title: Inhibition of Gαi activity by Gβγ is mediated by PI 3-kinase-γ- and cSrc-dependent tyrosine phosphorylation of Gαi and recruitment of RGS12.
abstract: Others and we have characterized several Gβγ-dependent effectors in smooth muscle, including G protein-coupled receptor kinase 2 (GRK2), PLCβ3, and phosphatidylinositol (PI) 3-kinase-γ, and have identified various signaling targets downstream of PI 3-kinase-γ, including cSrc, integrin-linked kinase, and Rac1-Cdc42/p21-activated kinase/p38 MAP kinase. This study identified a novel mechanism whereby Gβγ acting via PI 3-kinase-γ and cSrc exerts an inhibitory influence on Gαi activity. The Gi2-coupled δ-opioid receptor agonist d-penicillamine (2,5)-enkephalin (DPDPE) activated cSrc, stimulated tyrosine phosphorylation of Gαi2, and induced regulator of G protein signaling 12 (RGS12) association; all three events were blocked by PI 3-kinase (LY294002) and cSrc (PP2) inhibitors and by expression of the COOH-terminal sequence of GRK2-(495-689), a Gβγ-scavenging peptide. Inhibition of forskolin-stimulated cAMP and muscle relaxation by DPDPE was augmented by PP2, LY294002, and a selective PI 3-kinase-γ inhibitor, AS-605420. Expression of tyrosine-deficient (Y69F, Y231F, or Y321F) Gαi2 mutant or knockdown of RGS12 blocked Gαi2 phosphorylation and Gαi2-RGS12 association and caused greater inhibition of cAMP. Parallel studies using somatostatin, cyclopentyl adenosine, or ACh to activate, respectively, Gi1-coupled somatostatin (sstr3) receptors, and Gi3-coupled adenosine A1 or muscarinic m2 receptors elicited cSrc activation, Gαi1 or Gαi3 phosphorylation, Gαi1-RGS12 or Gαi3-RGS12 association, and inhibition of cAMP. Inhibition of cAMP and muscle relaxation was greatly increased by AS-605240 and PP2. The results demonstrate that Gβγ-dependent tyrosine phosphorylation of Gαi1/2/3 by cSrc facilitated recruitment of RGS12, a Gαi-specific RGS protein with a unique phosphotyrosine-binding domain, resulting in rapid deactivation of Gαi and facilitation of smooth muscle relaxation.
authors.affiliation: Department of Physiology and Biophysics, Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Jiean
authors.initials: J
authors.lastname: Huang
authors.forename: Ancy D
authors.initials: AD
authors.lastname: Nalli
authors.forename: Sunila
authors.initials: S
authors.lastname: Mahavadi
authors.forename: Divya P
authors.initials: DP
authors.lastname: Kumar
authors.forename: Karnam S
authors.initials: KS
authors.lastname: Murthy
mesh_terms: D058907:Adenosine A1 Receptor Agonists / Q000494:pharmacology; D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D025461:Feedback, Physiological; D051960:GTP-Binding Protein alpha Subunit, Gi2 / Q000235:genetics / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D018721:Muscarinic Agonists / Q000494:pharmacology; D009126:Muscle Relaxation / Q000187:drug effects; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011817:Rabbits; D043682:Receptor, Adenosine A1 / Q000187:drug effects / Q000378:metabolism; D043585:Receptor, Muscarinic M2 / Q000819:agonists / Q000378:metabolism; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000378:metabolism; D017481:Receptors, Somatostatin / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction; D013270:Stomach / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D014162:Transfection; D014443:Tyrosine
chemical_list: D058907:Adenosine A1 Receptor Agonists; D000701:Analgesics, Opioid; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D018721:Muscarinic Agonists; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D020710:RGS Proteins; D043682:Receptor, Adenosine A1; D043585:Receptor, Muscarinic M2; D017465:Receptors, Opioid, delta; D017481:Receptors, Somatostatin; C103631:somatostatin receptor 3; D014443:Tyrosine; D000242:Cyclic AMP; D058539:Phosphatidylinositol 3-Kinase; D016392:Proto-Oncogene Proteins pp60(c-src); D051960:GTP-Binding Protein alpha Subunit, Gi2
keywords: G protein activation; Gαi proteins; RGS proteins; smooth muscle; tyrosine phosphorylation