterotrimers. N-VDCCs may be novel effectors of Na(+)ion, regulated by the Na(+) concentration via Gbetagamma.

This study investigated regulation of L-type calcium channels (Cav1.2b) by acetylcholine (ACh) in rabbit portal vein myocytes. Whole-cell currents were recorded using 5 mmol/L barium as charge carrier. ACh (10 micromol/L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4-DAMP (100 nmol/L) but was blocked by the M2 receptor antagonist methoctramine (5 micromol/L). The classical and novel protein kinase C (PKC) antagonist calphostin C (50 nmol/L) abolished ACh responses, whereas the classical PKC antagonist Gö6976 (200 nmol/L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 micromol/L), by the c-Src inhibitor PP2 (10 micromol/L) (but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G-protein subunit Gbetagamma. Cells dialyzed with c-Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c-Src is downstream of PI3K. Phorbol 12,13-dibutyrate (PDBu, 0.1 micromol/L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Gö6976. The PDBu response was also reduced by PP2, and the PP2-insensitive component was blocked by Gö6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to Gbetagamma, PI3K, a novel PKC, and c-Src. PDBu stimulates the novel PKC/c-Src pathway along with a second pathway that is independent of c-Src and involves a classical PKC.

Objective: Endopeptidase EC3.4.24.15 (EP24.15)- and EC3.4.24.16 (EP24.16)-specific peptide hydrolysis plays an important role in endothelium-mediated vasoregulation. Given the significant influence of hemodynamic forces on vascular homeostasis and pathology, we postulated that these related peptidases may be mechanosensitive. The objective of this study, therefore, was to investigate the putative role of cyclic strain in regulating the expression and enzymatic activity of EP24.15 and EP24.16 in bovine aortic endothelial cells (BAECs)., Methods and Results: BAECs were cultured under conditions of defined cyclic strain (0% to 10% stretch, 60 cycles/min, 0 to 24 hours). Strain significantly increased EP24.15 and EP24.16 soluble activity in a force- and time-dependent manner, with elevations of 2.3+/-0.4- and 1.9+/-0.3-fold for EP24.15 and EP24.16, respectively, after 24 hours at 10% strain. Pharmacological agents and dominant-negative G protein mutants used to selectively disrupt Gi(alpha)- and Gbetagamma-mediated signaling pathways attenuated strain-dependent (24 hours, 5%) increases for both enzymes. Differences in the inhibitory profile for both enzymes were also noted, with EP24.15 displaying greater sensitivity to Gi(alpha2/3) inhibition and EP24.16 exhibiting greater sensitivity to Gi(alpha1/2) and Gbetagamma inhibition. Cyclic strain also increased levels of secreted EP24.15 and EP24.16 activity by 2.6+/-0.02- and 3.6+/-0.2-fold, respectively, in addition to mRNA levels for both enzymes (EP24.15 +42%, EP24.16 +56%)., Conclusions: Our findings suggest that cyclic strain putatively regulates both the mRNA expression and enzymatic function of EP24.15 and EP24.16 in BAECs via alternate Gi protein signaling pathways.

Prostate cancer starts as androgen-dependent malignancy and responds initially to androgen ablative therapy. Beneficial effects of androgen ablation, however, are often temporary and the cancer reappears as androgen-independent tumor, suggesting the existence of additional factors responsible for progression of the disease. Attention has focused on receptor tyrosine kinases as the growth mediators of androgen-independent prostate cancer; overexpression of epidermal growth factor receptors or their ligand heparin-bound epidermal growth factor, for example, promotes transition to androgen independence. Emerging data demonstrate involvement of another class of cell membrane-anchored receptors, the heterotrimeric guanine-binding (G) protein-coupled receptors (GPCRs) in prostate cancer. In vitro, stimulation of many endogenous GPCRs induces mitogenic signaling and growth of prostate cancer cells. The GPCRs transduce mitogenic signals via activated G proteins in the form of Galpha-GTP and Gbetagamma subunits. Here, we show that expression of a Gbetagamma inhibitor peptide derived from carboxy terminus of G protein-coupled receptor kinase 2 obliterates serum-regulated prostate cancer cell growth in vitro and prevents prostate tumor formation in vivo. We also demonstrate that inhibition of Gbetagamma signaling retards growth of existing prostate tumors by inducing cell death. These data establish a central role for heterotrimeric G proteins in prostate cancer and suggest targeted inhibition of Gbetagamma signaling may serve as specific molecular therapy tool to limit pathologic growth of advanced prostate cancer.

The most common form of Ca(2+) signaling by Gq-coupled receptors entails activation of PLCbeta2 by Galphaq to generate IP(3) and evoke Ca(2+) release from the ER. Another form of Ca(2+) signaling by G protein-coupled receptors involves activation of Gi to release Gbetagamma, which activates PLCbeta1. Whether Gbetagamma has additional roles in Ca(2+) signaling is unknown. Introduction of Gbetagamma into cells activated Ca(2+) release from the IP(3) Ca(2+) pool and Ca(2) oscillations. This can be due to activation of PLCbeta1 or direct activation of the IP(3)R by Gbetagamma. We report here that Gbetagamma potently activates the IP(3) receptor. Thus, Gbetagamma-triggered [Ca(2+)](i) oscillations are not affected by inhibition of PLCbeta. Coimmunoprecipitation and competition experiments with Gbetagamma scavengers suggest binding of Gbetagamma to IP(3) receptors. Furthermore, Gbetagamma inhibited IP(3) binding to IP(3) receptors. Notably, Gbetagamma activated single IP(3)R channels in native ER as effectively as IP(3). The physiological significance of this form of signaling is demonstrated by the reciprocal sensitivity of Ca(2+) signals evoked by Gi- and Gq-coupled receptors to Gbetagamma scavenging and PLCbeta inhibition. We propose that gating of IP(3)R by Gbetagamma is a new mode of Ca(2+) signaling with particular significance for Gi-coupled receptors.