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. MurthyEmail author
Original Paper


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.


Phospholipase-C Muscle relaxation Nitric oxide Muscle contraction G protein 



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


  1. 1.
    Somlyo, A. V., Khromov, A. S., Webb, M. R., Ferenzi, M. A., Trenthanm, D. R., He, Z. H., et al. (2004). Smooth muscle myosin: Regulation and properties. Philosophical Transactions of the Royal Society B: Biological Sciences, 29, 1921–1930.Google Scholar
  2. 2.
    Kamm, K. E., & Stull, J. T. (2001). Dedicated myosin light kinases with diverse cellular functions. Journal of Biological Chemistry, 276, 4527–4530.PubMedCrossRefGoogle Scholar
  3. 3.
    de Godoy, M. A., & Rattan, S. (2011). Role of rho kinase in the functional and dysfunctional tonic smooth muscle. Trends in Pharmacological Sciences, 32, 384–393.PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Murthy, K. S. (2006). Signaling for contraction and relaxation in smooth muscle of the gut. Annual Review of Physiology, 68, 345–374.PubMedCrossRefGoogle Scholar
  5. 5.
    Murthy, K. S., & Makhlouf, G. M. (1995). Functional characterization of phosphoinositide-specific phospholipase C-β1 and -β3 in intestinal smooth muscle. American Journal of Physiology, 269, C969–C978.PubMedGoogle Scholar
  6. 6.
    Murthy, K. S., & Makhlouf, G. M. (1998). Coexpression of ligand-gated P2X and G protein-coupled P2Y receptors in smooth muscle. Preferential activation of P2Y receptors coupled to phospholipase C (PLC)-beta1 via Gα/11 and to PLC-β3 via Gβγi3. Journal of Biological Chemistry, 273, 4695–4704.PubMedCrossRefGoogle Scholar
  7. 7.
    Murthy, K. S., & Makhlouf, G. M. (1995). Adenosine A1 receptor-mediated activation of phospholipase C-beta 3 in intestinal muscle: dual requirement for alpha and beta gamma subunits of Gi3. Molecular Pharmacology, 47, 1172–1179.PubMedGoogle Scholar
  8. 8.
    Murthy, K. S., Coy, D. H., & Makhlouf, G. M. (1996). Somatostatin receptor-mediated signaling in smooth muscle. Activation of phospholipase C-beta3 by Gbetagamma and inhibition of adenylyl cyclase by Galphai1 and Galphao. Journal of Biological Chemistry, 271, 23458–23463.PubMedCrossRefGoogle Scholar
  9. 9.
    Murthy, K. S., & Makhlouf, G. M. (1996). Opioid µ, δ and κ receptor-induced activation of phospholipase C-β3 and inhibition of adenylyl cyclase is mediated by Gi2 and Go in smooth muscle. Molecular Pharmacology, 50, 870–877.PubMedGoogle Scholar
  10. 10.
    Berstein, G., Blank, J. L., Jhon, D. Y., Exton, J. H., Rhee, S. G., & Ross, E. M. (1992). Phospholipase C-β1 is a GTPase-activating protein for Gq/11, its physiologic regulator. Cell, 70, 411–418.PubMedCrossRefGoogle Scholar
  11. 11.
    Chidiac, P., & Ross, E. M. (1999). Phospholipase C-β1 directly accelerates GTP hydrolysis by Galphaq and acceleration is inhibited by Gbeta gamma subunits. Journal of Biological Chemistry, 274, 19639–19643.PubMedCrossRefGoogle Scholar
  12. 12.
    Berman, B., & Gilman, A. G. (1998). Mammalian RGS proteins: Barbarians at the gate. Journal of Biological Chemistry, 273, 1269–1272.PubMedCrossRefGoogle Scholar
  13. 13.
    Hollinger, S., & Hepler, J. R. (2002). Cellular regulation of RGS proteins: Modulators and integrators of G protein signaling. Pharmacological Reviews, 54, 527–559.PubMedCrossRefGoogle Scholar
  14. 14.
    Kach, J., Sethakorn, N., & Dulin, N. O. (2012). A finer tuning of G-protein signaling through regulated control of RGS proteins. American Journal of Physiology Heart and Circulatory Physiology, 303, H19–H35.PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Xu, X., Zeng, W., Popov, S., Berman, D. M., Davignon, I., Yu, K., et al. (1999). RGS proteins determine signaling specificity of Gq-coupled receptors. Journal of Biological Chemistry, 274, 3549–3556.PubMedCrossRefGoogle Scholar
  16. 16.
    Litosch, I. (2002). Novel mechanisms for feedback regulation of phospholipase C-beta activity. IUBMB Life, 54, 253–260.PubMedCrossRefGoogle Scholar
  17. 17.
    Rebecchi, M. J., & Pentyala, S. N. (2000). Structure, function, and control of phosphoinositide-specific phospholipase C. Physiological Reviews, 80, 1291–1335.PubMedGoogle Scholar
  18. 18.
    Rhee, S. G. (2001). Regulation of phosphoinositide-specific phospholipase C. Annual Review of Biochemistry, 70, 281–312.PubMedCrossRefGoogle Scholar
  19. 19.
    Liu, M., & Simon, M. I. (1996). Regulation by cAMP-dependent protein kinase of a G protein-mediated phospholipase C. Nature, 382, 83–87.PubMedCrossRefGoogle Scholar
  20. 20.
    Ali, H., Fisher, I., Haribabu, B., Richardson, R. M., & Snyderman, R. (1997). Role of phospholipase Cbeta3 phosphorylation in the desensitization of cellular responses to platelet-activating factor. Journal of Biological Chemistry, 272, 11706–11709.PubMedCrossRefGoogle Scholar
  21. 21.
    Yue, C., Dodge, K. L., Weber, G., & Sanborn, B. M. (1998). Phosphorylation of serine 1105 by protein kinase A inhibits phospholipase Cbeta3 stimulation by Galphaq. Journal of Biological Chemistry, 273, 18023–18027.PubMedCrossRefGoogle Scholar
  22. 22.
    Yue, C., Ku, C. Y., Liu, M., Simon, M. I., & Sanborn, B. M. (2000). Molecular mechanism of the inhibition of phospholipase C beta 3 by protein kinase C. Journal of Biological Chemistry, 275, 30220–30225.PubMedCrossRefGoogle Scholar
  23. 23.
    Ryu, S. H., Kim, U. H., Wahl, M. I., Brown, A. B., Carpenter, G., Huang, K. P., et al. (1990). Feedback regulation of phospholipase C-beta by protein kinase C. Journal of Biological Chemistry, 265, 17941–17945.PubMedGoogle Scholar
  24. 24.
    Xu, A., Suh, P. G., Marmy-Conus, N., Pearson, R. B., Seok, O. Y., Cocco, L., et al. (2001). Phosphorylation of nuclear phospholipase C beta1 by extracellular signal-regulated kinase mediates the mitogenic action of insulin-like growth factor I. Molecular and Cellular Biology, 21, 2981–2990.PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Ishii, M., Fujita, S., Yamada, M., Hosaka, Y., & Kurachi, Y. (2005). Phosphatidylinositol 3,4,5-trisphosphate and Ca2+/calmodulin competitively bind to the regulators of G-protein-signalling (RGS) domain of RGS4 and reciprocally regulate its action. Biochemical Journal, 385, 65–73.PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Tokudome, T., Kishimoto, I., Horio, T., Arai, Y., Schwenke, D. O., Hino, J., et al. (2008). Regulator of G-protein signaling subtype 4 mediates antihypertrophic effect of locally secreted natriuretic peptides in the heart. Circulation, 117, 2329–2339.PubMedCrossRefGoogle Scholar
  27. 27.
    Huang, J., Zhou, H., Mahavadi, S., Sriwai, W., & Murthy, K. S. (2007). Inhibition of Galphaq-dependent PLC-beta1 activity by PKG and PKA is mediated by phosphorylation of RGS4 and GRK2. American Journal of Physiology: Cell Physiology, 292, C200–C208.PubMedCrossRefGoogle Scholar
  28. 28.
    Chen, C., Wang, H., Fong, C. W., & Lin, S. C. (2001). Multiple phosphorylation sites in RGS16 differentially modulate its GAP activity. FEBS Letters, 504, 16–22.PubMedCrossRefGoogle Scholar
  29. 29.
    Derrien, A., & Druey, K. M. (2001). RGS16 function is regulated by epidermal growth factor receptor-mediated tyrosine phosphorylation. Journal of Biological Chemistry, 276, 48532–48538.PubMedGoogle Scholar
  30. 30.
    Derrien, A., Zheng, B., Osterhout, J. L., Ma, Y. C., Milligan, G., Farquhar, M. G., et al. (2003). Src-mediated RGS16 tyrosine phosphorylation promotes RGS16 stability. Journal of Biological Chemistry, 278, 16107–16116.PubMedCrossRefGoogle Scholar
  31. 31.
    Osei-Owusu, P., Sun, X., Drenan, R. M., Steinberg, T. H., & Blumer, K. J. (2007). Regulation of RGS2 and second messenger signaling in vascular smooth muscle cells by cGMP-dependent protein kinase. Journal of Biological Chemistry, 282, 31656–31665.PubMedCrossRefGoogle Scholar
  32. 32.
    Tang, K. M., Wang, G. R., Lu, P., Karas, R. H., Aronovitz, M., Heximer, S. P., et al. (2003). Regulator of G protein signaling-2 mediate vascular smooth muscle relaxation and blood pressure. Nature Medicine, 9, 1506–1512.PubMedCrossRefGoogle Scholar
  33. 33.
    Sun, X., Kaltenbronn, K. M., Steinberg, T. H., & Blumer, K. J. (2005). RGS2 is a mediator of nitric oxide action on blood pressure and vasoconstrictor signaling. Molecular Pharmacology, 67, 631–639.PubMedCrossRefGoogle Scholar
  34. 34.
    Cunningham, M. L., Waldo, G. L., Hollinger, S., Hepler, J. R., & Harden, T. K. (2001). Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of Galpha 11 signaling. Journal of Biological Chemistry, 276, 5438–5444.PubMedCrossRefGoogle Scholar
  35. 35.
    Obst, M., Tank, J., Plehm, R., Blumer, K. J., Diedrich, A., Jordan, J., et al. (2006). NO-dependent blood pressure regulation in RGS2-deficient mice. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology, 290, R1012–R1019.PubMedPubMedCentralGoogle Scholar
  36. 36.
    Gross, V., Tank, J., Obst, M., Plehm, R., Blumer, K. J., Diedrich, A., et al. (2005). Autonomic nervous system and blood pressure regulation in RGS2-deficient mice. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology, 288, R1134–R1142.PubMedCrossRefGoogle Scholar
  37. 37.
    Heximer, S. P., Knutsen, R. H., Sun, X., Kaltenbronn, K. M., Rhee, M. H., Peng, N., et al. (2003). Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice. Journal of Clinical Investigation, 111, 1259.PubMedCrossRefGoogle Scholar
  38. 38.
    Zhou, H., & Murthy, K. S. (2004). Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2. American Journal of Physiology: Cell Physiology, 286, C1130–C1138.PubMedCrossRefGoogle Scholar
  39. 39.
    Sriwai, W., Mahavadi, S., Al-Shboul, O., Grider, J. R., & Murthy, K. S. (2013). Distinctive G protein-dependent signaling by protease-activated receptor 2 (PAR2) in smooth muscle: feedback inhibition of RhoA by cAMP-independent PKA. PLoS ONE, 8, e66743.PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Gilchrist, A., Bunemann, M., Li, A., Hosey, M. M., & Hamm, H. E. (1999). A dominant-negative strategy for studying roles of G proteins in vivo. Journal of Biological Chemistry, 274, 6610–6616.PubMedCrossRefGoogle Scholar
  41. 41.
    Gilchrist, A., Vanhauwe, J., Li, A., Thoma, T. T., Voyno-Yasenetskaya, T., Hosey, M. M., et al. (2001). Gα minigenes expressing C-terminal peptides serve a specific inhibitors of thrombin-mediated endothelial activation. Journal of Biological Chemistry, 276, 25672–25679.PubMedCrossRefGoogle Scholar
  42. 42.
    Berridge, M. J., Downes, C. P., & Hanley, M. R. (1982). Lithium amplifies agonist dependent phosphatidylinositol responses in brain and salivary glands. Biochemical Journal, 206, 587–595.PubMedPubMedCentralGoogle Scholar
  43. 43.
    Poggioli, J., & Putney, J. W, Jr. (1982). Net calcium fluxes in rat parotid acinar cells: Evidence for a hormone-sensitive calcium pool in or near the plasma membrane. Pflugers Archiv: European Journal of Physiology, 392, 239–243.PubMedCrossRefGoogle Scholar
  44. 44.
    Murthy, K. S., Severi, C., Grider, J. R., & Makhlouf, G. M. (1993). Inhibition of IP3 and IP3-dependent Ca2+ mobilization by cyclic nucleotides in isolated gastric muscle cells. American Journal of Physiology, 264, G967–G974.PubMedGoogle Scholar
  45. 45.
    Murthy, K. S., & Makhlouf, G. M. (1995). Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cells. American Journal of Physiology, 268, C171–C180.PubMedGoogle Scholar
  46. 46.
    Welch, E. J., Jones, B. W., & Scott, J. D. (2010). Networking with AKAPs: Context-dependent regulation of anchored enzymes. Molecular Intervention, 10, 86–97.CrossRefGoogle Scholar
  47. 47.
    Hausken, Z. E., Dell’Acqua, M. L., Coghlan, V. M., & Scott, J. D. (1996). Mutational analysis of the A-kinase anchoring protein (AKAP-binding site on RII: Classification of side chain determinants for anchoring and isoform selective association with AKAPs. Journal of Biological Chemistry, 271, 29016–29022.PubMedCrossRefGoogle Scholar
  48. 48.
    Ogier-Denis, E., Petiot, A., Bauvy, C., & Codogno, P. (1997). Control of the expression and activity of the Galpha-interacting protein (GAIP) in human intestinal cells. Journal of Biological Chemistry, 272, 24599–24603.PubMedCrossRefGoogle Scholar
  49. 49.
    Komalavilas, P., & Lincoln, T. M. (1996). Phosphorylation of the inositol 1,4,5-trisphosphate receptor. cGMP-dependent protein kinase mediates cAMP and cGMP dependent phosphorylation in intact rat aorta. Journal of Biological Chemistry, 271, 21933–21938.PubMedCrossRefGoogle Scholar
  50. 50.
    Zhou, H., & Murthy, K. S. (2003). Selective phosphorylation of IP3 receptor type I (IP3RI) in vivo by cGMP-dependent protein kinase in gastric smooth muscle. American Journal of Physiology: Gastrointestinal and Liver Physiology, 284, G221–G230.PubMedGoogle Scholar
  51. 51.
    Coyler, J. (1998). Phosphorylation states of phospholamban. Annals of the New York Academy of Sciences, 853, 79–91.CrossRefGoogle Scholar
  52. 52.
    Lounsbury, K. M., Schlegel, B., Poncz, M., Brass, L. F., & Manning, D. R. (1993). Analysis of Gz alpha by site-directed mutagenesis. Sites and specificity of protein kinase C-dependent phosphorylation. Journal of Biological Chemistry, 268, 3494–3498.PubMedGoogle Scholar
  53. 53.
    Jhon, D. Y., Lee, H. H., Park, D., Lee, C. W., Lee, K. H., Yoo, O. J., et al. (1993). Cloning, sequencing, purification and Gq-dependent activation of phospholipase C-β3. Journal of Biological Chemistry, 268, 6654–6661.PubMedGoogle Scholar
  54. 54.
    Xiong, Z., & Sperelakis, N. (1995). Regulation of L-type calcium channels of vascular smooth muscle cells. Journal of Molecular and Cellular Cardiology, 27, 75–91.PubMedCrossRefGoogle Scholar
  55. 55.
    Fukao, M., Mason, H. S., Britton, F. C., Kenyon, J. L., Horowitz, B., & Keef, K. D. (1999). Cyclic GMP-dependent protein kinase activates cloned BKCa channels expressed in mammalian cells by direct phosphorylation at serine 1072. Journal of Biological Chemistry, 274, 10927–10935.PubMedCrossRefGoogle Scholar
  56. 56.
    Ku, C. Y., & Sanborn, B. M. (2002). Progesterone prevents the pregnancy-related decline in protein kinase A association with rat myometrial plasma membrane and A-kinase anchoring protein. Biology of Reproduction, 67, 605–609.PubMedCrossRefGoogle Scholar
  57. 57.
    Kehrl, J. H., & Sinnarajah, S. (2002). RGS2: A multifunctional regulator of G-protein signaling. International Journal of Biochemistry & Cell Biology, 34, 432–438.CrossRefGoogle Scholar
  58. 58.
    Heximer, S. P., Watson, N., Linder, M. E., Blumer, K. J., & Hepler, J. R. (1997). RGS2/G0S8 is a selective inhibitor of Gqalpha function. Proceedings of the National Academy of Sciences of the United States of America, 94, 14389–14393.PubMedCrossRefPubMedCentralGoogle Scholar
  59. 59.
    Han, J., Mark, M. D., Li, X., Xie, M., Waka, S., Rettig, J., et al. (2006). RGS2 determines short-term synaptic plasticity in hippocampal neurons by regulating Gi/o-mediated inhibition of presynaptic Ca2+ channels. Neuron, 51, 575–586.PubMedCrossRefGoogle Scholar
  60. 60.
    Anger, T., Klintworth, N., Stumpf, C., Daniel, W. G., Mende, U., & Garlichs, C. D. (2007). RGS protein specificity towards Gq- and Gi/o-mediated ERK 1/2 and Akt activation, in vitro. Journal of Biochemistry and Molecular Biology, 40, 899–910.PubMedCrossRefGoogle Scholar
  61. 61.
    Roy, A. A., Nunn, C., Ming, H., Zou, M. X., Penninger, J., Kirshenbaum, L. A., et al. (2006). Up-regulation of endogenous RGS2 mediates cross-desensitization between Gs and Gq signaling in osteoblasts. Journal of Biological Chemistry, 281, 32684–32693.PubMedCrossRefGoogle Scholar

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
    Email author
  1. 1.Department of Physiology, VCU Program in Enteric Neuromuscular SciencesVirginia Commonwealth UniversityRichmondUSA

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