Insulin and Insulin-Like Growth Factor I Mediated Phosphorylations in Mouse Neuroblastoma N18 Cells: A Model for Studying Insulin and IGF-I Action on Neural Tissue

  • Martin Adamo
  • Joshua Shemer
  • Derek LeRoith


Insulin and insulin-like growth factor I are traditionally believed to regulate metabolic and growth activities in peripheral target tissues (1). However, since both peptides are present in the brain, they probably also function as neuropeptides (2). Additional evidence for their ability to function on nervous tissue is the finding that specific insulin and IGF-I receptors are present throughout the brain (3,4). These receptors exhibit structures typical of insulin and IGF-I receptors, which are heterotetramers consisting of 2 alpha-subunits which bind the ligand and two beta-subunits which are autophosphorylated as a result of ligand binding. The subunits are covalently linked via disulfide bridges (5,6,7). An important difference between brain and peripheral insulin and IGF-I receptors is the size of the receptor alpha-subunit, which has been found to be approximately lOkDa smaller in receptors from the CNS (8,9). The basis for this difference lies primarily in the extent and pattern of alpha-subunit glycosylation (10,11). Upon autophosphoryl-ation, insulin and IGF-I beta-subunits act as tyrosine kinases, toward endogenous and exogenous substrates, and considerable evidence has accumulated to suggest that this cascade of phosphorylations is critical to insulin action (12,13,14).


Insulin Receptor Phosphoamino Acid Human Insulin Receptor Phosphoamino Acid Analysis Antiphosphotyrosine Antibody 
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  1. 1.
    J.F. Perdue, Chemistry, structure and function of insulin-like growth factors and their receptors: a review. Can. J. Biochem. Cell Biol. 62:1237 (1984).PubMedCrossRefGoogle Scholar
  2. 2.
    E. Recio-Pinto and D.N. Ishii, Insulin and related growth factors: effects on the nervous system and mechanism for neurite growth and regeneration. Neurochem. Int. 12:397 (1988)PubMedCrossRefGoogle Scholar
  3. 3.
    J. Havrankova, J. Roth, and M. Brownstein, Insulin receptors are widely distributed in the central nervous system of the rat. Nature 272:827 (1978).PubMedCrossRefGoogle Scholar
  4. 4.
    S. Gammeltoft, G. Hasselbacher, R. Humbel, M. Feldmann, E. Van Obberghen, Two types of receptor for insulin-like growth factors in mammalian brain. EMBO J. 4:3407 (1985).PubMedGoogle Scholar
  5. 5.
    A. Ullrich, J.R. Bell, E.Y. Chen, R. Herrera, L.M. Petruzzelli, T.J. Dull, A. Gray, L. Coussens, Y.-C. Liao, M. Tsubokawa, A. Mason, P.H. Seeburg, C. Grunfeld., O.M. Rosen and J. Ramachandran, Human insulin receptor and its relationship to the tyrosine kinase family of oncognes. Nature 313:756 (1985).PubMedCrossRefGoogle Scholar
  6. 6.
    Y. Ebina, L. Ellis, K. Jarnagin, M. Edery, L. Graf, E. Clauser, J.H. Ou, F. Masiarz, Y.W. Kan, I.D. Goldfine, R. Roth and W. Rutter, The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling. Cell 46:747 (1985).CrossRefGoogle Scholar
  7. 7.
    A. Ullrich, A. Gray, A.W. Tarn, F. Yang-Feng, M. Tsubokawa, C. Collins, W. Henzel, T. LeBon, S. Dathuria, E. Chen, S. Jacobs, U. Francke, J. Ramachandran and Y. Fujita-Yamaguchi, Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J. 5:2503 (1986).PubMedGoogle Scholar
  8. 8.
    C.C. Yip, M.L. Moule and C.W.T. Yeung, Characterization of insulin receptor subunits in brain and other tissues by photoaffinity labeling. Biochem. Biophys. Res. Coran. 96:1671 (1980).CrossRefGoogle Scholar
  9. 9.
    K.A. Heidenreich and D. Brandenberg, Oligosaccharide heterogeneity of insulin receptors. Comparison of N-linked glycosylation of insulin receptors in adipocytes and brain. Endocrinology 118:1835 (1986).PubMedCrossRefGoogle Scholar
  10. 10.
    K.A. Heidenreich, N.R. Zahniser, P. Berhanu, D. Brandenberg and J.M. Olefsky, Structural differences between insulin receptors in the brain and peripheral target tissues. _J. Biol. Chem. 258:8527 (1983).PubMedGoogle Scholar
  11. 11.
    K.A. Heidenreich, G.R. Freidenberg, D.P. Figlewicz and P.R. Gilmore, Evidence for a subtype of insulin-like growth factor I receptor in brain. Reg. Peptides 15:301 (1987).CrossRefGoogle Scholar
  12. 12.
    C.K. Chou, T.J. Dull, D.S. Russell, R. Gherzi, D. Lebwohl, A. Ullrich, O.M. Rosen, Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate post-receptor effects of insulin, J. Biol. Chem. 262:1842 (1987).PubMedGoogle Scholar
  13. 13.
    Y. Ebina, E. Araki, M. Taira, F. Shimada, M. Mori, C.S. Craik, K. Siddle, S.B. Pierce, R.A. Roth and W.J. Rutter, Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody stimulated glucose uptake and receptor kinase activity: introduction of an inhibitory monoclonal antibody into mammalian cells blocks the rapid effects of insulin. Proc. Natl. Acad. Sci. U.S.A. 84:41 (1987).CrossRefGoogle Scholar
  14. 14.
    D.O. Morgan and R.A. Roth, Acute insulin action requires insulin receptor kinase activity: introduction of an inhibitory monoclonal antibody into mammalian cells blocks the rapid effects of insulin. Proc Natl. Acad. Sci. U.S.A. 84:41 (1987).PubMedCrossRefGoogle Scholar
  15. 15.
    R.W. Rees-Jones, S.A. Hendricks, M. Quarum, and J. Roth, The insulin receptors of rat brain are coupled to tyrosine kinase activity. J. Biol. Chem. 260:4461 (1984).Google Scholar
  16. 16.
    W.L. Lowe Jr., F.J. Boyd, D.W. Clarke, M.K. Raizada, C. Hart and D. LeRoith, Development of brain insulin receptors: structural and functional studies of insulin receptors from whole brain and primary cell cultures. Endocrinology 119:25 (1986).PubMedCrossRefGoogle Scholar
  17. 17.
    J. Shemer, M.K. Raizada, B.A. Masters, A. Ota, and D. LeRoith, Insulin-like growth factor I receptors in neuronal and glial cells. Characterization and biological effects in primary culture. J. Biol. Chem. 262:7693 (1987).PubMedGoogle Scholar
  18. 18.
    A. Ota, G.L. Wilson, O. Spilberg, R. Pruss and D. LeRoith, Functional insulin-like growth factor I receptors are expressed by neural-derived continuous cell lines. Endocrinology 122:145 (1988).PubMedCrossRefGoogle Scholar
  19. 19.
    J. Shemer, M. Adamo, G.L. Wilson, D. Heffez, Y. Zick and D. LeRoith, Insulin and insulin-like growth factor 1 stimulate a common endo-geneous phosphoprotein substrate (ppl85) in intact neuroblastoma cells. J. Biol. Chem. 262:15476 (1987).PubMedGoogle Scholar
  20. 20.
    M.F. White, R. Maron and C.R. Kahn, Insulin rapidly stimulates tyrosine phosphorylation of a Mr 185,000 protein in intact cells. Nature 318:183 (1985).PubMedCrossRefGoogle Scholar
  21. 21.
    T. Kadowaki, S. Kayasu, E. Nishida, K. Tobe, T. Izumi, F. Takaku, H. Sakai, I. Yahara, and M. Kasuga, Tyrosine phosphorylation of common and specific sets of cellular proteins rapidly induced by insulin, insulin-like growth factor I, and epidermal growth factor in an intact cell. J. Biol. Chem. 262:7342 (1987).PubMedGoogle Scholar
  22. 22.
    J.A. Cooper, B.M. Sefton and T. Hunter, Detection and quantification of phosphotyrosine in proteins.Meth. Enzymol. 99:387 (1983).PubMedCrossRefGoogle Scholar
  23. 23.
    A. Ota, G.L. Wilson and D. LeRoith, Insulin-like growth factor I receptors on mouse neuroblastoma cells. Two beta subunits are derived from differences in glycosylation. Eur. J. Biochem. 174:521 (1988).PubMedCrossRefGoogle Scholar
  24. 24.
    M. Kasuga, N. Sasaki, C.R. Kahn, S.P. Nissley and M.M. Rechler. Antireceptor antibodies as probes of insulin-like growth factor receptor structure. J. Clin. Invest. 72:1459 (1983).PubMedCrossRefGoogle Scholar
  25. 25.
    A. Ota, J. Shemer, R.M. Pruss, W.L. Lowe Jr., and D. LeRoith, Characterization of the altered oligosaccharide composition of the insulin receptor on neural derived cells. Brain Res. 443:1 (1988).PubMedCrossRefGoogle Scholar
  26. 26.
    M. Kasuga, F.A. Karlsson, and C.R. Kahn, Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor. Science 215:185 (1982).PubMedCrossRefGoogle Scholar
  27. 27.
    L.M. Petruzzelli, L. Stadtmauer, R. Herrera, M. Makowske, S. Ganguly, D. Tabarini, H. Lee, Y. Olowe, and O.M. Rosen, The insulin receptor as a tyrosine-specific protein kinase, in: “Mechanisms of Receptor Regulation” G. Poste and S.T. Crooke, eds. Plenum, New York and London (1985).Google Scholar
  28. 28.
    M. F. White, E.W. Stegmann, T.J. Dull, A. Ullrich and C.R. Kahn, Characterization of an endogenous substrate of the insulin receptor in cultured cells. J. Biol. Chem. 262:9769 (1987).PubMedGoogle Scholar
  29. 29.
    M.F. White, J.N. Livingston, J.N. Bacher, V. Lauris, T.J. Dull, A. Ullrich, and C.R. Kahn, Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity. Cell 54:641 (1988).PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1989

Authors and Affiliations

  • Martin Adamo
    • 1
  • Joshua Shemer
    • 1
  • Derek LeRoith
    • 1
  1. 1.Diabetes BranchNIDDK, NIHBethesdaUSA

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