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. 2004 Jun 15;380(Pt 3):831–836. doi: 10.1042/BJ20031659

Ligand-dependent autophosphorylation of the insulin receptor is positively regulated by Gi-proteins.

J Kreuzer 1, B Nürnberg 1, H I Krieger-Brauer 1
PMCID: PMC1224225  PMID: 15025562

Abstract

Previously, we have shown that the human insulin receptor (IR) interacts with G(i)2, independent of tyrosine kinase activity and stimulates NADPH oxidase via the Galpha subunit of G(i)2. We have now investigated the regulatory role of G(i)2-proteins in IR function. For the experiments, isolated IRs from plasma membranes of human fat cells were used. The activation of IR autophosphorylation by insulin was blocked by G-protein inactivation through GDPbetaS (guanosine 5'-[beta-thio]disphosphate). Consistently, activation of G-proteins by micromolar concentrations of GTPgammaS (guanosine 5'-[gamma-thio]triphosphate) induced receptor autophosphorylation 5-fold over baseline and increased insulin-induced autophosphorylation by 3-fold. In the presence of 10 microM GTPgammaS, insulin was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. Pretreatment of the plasma membranes with pertussis toxin prevented insulin- and GTPgammaS-induced autophosphorylation, but did not disrupt the IR-G(i)2 complex. The functional nature of the IR-G(i)2 complex was made evident by insulin's ability to increase association of G(i)2 with the IR. This leads to an augmentation of maximal receptor autophosphorylation induced by insulin and GTPgammaS. The specificity of this mechanism was further demonstrated by the use of isolated preactivated G-proteins. Addition of G(i)2alpha and Gbetagamma mimicked maximal response of insulin, whereas Galphas or Galphao had no stimulatory effect. These results define a novel mechanism by which insulin signalling mediates tyrosine kinase activity and autophosphorylation of the IR through recruitment of G(i)-proteins.

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Selected References

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  1. Ahmad F., Considine R. V., Goldstein B. J. Increased abundance of the receptor-type protein-tyrosine phosphatase LAR accounts for the elevated insulin receptor dephosphorylating activity in adipose tissue of obese human subjects. J Clin Invest. 1995 Jun;95(6):2806–2812. doi: 10.1172/JCI117985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anger Thomas, Zhang Wei, Mende Ulrike. Differential contribution of GTPase activation and effector antagonism to the inhibitory effect of RGS proteins on Gq-mediated signaling in vivo. J Biol Chem. 2003 Nov 20;279(6):3906–3915. doi: 10.1074/jbc.M309496200. [DOI] [PubMed] [Google Scholar]
  3. Bence K., Ma W., Kozasa T., Huang X. Y. Direct stimulation of Bruton's tyrosine kinase by G(q)-protein alpha-subunit. Nature. 1997 Sep 18;389(6648):296–299. doi: 10.1038/38520. [DOI] [PubMed] [Google Scholar]
  4. Berman D. M., Wilkie T. M., Gilman A. G. GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein alpha subunits. Cell. 1996 Aug 9;86(3):445–452. doi: 10.1016/s0092-8674(00)80117-8. [DOI] [PubMed] [Google Scholar]
  5. Booth Ronald A., Cummings Cathy, Tiberi Mario, Liu X. Johné. GIPC participates in G protein signaling downstream of insulin-like growth factor 1 receptor. J Biol Chem. 2002 Jan 7;277(8):6719–6725. doi: 10.1074/jbc.M108033200. [DOI] [PubMed] [Google Scholar]
  6. Béréziat Veronique, Kasus-Jacobi Anne, Perdereau Dominique, Cariou Bertrand, Girard Jean, Burnol Anne-Françoise. Inhibition of insulin receptor catalytic activity by the molecular adapter Grb14. J Biol Chem. 2001 Nov 28;277(7):4845–4852. doi: 10.1074/jbc.M106574200. [DOI] [PubMed] [Google Scholar]
  7. Böni-Schnetzler M., Scott W., Waugh S. M., DiBella E., Pilch P. F. The insulin receptor. Structural basis for high affinity ligand binding. J Biol Chem. 1987 Jun 15;262(17):8395–8401. [PubMed] [Google Scholar]
  8. Calera M. R., Vallega G., Pilch P. F. Dynamics of protein-tyrosine phosphatases in rat adipocytes. J Biol Chem. 2000 Mar 3;275(9):6308–6312. doi: 10.1074/jbc.275.9.6308. [DOI] [PubMed] [Google Scholar]
  9. Chen J. F., Guo J. H., Moxham C. M., Wang H. Y., Malbon C. C. Conditional, tissue-specific expression of Q205L G alpha i2 in vivo mimics insulin action. J Mol Med (Berl) 1997 Apr;75(4):283–289. doi: 10.1007/s001090050113. [DOI] [PubMed] [Google Scholar]
  10. Codina J., Hildebrandt J. D., Birnbaumer L., Sekura R. D. Effects of guanine nucleotides and Mg on human erythrocyte Ni and Ns, the regulatory components of adenylyl cyclase. J Biol Chem. 1984 Sep 25;259(18):11408–11418. [PubMed] [Google Scholar]
  11. Dalle S., Ricketts W., Imamura T., Vollenweider P., Olefsky J. M. Insulin and insulin-like growth factor I receptors utilize different G protein signaling components. J Biol Chem. 2001 Feb 8;276(19):15688–15695. doi: 10.1074/jbc.M010884200. [DOI] [PubMed] [Google Scholar]
  12. Davis H. W., McDonald J. M. Insulin receptor function is inhibited by guanosine 5'-[gamma-thio]triphosphate (GTP[S]). Biochem J. 1990 Sep 1;270(2):401–407. doi: 10.1042/bj2700401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guo J. H., Wang H. Y., Malbon C. C. Conditional, tissue-specific expression of Q205L Galphai2 in vivo mimics insulin activation of c-Jun N-terminal kinase and p38 kinase. J Biol Chem. 1998 Jun 26;273(26):16487–16493. doi: 10.1074/jbc.273.26.16487. [DOI] [PubMed] [Google Scholar]
  14. Hallak H., Seiler A. E., Green J. S., Ross B. N., Rubin R. Association of heterotrimeric G(i) with the insulin-like growth factor-I receptor. Release of G(betagamma) subunits upon receptor activation. J Biol Chem. 2000 Jan 28;275(4):2255–2258. doi: 10.1074/jbc.275.4.2255. [DOI] [PubMed] [Google Scholar]
  15. Hawley D. M., Maddux B. A., Patel R. G., Wong K. Y., Mamula P. W., Firestone G. L., Brunetti A., Verspohl E., Goldfine I. D. Insulin receptor monoclonal antibodies that mimic insulin action without activating tyrosine kinase. J Biol Chem. 1989 Feb 15;264(5):2438–2444. [PubMed] [Google Scholar]
  16. Imamura T., Vollenweider P., Egawa K., Clodi M., Ishibashi K., Nakashima N., Ugi S., Adams J. W., Brown J. H., Olefsky J. M. G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes. Mol Cell Biol. 1999 Oct;19(10):6765–6774. doi: 10.1128/mcb.19.10.6765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jiang Y., Ma W., Wan Y., Kozasa T., Hattori S., Huang X. Y. The G protein G alpha12 stimulates Bruton's tyrosine kinase and a rasGAP through a conserved PH/BM domain. Nature. 1998 Oct 22;395(6704):808–813. doi: 10.1038/27454. [DOI] [PubMed] [Google Scholar]
  18. Kanaho Y., Chang P. P., Moss J., Vaughan M. Mechanism of inhibition of transducin guanosine triphosphatase activity by vanadate. J Biol Chem. 1988 Nov 25;263(33):17584–17589. [PubMed] [Google Scholar]
  19. Kanzaki M., Watson R. T., Artemyev N. O., Pessin J. E. The trimeric GTP-binding protein (G(q)/G(11)) alpha subunit is required for insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes. J Biol Chem. 2000 Mar 10;275(10):7167–7175. doi: 10.1074/jbc.275.10.7167. [DOI] [PubMed] [Google Scholar]
  20. Katada T., Bokoch G. M., Northup J. K., Ui M., Gilman A. G. The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Properties and function of the purified protein. J Biol Chem. 1984 Mar 25;259(6):3568–3577. [PubMed] [Google Scholar]
  21. Kellerer M., Obermaier-Kusser B., Pröfrock A., Schleicher E., Seffer E., Mushack J., Ermel B., Häring H. U. Insulin activates GTP binding to a 40 kDa protein in fat cells. Biochem J. 1991 May 15;276(Pt 1):103–108. doi: 10.1042/bj2760103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kiely Patrick A., Sant Anagha, O'Connor Rosemary. RACK1 is an insulin-like growth factor 1 (IGF-1) receptor-interacting protein that can regulate IGF-1-mediated Akt activation and protection from cell death. J Biol Chem. 2002 Apr 18;277(25):22581–22589. doi: 10.1074/jbc.M201758200. [DOI] [PubMed] [Google Scholar]
  23. Kreuzer J., Viedt C., Brandes R. P., Seeger F., Rosenkranz A. S., Sauer H., Babich A., Nürnberg B., Kather H., Krieger-Brauer H. I. Platelet-derived growth factor activates production of reactive oxygen species by NAD(P)H oxidase in smooth muscle cells through Gi1,2. FASEB J. 2002 Nov 1;17(1):38–40. doi: 10.1096/fj.01-1036fje. [DOI] [PubMed] [Google Scholar]
  24. Krieger-Brauer H. I., Kather H. Antagonistic effects of different members of the fibroblast and platelet-derived growth factor families on adipose conversion and NADPH-dependent H2O2 generation in 3T3 L1-cells. Biochem J. 1995 Apr 15;307(Pt 2):549–556. doi: 10.1042/bj3070549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Krieger-Brauer H. I., Kather H. Human fat cells possess a plasma membrane-bound H2O2-generating system that is activated by insulin via a mechanism bypassing the receptor kinase. J Clin Invest. 1992 Mar;89(3):1006–1013. doi: 10.1172/JCI115641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Krieger-Brauer H. I., Kather H. The stimulus-sensitive H2O2-generating system present in human fat-cell plasma membranes is multireceptor-linked and under antagonistic control by hormones and cytokines. Biochem J. 1995 Apr 15;307(Pt 2):543–548. doi: 10.1042/bj3070543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Krieger-Brauer H. I., Medda P. K., Kather H. Insulin-induced activation of NADPH-dependent H2O2 generation in human adipocyte plasma membranes is mediated by Galphai2. J Biol Chem. 1997 Apr 11;272(15):10135–10143. doi: 10.1074/jbc.272.15.10135. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Lammers R., Møler N. P., Ullrich A. The transmembrane protein tyrosine phosphatase alpha dephosphorylates the insulin receptor in intact cells. FEBS Lett. 1997 Mar 3;404(1):37–40. doi: 10.1016/s0014-5793(97)00080-x. [DOI] [PubMed] [Google Scholar]
  30. Lou X., Yano H., Lee F., Chao M. V., Farquhar M. G. GIPC and GAIP form a complex with TrkA: a putative link between G protein and receptor tyrosine kinase pathways. Mol Biol Cell. 2001 Mar;12(3):615–627. doi: 10.1091/mbc.12.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lowry William E., Huang Xin-Yun. G Protein beta gamma subunits act on the catalytic domain to stimulate Bruton's agammaglobulinemia tyrosine kinase. J Biol Chem. 2001 Nov 6;277(2):1488–1492. doi: 10.1074/jbc.M110390200. [DOI] [PubMed] [Google Scholar]
  32. Ma Y. C., Huang J., Ali S., Lowry W., Huang X. Y. Src tyrosine kinase is a novel direct effector of G proteins. Cell. 2000 Sep 1;102(5):635–646. doi: 10.1016/s0092-8674(00)00086-6. [DOI] [PubMed] [Google Scholar]
  33. Mattera R., Codina J., Sekura R. D., Birnbaumer L. The interaction of nucleotides with pertussis toxin. Direct evidence for a nucleotide binding site on the toxin regulating the rate of ADP-ribosylation of Ni, the inhibitory regulatory component of adenylyl cyclase. J Biol Chem. 1986 Aug 25;261(24):11173–11179. [PubMed] [Google Scholar]
  34. Moxham C. M., Malbon C. C. Insulin action impaired by deficiency of the G-protein subunit G ialpha2. Nature. 1996 Feb 29;379(6568):840–844. doi: 10.1038/379840a0. [DOI] [PubMed] [Google Scholar]
  35. Müller-Wieland D., White M. F., Behnke B., Gebhardt A., Neumann S., Krone W., Kahn C. R. Pertussis toxin inhibits autophosphorylation and activation of the insulin receptor kinase. Biochem Biophys Res Commun. 1991 Dec 31;181(3):1479–1485. doi: 10.1016/0006-291x(91)92106-t. [DOI] [PubMed] [Google Scholar]
  36. Ottensmeyer F. P., Beniac D. R., Luo R. Z., Yip C. C. Mechanism of transmembrane signaling: insulin binding and the insulin receptor. Biochemistry. 2000 Oct 10;39(40):12103–12112. doi: 10.1021/bi0015921. [DOI] [PubMed] [Google Scholar]
  37. Rothenberg P. L., Kahn C. R. Insulin inhibits pertussis toxin-catalyzed ADP-ribosylation of G-proteins. Evidence for a novel interaction between insulin receptors and G-proteins. J Biol Chem. 1988 Oct 25;263(30):15546–15552. [PubMed] [Google Scholar]
  38. Sánchez-Margalet V., González-Yanes C., Santos-Alvarez J., Najib S. Insulin activates G alpha il,2 protein in rat hepatoma (HTC) cell membranes. Cell Mol Life Sci. 1999 Jan;55(1):142–147. doi: 10.1007/s000180050279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ullrich A., Bell J. R., Chen E. Y., Herrera R., Petruzzelli L. M., Dull T. J., Gray A., Coussens L., Liao Y. C., Tsubokawa M. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. 1985 Feb 28-Mar 6Nature. 313(6005):756–761. doi: 10.1038/313756a0. [DOI] [PubMed] [Google Scholar]
  40. Zeng Huiyan, Zhao Dezheng, Yang Suping, Datta Kaustubh, Mukhopadhyay Debabrata. Heterotrimeric G alpha q/G alpha 11 proteins function upstream of vascular endothelial growth factor (VEGF) receptor-2 (KDR) phosphorylation in vascular permeability factor/VEGF signaling. J Biol Chem. 2003 Apr 1;278(23):20738–20745. doi: 10.1074/jbc.M209712200. [DOI] [PubMed] [Google Scholar]

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