Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Sep 1;326(Pt 2):545–551. doi: 10.1042/bj3260545

Co-transfection with protein kinase D confers phorbol-ester-mediated inhibition on glucagon-stimulated cAMP accumulation in COS cells transfected to overexpress glucagon receptors.

E S Tobias 1, E Rozengurt 1, J M Connell 1, M D Houslay 1
PMCID: PMC1218703  PMID: 9291130

Abstract

Glucagon elicited a profound increase in the intracellular cAMP concentration of COS-7 cells which had been transiently transfected with a cDNA encoding the rat glucagon receptor and under conditions where cAMP phosphodiesterase activity was fully inhibited. This was achieved in a dose-dependent fashion with an EC50 of 1.8+/-0.4 nM glucagon. In contrast with previous observations made using hepatocytes [Heyworth, Whetton, Kinsella and Houslay (1984) FEBS Lett. 170, 38-42], treatment of transfected COS-7 cells with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels. PMA-mediated inhibition was not conferred by treatment with okadaic acid, nor by co-transfecting cells with cDNAs encoding various protein kinase C isoforms (PKC-alpha, PKC-betaII and PKC-epsilon) or with the PMA-activated G-protein-receptor kinases GRK2 and GRK3. In contrast, PMA induced the marked inhibition of glucagon-stimulated cAMP production in COS-7 cells that had been co-transfected with a cDNA encoding protein kinase D (PKD). Such inhibition was not due to an action on the catalytic unit of adenylate cyclase, as forskolin-stimulated cAMP production was unchanged by PMA treatment of COS cells that had been co-transfected with both the glucagon receptor and PKD. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS cells, whereas transcripts for GRK3 were not found in either cell type. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase.

Full Text

The Full Text of this article is available as a PDF (359.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beavo J. A., Reifsnyder D. H. Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors. Trends Pharmacol Sci. 1990 Apr;11(4):150–155. doi: 10.1016/0165-6147(90)90066-H. [DOI] [PubMed] [Google Scholar]
  2. Benovic J. L., DeBlasi A., Stone W. C., Caron M. G., Lefkowitz R. J. Beta-adrenergic receptor kinase: primary structure delineates a multigene family. Science. 1989 Oct 13;246(4927):235–240. doi: 10.1126/science.2552582. [DOI] [PubMed] [Google Scholar]
  3. Benovic J. L., Onorato J. J., Arriza J. L., Stone W. C., Lohse M., Jenkins N. A., Gilbert D. J., Copeland N. G., Caron M. G., Lefkowitz R. J. Cloning, expression, and chromosomal localization of beta-adrenergic receptor kinase 2. A new member of the receptor kinase family. J Biol Chem. 1991 Aug 15;266(23):14939–14946. [PubMed] [Google Scholar]
  4. Berry M. N., Friend D. S. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 1969 Dec;43(3):506–520. doi: 10.1083/jcb.43.3.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birnbaumer L., Abramowitz J., Brown A. M. Receptor-effector coupling by G proteins. Biochim Biophys Acta. 1990 May 7;1031(2):163–224. doi: 10.1016/0304-4157(90)90007-y. [DOI] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Brown B. L., Ekins R. P., Albano J. D. Saturation assay for cyclic AMP using endogenous binding protein. Adv Cyclic Nucleotide Res. 1972;2:25–40. [PubMed] [Google Scholar]
  8. Burcelin R., Li J., Charron M. J. Cloning and sequence analysis of the murine glucagon receptor-encoding gene. Gene. 1995 Oct 27;164(2):305–310. doi: 10.1016/0378-1119(95)00472-i. [DOI] [PubMed] [Google Scholar]
  9. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  10. Chuang T. T., LeVine H., 3rd, De Blasi A. Phosphorylation and activation of beta-adrenergic receptor kinase by protein kinase C. J Biol Chem. 1995 Aug 4;270(31):18660–18665. doi: 10.1074/jbc.270.31.18660. [DOI] [PubMed] [Google Scholar]
  11. Cohen P., Holmes C. F., Tsukitani Y. Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci. 1990 Mar;15(3):98–102. doi: 10.1016/0968-0004(90)90192-e. [DOI] [PubMed] [Google Scholar]
  12. García-Sáinz J. A., Mendlovic F., Martínez-Olmedo M. A. Effects of phorbol esters on alpha 1-adrenergic-mediated and glucagon-mediated actions in isolated rat hepatocytes. Biochem J. 1985 May 15;228(1):277–280. doi: 10.1042/bj2280277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gibson T. J., Hyvönen M., Musacchio A., Saraste M., Birney E. PH domain: the first anniversary. Trends Biochem Sci. 1994 Sep;19(9):349–353. doi: 10.1016/0968-0004(94)90108-2. [DOI] [PubMed] [Google Scholar]
  14. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  15. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  16. Heyworth C. M., Houslay M. D. Challenge of hepatocytes by glucagon triggers a rapid modulation of adenylate cyclase activity in isolated membranes. Biochem J. 1983 Jul 15;214(1):93–98. doi: 10.1042/bj2140093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Heyworth C. M., Wallace A. V., Houslay M. D. Insulin and glucagon regulate the activation of two distinct membrane-bound cyclic AMP phosphodiesterases in hepatocytes. Biochem J. 1983 Jul 15;214(1):99–110. doi: 10.1042/bj2140099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heyworth C. M., Whetton A. D., Kinsella A. R., Houslay M. D. The phorbol ester, TPA inhibits glucagon-stimulated adenylate cyclase activity. FEBS Lett. 1984 May 7;170(1):38–42. doi: 10.1016/0014-5793(84)81364-2. [DOI] [PubMed] [Google Scholar]
  19. Heyworth C. M., Wilson S. P., Gawler D. J., Houslay M. D. The phorbol ester TPA prevents the expression of both glucagon desensitisation and the glucagon-mediated block of insulin stimulation of the peripheral plasma membrane cyclic AMP phosphodiesterase in rat hepatocytes. FEBS Lett. 1985 Aug 5;187(2):196–200. doi: 10.1016/0014-5793(85)81241-2. [DOI] [PubMed] [Google Scholar]
  20. Hoey M., Houslay M. D. Identification and selective inhibition of four distinct soluble forms of cyclic nucleotide phosphodiesterase activity from kidney. Biochem Pharmacol. 1990 Jul 15;40(2):193–202. doi: 10.1016/0006-2952(90)90678-e. [DOI] [PubMed] [Google Scholar]
  21. Houslay M. D. 'Crosstalk': a pivotal role for protein kinase C in modulating relationships between signal transduction pathways. Eur J Biochem. 1991 Jan 1;195(1):9–27. doi: 10.1111/j.1432-1033.1991.tb15671.x. [DOI] [PubMed] [Google Scholar]
  22. Houslay M. D. Insulin, glucagon and the receptor-mediated control of cyclic AMP concentrations in liver. Twenty-second Colworth medal lecture. Biochem Soc Trans. 1986 Apr;14(2):183–193. doi: 10.1042/bst0140183. [DOI] [PubMed] [Google Scholar]
  23. Hug H., Sarre T. F. Protein kinase C isoenzymes: divergence in signal transduction? Biochem J. 1993 Apr 15;291(Pt 2):329–343. doi: 10.1042/bj2910329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell. 1995 Jan 27;80(2):225–236. doi: 10.1016/0092-8674(95)90405-0. [DOI] [PubMed] [Google Scholar]
  25. Jelinek L. J., Lok S., Rosenberg G. B., Smith R. A., Grant F. J., Biggs S., Bensch P. A., Kuijper J. L., Sheppard P. O., Sprecher C. A. Expression cloning and signaling properties of the rat glucagon receptor. Science. 1993 Mar 12;259(5101):1614–1616. doi: 10.1126/science.8384375. [DOI] [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Livingstone C., McLellan A. R., McGregor M. A., Wilson A., Connell J. M., Small M., Milligan G., Paterson K. R., Houslay M. D. Altered G-protein expression and adenylate cyclase activity in platelets of non-insulin-dependent diabetic (NIDDM) male subjects. Biochim Biophys Acta. 1991 Feb 22;1096(2):127–133. doi: 10.1016/0925-4439(91)90050-j. [DOI] [PubMed] [Google Scholar]
  28. MacNeil D. J., Occi J. L., Hey P. J., Strader C. D., Graziano M. P. Cloning and expression of a human glucagon receptor. Biochem Biophys Res Commun. 1994 Jan 14;198(1):328–334. doi: 10.1006/bbrc.1994.1046. [DOI] [PubMed] [Google Scholar]
  29. Marchmont R. J., Houslay M. D. Insulin controls the cyclic AMP-dependent phosphorylation of integral and peripheral proteins associated with the rat liver plasma membrane. FEBS Lett. 1980 Aug 25;118(1):18–24. doi: 10.1016/0014-5793(80)81209-9. [DOI] [PubMed] [Google Scholar]
  30. Marcus R., Aurbach G. D. Bioassay of parathyroid hormone in vitro with a stable preparation of adenyl cyclase from rat kidney. Endocrinology. 1969 Nov;85(5):801–810. doi: 10.1210/endo-85-5-801. [DOI] [PubMed] [Google Scholar]
  31. McPhee I., Pooley L., Lobban M., Bolger G., Houslay M. D. Identification, characterization and regional distribution in brain of RPDE-6 (RNPDE4A5), a novel splice variant of the PDE4A cyclic AMP phosphodiesterase family. Biochem J. 1995 Sep 15;310(Pt 3):965–974. doi: 10.1042/bj3100965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Melson G. L., Chase L. R., Aurbach G. D. Parathyroid hormone-sensitive adenyl cyclase in isolated renal tubules. Endocrinology. 1970 Mar;86(3):511–518. doi: 10.1210/endo-86-3-511. [DOI] [PubMed] [Google Scholar]
  33. Murphy G. J., Hruby V. J., Trivedi D., Wakelam M. J., Houslay M. D. The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism. Biochem J. 1987 Apr 1;243(1):39–46. doi: 10.1042/bj2430039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Newlands C., Houslay M. D. Treatment of intact hepatocytes with synthetic diacyl glycerols mimics the ability of glucagon to cause the desensitization of adenylate cyclase. FEBS Lett. 1991 Sep 9;289(2):129–132. doi: 10.1016/0014-5793(91)81051-9. [DOI] [PubMed] [Google Scholar]
  35. Ono Y., Fujii T., Ogita K., Kikkawa U., Igarashi K., Nishizuka Y. Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties. Proc Natl Acad Sci U S A. 1989 May;86(9):3099–3103. doi: 10.1073/pnas.86.9.3099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Parker P. J., Cook P. P., Olivier A. R., Pears C., Ways D. K., Webster C. Protein kinase C. Biochem Soc Trans. 1992 May;20(2):415–418. doi: 10.1042/bst0200415. [DOI] [PubMed] [Google Scholar]
  37. Parker P. J., Kour G., Marais R. M., Mitchell F., Pears C., Schaap D., Stabel S., Webster C. Protein kinase C--a family affair. Mol Cell Endocrinol. 1989 Aug;65(1-2):1–11. doi: 10.1016/0303-7207(89)90159-7. [DOI] [PubMed] [Google Scholar]
  38. Ponte P., Ng S. Y., Engel J., Gunning P., Kedes L. Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA. Nucleic Acids Res. 1984 Feb 10;12(3):1687–1696. doi: 10.1093/nar/12.3.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Premont R. T., Inglese J., Lefkowitz R. J. Protein kinases that phosphorylate activated G protein-coupled receptors. FASEB J. 1995 Feb;9(2):175–182. doi: 10.1096/fasebj.9.2.7781920. [DOI] [PubMed] [Google Scholar]
  40. Refsnes M., Johansen E. J., Christoffersen T. Glucagon-induced refractoriness of hepatocyte adenylate cyclase: comparison of homologous and heterologous components and evidence against a role of cAMP. Pharmacol Toxicol. 1989 May;64(5):397–403. doi: 10.1111/j.1600-0773.1989.tb00675.x. [DOI] [PubMed] [Google Scholar]
  41. Rozengurt E., Sinnett-Smith J., Van Lint J., Valverde A. M. Protein kinase D (PKD): a novel target for diacylglycerol and phorbol esters. Mutat Res. 1995 Dec;333(1-2):153–160. doi: 10.1016/0027-5107(95)00141-7. [DOI] [PubMed] [Google Scholar]
  42. Savage A., Zeng L., Houslay M. D. A role for protein kinase C-mediated phosphorylation in eliciting glucagon desensitization in rat hepatocytes. Biochem J. 1995 Apr 1;307(Pt 1):281–285. doi: 10.1042/bj3070281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shakur Y., Pryde J. G., Houslay M. D. Engineered deletion of the unique N-terminal domain of the cyclic AMP-specific phosphodiesterase RD1 prevents plasma membrane association and the attainment of enhanced thermostability without altering its sensitivity to inhibition by rolipram. Biochem J. 1993 Jun 15;292(Pt 3):677–686. doi: 10.1042/bj2920677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sonne O., Berg T., Christoffersen T. Binding of 125I-labeled glucagon and glucagon-stimulated accumulation of adenosine 3':5'-monophosphate in isolated intact rat hepatocytes. Evidence for receptor heterogeneity. J Biol Chem. 1978 May 10;253(9):3203–3210. [PubMed] [Google Scholar]
  45. Svoboda M., Ciccarelli E., Tastenoy M., Cauvin A., Stiévenart M., Christophe J. Small introns in a hepatic cDNA encoding a new glucagon-like peptide 1-type receptor. Biochem Biophys Res Commun. 1993 Mar 15;191(2):479–486. doi: 10.1006/bbrc.1993.1243. [DOI] [PubMed] [Google Scholar]
  46. Svoboda M., Ciccarelli E., Tastenoy M., Robberecht P., Christophe J. A cDNA construct allowing the expression of rat hepatic glucagon receptors. Biochem Biophys Res Commun. 1993 Apr 15;192(1):135–142. doi: 10.1006/bbrc.1993.1392. [DOI] [PubMed] [Google Scholar]
  47. Tang E. Y., Parker P. J., Beattie J., Houslay M. D. Diabetes induces selective alterations in the expression of protein kinase C isoforms in hepatocytes. FEBS Lett. 1993 Jul 12;326(1-3):117–123. doi: 10.1016/0014-5793(93)81774-t. [DOI] [PubMed] [Google Scholar]
  48. Thompson W. J., Appleman M. M. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry. 1971 Jan 19;10(2):311–316. [PubMed] [Google Scholar]
  49. Valverde A. M., Sinnett-Smith J., Van Lint J., Rozengurt E. Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8572–8576. doi: 10.1073/pnas.91.18.8572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Van Lint J. V., Sinnett-Smith J., Rozengurt E. Expression and characterization of PKD, a phorbol ester and diacylglycerol-stimulated serine protein kinase. J Biol Chem. 1995 Jan 20;270(3):1455–1461. doi: 10.1074/jbc.270.3.1455. [DOI] [PubMed] [Google Scholar]
  51. Zugaza J. L., Sinnett-Smith J., Van Lint J., Rozengurt E. Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway. EMBO J. 1996 Nov 15;15(22):6220–6230. [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES