Huang J, Nalli AD, Mahavadi S, Kumar DP, Murthy KS. Inhibition of G␣ i activity by G␥ is mediated by PI 3-kinase-␥-and cSrc-dependent tyrosine phosphorylation of G␣i and recruitment of RGS12. Am J Physiol Gastrointest Liver Physiol 306: G802-G810, 2014. First published February 27, 2014 doi:10.1152/ajpgi.00440.2013.-Others and we have characterized several G␥-dependent effectors in smooth muscle, including G protein-coupled receptor kinase 2 (GRK2), PLC 3, and phosphatidylinositol (PI) 3-kinase-␥, and have identified various signaling targets downstream of PI 3-kinase-␥, including cSrc, integrin-linked kinase, and Rac1-Cdc42/p21-activated kinase/p38 MAP kinase. This study identified a novel mechanism whereby G␥ acting via PI 3-kinase-␥ and cSrc exerts an inhibitory influence on G␣ i activity. The Gi2-coupled ␦-opioid receptor agonist D-penicillamine (2,5)-enkephalin (DPDPE) activated cSrc, stimulated tyrosine phosphorylation of G␣ 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 G␥-scavenging peptide. Inhibition of forskolinstimulated cAMP and muscle relaxation by DPDPE was augmented by PP2, LY294002, and a selective PI 3-kinase-␥ inhibitor, AS-605420. Expression of tyrosine-deficient (Y69F, Y231F, or Y321F) G␣i2 mutant or knockdown of RGS12 blocked G␣i2 phosphorylation and G␣i2-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, G␣i1 or G␣i3 phosphorylation, G␣i1-RGS12 or G␣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 G␥-dependent tyrosine phosphorylation of G␣i1/2/3 by cSrc facilitated recruitment of RGS12, a G␣i-specific RGS protein with a unique phosphotyrosine-binding domain, resulting in rapid deactivation of G␣i and facilitation of smooth muscle relaxation.