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. 1990 Jun 1;268(2):449–457. doi: 10.1042/bj2680449

Hormonal regulation of Gi2 alpha-subunit phosphorylation in intact hepatocytes.

M Bushfield 1, G J Murphy 1, B E Lavan 1, P J Parker 1, V J Hruby 1, G Milligan 1, M D Houslay 1
PMCID: PMC1131453  PMID: 2114093

Abstract

Hepatocytes contain the Gi2 and Gi3 forms of the 'Gi-family' of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the alpha-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity ('Gi'-function). The immunoprecipitation of phosphorylated Gi-2 alpha-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a 'Gi-like' G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the alpha-subunits of either Gi3 or Gs was observed. alpha-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of alpha-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of alpha-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of alpha-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 microM) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated alpha-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [gamma-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated alpha-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of alpha-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of alpha-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.

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Selected References

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

  1. Blackmore P. F., Exton J. H. Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate. J Biol Chem. 1986 Aug 25;261(24):11056–11063. [PubMed] [Google Scholar]
  2. Bocckino S. B., Blackmore P. F., Exton J. H. Stimulation of 1,2-diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and angiotensin II. J Biol Chem. 1985 Nov 15;260(26):14201–14207. [PubMed] [Google Scholar]
  3. Bregman M. D., Trivedi D., Hruby V. J. Glucagon amino groups. Evaluation of modifications leading to antagonism and agonism. J Biol Chem. 1980 Dec 25;255(24):11725–11731. [PubMed] [Google Scholar]
  4. Carlson K. E., Brass L. F., Manning D. R. Thrombin and phorbol esters cause the selective phosphorylation of a G protein other than Gi in human platelets. J Biol Chem. 1989 Aug 5;264(22):13298–13305. [PubMed] [Google Scholar]
  5. Creba J. A., Downes C. P., Hawkins P. T., Brewster G., Michell R. H., Kirk C. J. Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones. Biochem J. 1983 Jun 15;212(3):733–747. doi: 10.1042/bj2120733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crouch M. F., Lapetina E. G. A role for Gi in control of thrombin receptor-phospholipase C coupling in human platelets. J Biol Chem. 1988 Mar 5;263(7):3363–3371. [PubMed] [Google Scholar]
  7. Fong H. K., Yoshimoto K. K., Eversole-Cire P., Simon M. I. Identification of a GTP-binding protein alpha subunit that lacks an apparent ADP-ribosylation site for pertussis toxin. Proc Natl Acad Sci U S A. 1988 May;85(9):3066–3070. doi: 10.1073/pnas.85.9.3066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Garrison J. C., Johnsen D. E., Campanile C. P. Evidence for the role of phosphorylase kinase, protein kinase C, and other Ca2+-sensitive protein kinases in the response of hepatocytes to angiotensin II and vasopressin. J Biol Chem. 1984 Mar 10;259(5):3283–3292. [PubMed] [Google Scholar]
  9. Gawler D., Milligan G., Spiegel A. M., Unson C. G., Houslay M. D. Abolition of the expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes. Nature. 1987 May 21;327(6119):229–232. doi: 10.1038/327229a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Heyworth C. M., Hanski E., Houslay M. D. Islet-activating protein blocks glucagon desensitization in intact hepatocytes. Biochem J. 1984 Aug 15;222(1):189–194. doi: 10.1042/bj2220189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Heyworth C. M., Whetton A. D., Wong S., Martin B. R., Houslay M. D. Insulin inhibits the cholera-toxin-catalysed ribosylation of a Mr-25000 protein in rat liver plasma membranes. Biochem J. 1985 Jun 15;228(3):593–603. doi: 10.1042/bj2280593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Houslay M. D., Elliott K. R. Cholera toxin mediated activation of adenylate cyclase in intact rat hepatocytes. FEBS Lett. 1979 Aug 15;104(2):359–363. doi: 10.1016/0014-5793(79)80852-2. [DOI] [PubMed] [Google Scholar]
  15. Houslay M. D., Metcalfe J. C., Warren G. B., Hesketh T. R., Smith G. A. The glucagon receptor of rat liver plasma membrane can couple to adenylate cyclase without activating it. Biochim Biophys Acta. 1976 Jun 17;436(2):489–494. doi: 10.1016/0005-2736(76)90210-8. [DOI] [PubMed] [Google Scholar]
  16. Houslay M. D. Strategies for identifying functionally active domains upon cell surface receptors. Curr Opin Cell Biol. 1989 Aug;1(4):669–674. doi: 10.1016/0955-0674(89)90032-x. [DOI] [PubMed] [Google Scholar]
  17. Jakobs K. H., Bauer S., Watanabe Y. Modulation of adenylate cyclase of human platelets by phorbol ester. Impairment of the hormone-sensitive inhibitory pathway. Eur J Biochem. 1985 Sep 2;151(2):425–430. doi: 10.1111/j.1432-1033.1985.tb09119.x. [DOI] [PubMed] [Google Scholar]
  18. Jelsema C. L., Axelrod J. Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3623–3627. doi: 10.1073/pnas.84.11.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Johnson G. L., Bourne H. R. Influence of cholera toxin on the regulation of adenylate cyclase by GTP. Biochem Biophys Res Commun. 1977 Sep 23;78(2):792–798. doi: 10.1016/0006-291x(77)90249-2. [DOI] [PubMed] [Google Scholar]
  20. Jones D. T., Reed R. R. Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J Biol Chem. 1987 Oct 15;262(29):14241–14249. [PubMed] [Google Scholar]
  21. Katada T., Gilman A. G., Watanabe Y., Bauer S., Jakobs K. H. Protein kinase C phosphorylates the inhibitory guanine-nucleotide-binding regulatory component and apparently suppresses its function in hormonal inhibition of adenylate cyclase. Eur J Biochem. 1985 Sep 2;151(2):431–437. doi: 10.1111/j.1432-1033.1985.tb09120.x. [DOI] [PubMed] [Google Scholar]
  22. Krupinski J., Rajaram R., Lakonishok M., Benovic J. L., Cerione R. A. Insulin-dependent phosphorylation of GTP-binding proteins in phospholipid vesicles. J Biol Chem. 1988 Sep 5;263(25):12333–12341. [PubMed] [Google Scholar]
  23. 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]
  24. Matsuoka M., Itoh H., Kozasa T., Kaziro Y. Sequence analysis of cDNA and genomic DNA for a putative pertussis toxin-insensitive guanine nucleotide-binding regulatory protein alpha subunit. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5384–5388. doi: 10.1073/pnas.85.15.5384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Milligan G. Techniques used in the identification and analysis of function of pertussis toxin-sensitive guanine nucleotide binding proteins. Biochem J. 1988 Oct 1;255(1):1–13. doi: 10.1042/bj2550001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Milligan G., Unson C. G. Persistent activation of the alpha subunit of Gs promotes its removal from the plasma membrane. Biochem J. 1989 Jun 15;260(3):837–841. doi: 10.1042/bj2600837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mitchell F. M., Griffiths S. L., Saggerson E. D., Houslay M. D., Knowler J. T., Milligan G. Guanine-nucleotide-binding proteins expressed in rat white adipose tissue. Identification of both mRNAs and proteins corresponding to Gi1, Gi2 and Gi3. Biochem J. 1989 Sep 1;262(2):403–408. doi: 10.1042/bj2620403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Murphy G. J., Gawler D. J., Milligan G., Wakelam M. J., Pyne N. J., Houslay M. D. Glucagon desensitization of adenylate cyclase and stimulation of inositol phospholipid metabolism does not involve the inhibitory guanine nucleotide regulatory protein Gi, which is inactivated upon challenge of hepatocytes with glucagon. Biochem J. 1989 Apr 1;259(1):191–197. doi: 10.1042/bj2590191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. O'Brien R. M., Houslay M. D., Milligan G., Siddle K. The insulin receptor tyrosyl kinase phosphorylates holomeric forms of the guanine nucleotide regulatory proteins Gi and Go. FEBS Lett. 1987 Feb 23;212(2):281–288. doi: 10.1016/0014-5793(87)81361-3. [DOI] [PubMed] [Google Scholar]
  30. Parker P. J., Stabel S., Waterfield M. D. Purification to homogeneity of protein kinase C from bovine brain--identity with the phorbol ester receptor. EMBO J. 1984 May;3(5):953–959. doi: 10.1002/j.1460-2075.1984.tb01913.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Premont R. T., Iyengar R. Heterologous desensitization of the liver adenylyl cyclase: analysis of the role of G-proteins. Endocrinology. 1989 Sep;125(3):1151–1160. doi: 10.1210/endo-125-3-1151. [DOI] [PubMed] [Google Scholar]
  32. Price S. R., Tsai S. C., Adamik R., Angus C. W., Serventi I. M., Tsuchiya M., Moss J., Vaughan M. Expression of Go alpha mRNA and protein in bovine tissues. Biochemistry. 1989 May 2;28(9):3803–3807. doi: 10.1021/bi00435a027. [DOI] [PubMed] [Google Scholar]
  33. Pyne N. J., Murphy G. J., Milligan G., Houslay M. D. Treatment of intact hepatocytes with either the phorbol ester TPA or glucagon elicits the phosphorylation and functional inactivation of the inhibitory guanine nucleotide regulatory protein Gi. FEBS Lett. 1989 Jan 16;243(1):77–82. doi: 10.1016/0014-5793(89)81221-9. [DOI] [PubMed] [Google Scholar]
  34. Reimann E. M., Beham R. A. Catalytic subunit of cAMP-dependent protein kinase. Methods Enzymol. 1983;99:51–55. doi: 10.1016/0076-6879(83)99039-0. [DOI] [PubMed] [Google Scholar]
  35. Smith S. A., Elliott K. R., Pogson C. I. Differential effects of tryptophan on glucose synthesis in rats and guinea pigs. Biochem J. 1978 Dec 15;176(3):817–825. doi: 10.1042/bj1760817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Staddon J. M., Hansford R. G. Evidence indicating that the glucagon-induced increase in cytoplasmic free Ca2+ concentration in hepatocytes is mediated by an increase in cyclic AMP concentration. Eur J Biochem. 1989 Jan 15;179(1):47–52. doi: 10.1111/j.1432-1033.1989.tb14519.x. [DOI] [PubMed] [Google Scholar]
  37. Strassheim D., Milligan G., Houslay M. D. Diabetes abolishes the GTP-dependent, but not the receptor-dependent inhibitory function of the inhibitory guanine-nucleotide-binding regulatory protein (Gi) on adipocyte adenylate cyclase activity. Biochem J. 1990 Mar 1;266(2):521–526. doi: 10.1042/bj2660521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wakelam M. J., Murphy G. J., Hruby V. J., Houslay M. D. Activation of two signal-transduction systems in hepatocytes by glucagon. Nature. 1986 Sep 4;323(6083):68–71. doi: 10.1038/323068a0. [DOI] [PubMed] [Google Scholar]
  39. Walsh D. A., Ashby C. D., Gonzalez C., Calkins D., Fischer E. H. Krebs EG: Purification and characterization of a protein inhibitor of adenosine 3',5'-monophosphate-dependent protein kinases. J Biol Chem. 1971 Apr 10;246(7):1977–1985. [PubMed] [Google Scholar]
  40. Watanabe Y., Imaizumi T., Misaki N., Iwakura K., Yoshida H. Effects of phosphorylation of inhibitory GTP-binding protein by cyclic AMP-dependent protein kinase on its ADP-ribosylation by pertussis toxin, islet-activating protein. FEBS Lett. 1988 Aug 29;236(2):372–374. doi: 10.1016/0014-5793(88)80058-9. [DOI] [PubMed] [Google Scholar]
  41. Whetton A. D., Needham L., Dodd N. J., Heyworth C. M., Houslay M. D. Forskolin and ethanol both perturb the structure of liver plasma membranes and activate adenylate cyclase activity. Biochem Pharmacol. 1983 May 15;32(10):1601–1608. doi: 10.1016/0006-2952(83)90334-9. [DOI] [PubMed] [Google Scholar]
  42. Yatani A., Codina J., Brown A. M., Birnbaumer L. Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory protein Gk. Science. 1987 Jan 9;235(4785):207–211. doi: 10.1126/science.2432660. [DOI] [PubMed] [Google Scholar]
  43. Yatani A., Mattera R., Codina J., Graf R., Okabe K., Padrell E., Iyengar R., Brown A. M., Birnbaumer L. The G protein-gated atrial K+ channel is stimulated by three distinct Gi alpha-subunits. Nature. 1988 Dec 15;336(6200):680–682. doi: 10.1038/336680a0. [DOI] [PubMed] [Google Scholar]

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