Regulation of Gβγi-dependent PLC-β3 activity in smooth muscle: inhibitory phosphorylation of PLC-β3 by PKA and PKG and stimulatory phosphorylation of Gαi-GTPase-activating protein RGS2 by PKG.
In gastrointestinal smooth muscle, agonists that bind to Gi-coupled receptors activate preferentially PLC-β3 via Gβγ to stimulate phosphoinositide (PI) hydrolysis and generate inositol 1,4,5-trisphosphate (IP3) leading to IP3-dependent Ca(2+) release and muscle contraction. In the present study, we identified the mechanism of inhibition of PLC-β3-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 Gβγ-scavenging peptide, or Gαi minigene but not Gαq minigene. Isoproterenol and S-nitrosoglutathione (GSNO) induced phosphorylation of PLC-β3 and inhibited CPA-induced PI hydrolysis, Ca(2+) 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 Gαi-GTPase-activating protein, RGS2, and enhanced association of Gαi3-GTP and RGS2. The effect of GSNO on PI hydrolysis was partly reversed in cells (i) expressing constitutively active GTPase-resistant Gαi mutant (Q204L), (ii) phosphorylation-site-deficient RGS2 mutant (S46A/S64A), or (iii) siRNA for RGS2. We conclude that PKA and PKG inhibit Gβγi-dependent PLC-β3 activity by direct phosphorylation of PLC-β3. PKG, but not PKA, also inhibits PI hydrolysis indirectly by a mechanism involving phosphorylation of RGS2 and its association with Gαi-GTP. This allows RGS2 to accelerate Gαi-GTPase activity, enhance Gαβγi trimer formation, and inhibit Gβγi-dependent PLC-β3 activity.
Department of Physiology, VCU Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, VA, 23298-0711, USA.
Ancy D
AD
Nalli
Divya P
DP
Kumar
Othman
O
Al-Shboul
Sunila
S
Mahavadi
John F
JF
Kuemmerle
John R
JR
Grider
Karnam S
KS
Murthy
D000241:Adenosine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000378:metabolism* / Q000502:physiology; D010710:Phosphates / Q000378:metabolism; D054799:Phospholipase C beta / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D020710:RGS Proteins / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D012694:Serine / Q000378:metabolism
D010710:Phosphates; D020710:RGS Proteins; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D054799:Phospholipase C beta; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000241:Adenosine; D002118:Calcium