Inhibition of Gαq-dependent PLC-β1 activity by PKG and PKA is mediated by phosphorylation of RGS4 and GRK2

In smooth muscle of the gut, G(q)-coupled receptor agonists activate preferentially PLC-beta 1 to stimulate phosphoinositide (PI) hydrolysis and inositol 1,4,5-trisphosphate (IP3) generation and induce IP3-dependent Ca2+ release. Inhibition of Ca2+ mobilization by cAMP- (PKA) and cGMP-dependent (PKG) protein kinases reflects inhibition of PI hydrolysis by both kinases and PKG-specific inhibitory phosphorylation of IP3 receptor type I. The mechanism of inhibition of PLC-beta 1-dependent PI hydrolysis has not been established. Neither G(q) nor PLC-beta 1 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 G alpha(q)center dot GTP and intrinsic G alpha(q)center dot 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:G alpha q center dot 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 G alpha(q)(G188S), a G alpha(q) mutant that binds GRK2 but not RGS4. The results demonstrate that inhibition of PLC-beta 1-dependent PI hydrolysis by PKA is mediated via stimulatory phosphorylation of RGS4 and GRK2, leading to rapid inactivation of G alpha(q)center dot GTP. PKG acts only via phosphorylation of RGS4.