title: Inhibition of Galphaq-dependent PLC-beta1 activity by PKG and PKA is mediated by phosphorylation of RGS4 and GRK2.
abstract: In smooth muscle of the gut, G(q)-coupled receptor agonists activate preferentially PLC-beta1 to stimulate phosphoinositide (PI) hydrolysis and inositol 1,4,5-trisphosphate (IP(3)) generation and induce IP(3)-dependent Ca(2+) release. Inhibition of Ca(2+) mobilization by cAMP- (PKA) and cGMP-dependent (PKG) protein kinases reflects inhibition of PI hydrolysis by both kinases and PKG-specific inhibitory phosphorylation of IP(3) receptor type I. The mechanism of inhibition of PLC-beta1-dependent PI hydrolysis has not been established. Neither G(q) nor PLC-beta1 was directly phosphorylated by PKA or PKG in gastric smooth muscle cells. However, both kinases 1) phosphorylated regulator of G protein signaling 4 (RGS4) and induced its translocation from cytosol to plasma membrane, 2) enhanced ACh-stimulated association of RGS4 and Galpha(q).GTP and intrinsic Galpha(q).GTPase activity, and 3) inhibited ACh-stimulated PI hydrolysis. RGS4 phosphorylation and inhibition of PI hydrolysis were blocked by selective PKA and PKG inhibitors. Expression of RGS4(S52A), which lacks a PKA/PKG phosphorylation site, blocked the increase in GTPase activity and the decrease in PI hydrolysis induced by PKA and PKG. Blockade of PKA-dependent effects was only partial. Selective phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which contains a RGS domain, by PKA augmented ACh-stimulated GRK2:Galpha(q).GTP association; both effects were blocked in cells expressing GRK2(S685A), which lacks a PKA phosphorylation site. Inhibition of PI hydrolysis induced by PKA was partly blocked in cells expressing GRK2(S685A) and completely blocked in cells coexpressing GRK2(S685A) and RGS4(S52A) or Galpha(q)(G188S), a Galpha(q) mutant that binds GRK2 but not RGS4. The results demonstrate that inhibition of PLC-beta1-dependent PI hydrolysis by PKA is mediated via stimulatory phosphorylation of RGS4 and GRK2, leading to rapid inactivation of Galpha(q).GTP. PKG acts only via phosphorylation of RGS4.
authors.affiliation: Medical College of Virginia Campus, Virginia Commonwealth University, PO Box 980551, Richmond, VA 23298, USA.
authors.forename: Jiean
authors.initials: J
authors.lastname: Huang
authors.forename: Huiping
authors.initials: H
authors.lastname: Zhou
authors.forename: Sunila
authors.initials: S
authors.lastname: Mahavadi
authors.forename: Wimolpak
authors.initials: W
authors.lastname: Sriwai
authors.forename: Karnam S
authors.initials: KS
authors.lastname: Murthy
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D054799:Phospholipase C beta; D010766:Phosphorylation; D020710:RGS Proteins / Q000378:metabolism*; D011817:Rabbits; D013270:Stomach; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D010716:Phosphatidylinositols; D020710:RGS Proteins; D009599:Nitroprusside; C098799:RGS4 protein; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D020558:GTP Phosphohydrolases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D007545:Isoproterenol; D000109:Acetylcholine