Regulation of G beta gamma(i)-Dependent PLC-beta 3 Activity in Smooth Muscle: Inhibitory Phosphorylation of PLC-beta 3 by PKA and PKG and Stimulatory Phosphorylation of G alpha(i)-GTPase-Activating Protein RGS2 by PKG

摘要

In gastrointestinal smooth muscle, agonists that bind to G(i)-coupled receptors activate preferentially PLC-beta 3 via G beta gamma to stimulate phosphoinositide (PI) hydrolysis and generate inositol 1,4,5-trisphosphate (IP3) leading to IP3-dependent Ca2+ release and muscle contraction. In the present study, we identified the mechanism of inhibition of PLC-beta 3-dependent PI hydrolysis by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). Cyclopentyl adenosine (CPA), an adenosine A(1) receptor agonist, caused an increase in PI hydrolysis in a concentration-dependent fashion; stimulation was blocked by expression of the carboxyl-terminal sequence of GRK2(495-689), a G beta gamma-scavenging peptide, or G alpha(i) mini-gene but not G alpha(q) minigene. Isoproterenol and S-nitrosoglutathione (GSNO) induced phosphorylation of PLC-beta 3 and inhibited CPA-induced PI hydrolysis, Ca2+ release, and muscle contraction. The effect of isoproterenol on all three responses was inhibited by PKA inhibitor, myristoylated PKI, or AKAP inhibitor, Ht-31, whereas the effect of GSNO was selectively inhibited by PKG inhibitor, Rp-cGMPS. GSNO, but not isoproterenol, also phosphorylated G alpha(i)-GTPase-activating protein, RGS2, and enhanced association of G alpha(i3)-GTP and RGS2. The effect of GSNO on PI hydrolysis was partly reversed in cells (i) expressing constitutively active GTPase-resistant G alpha(i) mutant (Q204L), (ii) phosphorylation-site-deficient RGS2 mutant (S46A/S64A), or (iii) siRNA for RGS2. We conclude that PKA and PKG inhibit G beta gamma(i)-dependent PLC-beta 3 activity by direct phosphorylation of PLC-beta 3. PKG, but not PKA, also inhibits PI hydrolysis indirectly by a mechanism involving phosphorylation of RGS2 and its association with G alpha(i)-GTP. This allows RGS2 to accelerate G(alpha i)-GTPase activity, enhance G alpha beta gamma(i) trimer formation, and inhibit G beta gamma(i)-dependent PLC-beta 3 activity.