Abstract:  

In gastrointestinal smooth muscle, agonists that bind to Gi-coupled receptors activate preferentially PLC-b3 via Gbc 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-b3-dependent PI hydrolysis by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). Cyclopentyl adenosine (CPA), an adenosine A1 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 Gbc-scavenging peptide, or Gai minigene but not Gaq minigene. Isoproterenol and S-nitrosoglutathione (GSNO) induced phosphorylation of PLC-b3 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 Gai-GTPase-activating protein, RGS2, and enhanced association of Gai3-GTP and RGS2. The effect of GSNO on PI hydrolysis was partly reversed in cells (i) expressing constitutively active GTPase-resistant Gai mutant (Q204L), (ii) phosphorylation-site-deficient RGS2 mutant (S46A/S64A), or (iii) siRNA for RGS2. We conclude that PKA and PKG inhibit Gbci-dependent PLC-b3 activity by direct phosphorylation of PLC-b3. PKG, but not PKA, also inhibits PI hydrolysis indirectly by a mechanism involving phosphorylation of RGS2 and its association with Gai-GTP. This allows RGS2 to accelerate Gai- GTPase activity, enhance Gabci trimer formation, and inhibit Gbci-dependent PLC-b3 activity.