Inhibition of Galpha_i activity by Gbetagamma is mediated by PI 3-kinase-7- and cSrc-dependent tyrosine phosphorylation of Galpha_i and recruitment of RGS12

摘要

Others and we have characterized several Gbetagamma-dependent effectors in smooth muscle, including G protein-coupled receptor kinase 2 (GRK2), PLCbeta_3, and phosphatidyl-inositol (PI) 3-kinase-gamma, and have identified various signaling targets downstream of PI 3-kinase-gamma, including cSrc, integrin-linked kinase, and Racl-Cdc42/p21-activated kinase/p38 MAP kinase. This study identified a novel mechanism whereby Gbetagamma acting via PI 3-kinase-7 and cSrc exerts an inhibitory influence on Galpha_i activity. The G_(i2)-coupled 8-opioid receptor agonist D-penicillamine (2,5)-enkephalin (DPDPE) activated cSrc, stimulated tyrosine phosphorylation of Galpha_(i2), 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 Gbetagamma-scavenging peptide. Inhibition of forskolin-stimulated cAMP and muscle relaxation by DPDPE was augmented by PP2, LY294002, and a selective PI 3-kinase-gamma inhibitor, AS-605420. Expression of tyrosine-deficient (Y69F, Y231F, or Y321F) Galpha_(i2) mutant or knockdown of RGS12 blocked Galpha_(i2) phosphorylation and Galpha_(i2)-RGS12 association and caused greater inhibition of cAMP. Parallel studies using somatostatin, cyclopentyl adenosine, or ACh to activate, respectively, G_(i1)-coupled somatostatin (sstr_3) receptors, and G_(i3)-coupled adenosine A_1 or muscarinic m_2 receptors elicited cSrc activation, Galpha_(i1) or Galpha_(i3) phosphorylation, Galpha_(i1)-RGS12 or Galpha_(i3)-RGS12 association, and inhibition of cAMP. Inhibition of cAMP and muscle relaxation was greatly increased by AS-605240 and PP2. The results demonstrate that Gbetagamma-dependent tyrosine phosphorylation of Galpha_(i1/2/3) by cSrc facilitated recruitment of RGS12, a Galpha_i-specific RGS protein with a unique phosphotyrosine-binding domain, resulting in rapid deactivation of Got; and facilitation of smooth muscle relaxation.