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Cell Biochemistry and Biophysics

, Volume 70, Issue 2, pp 867–880 | Cite as

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

  • Ancy D. Nalli
  • Divya P. Kumar
  • Othman Al-Shboul
  • Sunila Mahavadi
  • John F. Kuemmerle
  • John R. Grider
  • Karnam S. Murthy
Original Paper

Abstract

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 Ca2+ 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, 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 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.

Keywords

Phospholipase-C Muscle relaxation Nitric oxide Muscle contraction G protein 

Notes

Acknowledgments

This study was supported by Grants from the National Institutes of Diabetes, and Digestive and Kidney Diseases DK28300 and DK15564 to Karnam S. Murthy.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Ancy D. Nalli
    • 1
  • Divya P. Kumar
    • 1
  • Othman Al-Shboul
    • 1
  • Sunila Mahavadi
    • 1
  • John F. Kuemmerle
    • 1
  • John R. Grider
    • 1
  • Karnam S. Murthy
    • 1
  1. 1.Department of Physiology, VCU Program in Enteric Neuromuscular SciencesVirginia Commonwealth UniversityRichmondUSA

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