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G protein-coupled receptor-mediated activation of p110? by G?? is required for cellular transformation and invasiveness.

ABSTRACT: Synergistic activation by heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110? from other class IA phosphoinositide 3-kinases (PI3Ks). Activation of p110? is specifically implicated in various physiological and pathophysiological processes, such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110?-dependent functions, we identified the site in p110? that binds to the G?? subunit of G proteins. Mutation of this site eliminated G??-dependent activation of PI3K? (a dimer of p110? and the p85 regulatory subunit) in vitro and in cells, without affecting basal activity or phosphotyrosine peptide-mediated activation. Disrupting the p110?-G?? interaction by mutation or with a cell-permeable peptide inhibitor blocked the transforming capacity of PI3K? in fibroblasts and reduced the proliferation, chemotaxis, and invasiveness of PTEN-null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3K? could provide a therapeutic approach for tumors that depend on p110? for growth and metastasis.

SUBMITTER: Dbouk HA

PROVIDER: S-EPMC3979326 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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G protein-coupled receptor-mediated activation of p110β by Gβγ is required for cellular transformation and invasiveness.

Dbouk Hashem A HA Vadas Oscar O Shymanets Aliaksei A Burke John E JE Salamon Rachel S RS Khalil Bassem D BD Barrett Mathew O MO Waldo Gary L GL Surve Chinmay C Hsueh Christine C Perisic Olga O Harteneck Christian C Shepherd Peter R PR Harden T Kendall TK Smrcka Alan V AV Taussig Ronald R Bresnick Anne R AR Nürnberg Bernd B Williams Roger L RL Backer Jonathan M JM

Science signaling 20121204 253

Synergistic activation by heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110β from other class IA phosphoinositide 3-kinases (PI3Ks). Activation of p110β is specifically implicated in various physiological and pathophysiological processes, such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110β- ...[more]

PMID: 23211529

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Project description:Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gβγ subunits liberated following activation of Gαi/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gβγ released from Gαs and Gαq Activation of the Gs-coupled adenosine 2B receptor and the Gq-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 Gs-coupled prostaglandin-EP2 and Gq-coupled bradykinin B2 (BK2) receptors. The Gi/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 Gαi/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 Gαi/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 Gαi/o, we examined whether activation of Gs- and Gq-coupled receptors also influence TRPM3 via Gβγ. Our results demonstrate that activation of Gs- and Gq-coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gβγ liberation. We also demonstrated that Gs- and Gq-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.

Project description:BackgroundProinflammatory cytokine tumor necrosis factor-α (TNFα) induces β-adrenergic receptor (βAR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known.Methods and resultsTwo different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNFα showed that TNFα alone is sufficient to mediate βAR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling βAR desensitization independent of sympathetic overdrive. TNFα-mediated βAR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in β2AR-overexpressing human embryonic kidney 293 cells showed significant βAR desensitization, GRK2 upregulation, and recruitment to the βAR complex following TNFα. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated βAR phosphorylation and GRK2 recruitment on TNFα. Furthermore, TNFα-mediated βAR phosphorylation was not blocked with βAR antagonist propranolol. Additionally, TNFα administration in transgenic mice with cardiac overexpression of Gβγ-sequestering peptide βARK-ct could not prevent βAR desensitization or cardiac dysfunction showing that GRK2 recruitment to the βAR is Gβγ independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNFα-mediated βAR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNFα-mediated loss in contractility, showing that TNFα-induced βAR desensitization is GRK2 dependent.ConclusionsTNFα-induced βAR desensitization is mediated by GRK2 and is independent of Gβγ, uncovering a hitherto unknown cross-talk between TNFα and βAR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

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G protein-coupled receptor-mediated activation of p110? by G?? is required for cellular transformation and invasiveness.

Publications

G protein-coupled receptor-mediated activation of p110β by Gβγ is required for cellular transformation and invasiveness.

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