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. 1984 Sep 1;99(3):900–908. doi: 10.1083/jcb.99.3.900

Abnormality of insulin binding and receptor phosphorylation in an insulin-resistant melanoma cell line

PMCID: PMC2113397  PMID: 6381509

Abstract

The insulin receptor possesses an insulin-stimulated tyrosine-kinase activity; however, the significance of receptor phosphorylation in terms of the binding and signaling function of the receptor is unclear. To help clarify this problem, we have studied insulin binding and receptor phosphorylation in a Cloudman S91 melanoma cell line and two of its variants: the wild type (1A) in which insulin inhibits cell growth, an insulin-resistant variant (111) in which insulin neither stimulates or inhibits growth, and a variant (46) in which insulin stimulates cell growth. 125I-insulin binding to intact cells was similar for the wild-type 1A and insulin-stimulated variant 46. The insulin-resistant variant 111, in contrast, showed approximately 30% decrease in insulin binding. This was due to a decrease of receptor affinity with no major difference in receptor number. When the melanoma cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed. Phosphorylation was studied by incubation of the partially purified receptor with insulin and [gamma-32P]ATP, and the receptor was identified by immunoprecipitation and NaDodSO4 PAGE. Insulin stimulated phosphorylation of the 95,000-mol- wt beta-subunit of the receptor in all three cells types with similar kinetics. The amount of 32P incorporated into the beta-subunit in the insulin-resistant cell line 111 was approximately 50% of that observed with the two other cell lines. This difference was reflected throughout the entire dose-response curve (10(-9) M to 10(-6) M). Qualitatively similar results were obtained when phosphorylation was studied in the intact cell. Peptide mapping of the beta-subunit using tryptic digestion and reverse-phase high-performance liquid chromatography column separation indicated three sites of phosphorylation in receptor from the wild type and variant 46, but only two major sites of phosphorylation of variant 111. These data suggest that the insulin- resistant variant melanoma 111 possesses a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties, and also suggests a possible role of receptor phosphorylation in both the binding and the signaling function of the insulin receptor.

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Selected References

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  1. Avruch J., Leone G. R., Martin D. B. Effects of epinephrine and insulin on phosphopeptide metabolism in adipocytes. J Biol Chem. 1976 Mar 10;251(5):1511–1515. [PubMed] [Google Scholar]
  2. Avruch J., Nemenoff R. A., Blackshear P. J., Pierce M. W., Osathanondh R. Insulin-stimulated tyrosine phosphorylation of the insulin receptor in detergent extracts of human placental membranes. Comparison to epidermal growth factor-stimulated phosphorylation. J Biol Chem. 1982 Dec 25;257(24):15162–15166. [PubMed] [Google Scholar]
  3. Benjamin W. B., Clayton N. L. Action of insulin and catecholamines on the phosphorylation of proteins associated with the cytosol, membranes, and "fat cake" of rat fat cells. J Biol Chem. 1978 Mar 10;253(5):1700–1709. [PubMed] [Google Scholar]
  4. Buss J. E., Kudlow J. E., Lazar C. S., Gill G. N. Altered epidermal growth factor (EGF)-stimulated protein kinase activity in variant A431 cells with altered growth responses to EGF. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2574–2578. doi: 10.1073/pnas.79.8.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carpenter G., King L., Jr, Cohen S. Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor. J Biol Chem. 1979 Jun 10;254(11):4884–4891. [PubMed] [Google Scholar]
  6. Collett M. S., Erikson E., Purchio A. F., Brugge J. S., Erikson R. L. A normal cell protein similar in structure and function to the avian sarcoma virus transforming gene product. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3159–3163. doi: 10.1073/pnas.76.7.3159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collett M. S., Purchio A. F., Erikson R. L. Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine. Nature. 1980 May 15;285(5761):167–169. doi: 10.1038/285167a0. [DOI] [PubMed] [Google Scholar]
  8. Corbin J. D., Brostrom C. O., Alexander R. L., Krebs E. G. Adenosine 3',5'-monophosphate-dependent protein kinase from adipose tissue. J Biol Chem. 1972 Jun 25;247(12):3736–3743. [PubMed] [Google Scholar]
  9. DeMeyts P., Bainco A. R., Roth J. Site-site interactions among insulin receptors. Characterization of the negative cooperativity. J Biol Chem. 1976 Apr 10;251(7):1877–1888. [PubMed] [Google Scholar]
  10. Ek B., Westermark B., Wasteson A., Heldin C. H. Stimulation of tyrosine-specific phosphorylation by platelet-derived growth factor. Nature. 1982 Feb 4;295(5848):419–420. doi: 10.1038/295419a0. [DOI] [PubMed] [Google Scholar]
  11. Harrison L. C., Itin A. Purification of the insulin receptor from human placenta by chromatography on immobilized wheat germ lectin and receptor antibody. J Biol Chem. 1980 Dec 25;255(24):12066–12072. [PubMed] [Google Scholar]
  12. Hedo J. A., Harrison L. C., Roth J. Binding of insulin receptors to lectins: evidence for common carbohydrate determinants on several membrane receptors. Biochemistry. 1981 Jun 9;20(12):3385–3393. doi: 10.1021/bi00515a013. [DOI] [PubMed] [Google Scholar]
  13. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Häring H. U., Biermann E., Kemmler W. Coupling of insulin binding and insulin action on glucose transport in fat cells. Am J Physiol. 1981 May;240(5):E556–E565. doi: 10.1152/ajpendo.1981.240.5.E556. [DOI] [PubMed] [Google Scholar]
  15. Häring H. U., Kasuga M., Kahn C. R. Insulin receptor phosphorylation in intact adipocytes and in a cell-free system. Biochem Biophys Res Commun. 1982 Oct 29;108(4):1538–1545. doi: 10.1016/s0006-291x(82)80082-x. [DOI] [PubMed] [Google Scholar]
  16. Kahn C. R., Baird K. The fate of insulin bound to adipocytes. Evidence for compartmentalization and processing. J Biol Chem. 1978 Jul 25;253(14):4900–4906. [PubMed] [Google Scholar]
  17. Kahn R., Murray M., Pawelek J. Inhibition of proliferation of cloudman S91 melanoma cells by insulin and characterization of some insulin-resistant variants. J Cell Physiol. 1980 Apr;103(1):109–119. doi: 10.1002/jcp.1041030116. [DOI] [PubMed] [Google Scholar]
  18. Kasuga M., Fujita-Yamaguchi Y., Blithe D. L., Kahn C. R. Tyrosine-specific protein kinase activity is associated with the purified insulin receptor. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2137–2141. doi: 10.1073/pnas.80.8.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kasuga M., Fujita-Yamaguchi Y., Blithe D. L., White M. F., Kahn C. R. Characterization of the insulin receptor kinase purified from human placental membranes. J Biol Chem. 1983 Sep 25;258(18):10973–10980. [PubMed] [Google Scholar]
  20. Kasuga M., Karlsson F. A., Kahn C. R. Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor. Science. 1982 Jan 8;215(4529):185–187. doi: 10.1126/science.7031900. [DOI] [PubMed] [Google Scholar]
  21. Kasuga M., Zick Y., Blith D. L., Karlsson F. A., Häring H. U., Kahn C. R. Insulin stimulation of phosphorylation of the beta subunit of the insulin receptor. Formation of both phosphoserine and phosphotyrosine. J Biol Chem. 1982 Sep 10;257(17):9891–9894. [PubMed] [Google Scholar]
  22. Kasuga M., Zick Y., Blithe D. L., Crettaz M., Kahn C. R. Insulin stimulates tyrosine phosphorylation of the insulin receptor in a cell-free system. Nature. 1982 Aug 12;298(5875):667–669. doi: 10.1038/298667a0. [DOI] [PubMed] [Google Scholar]
  23. King G. L., Kahn C. R. Non-parallel evolution of metabolic and growth-promoting functions of insulin. Nature. 1981 Aug 13;292(5824):644–646. doi: 10.1038/292644a0. [DOI] [PubMed] [Google Scholar]
  24. Koontz J. W., Iwahashi M. Insulin as a potent, specific growth factor in a rat hepatoma cell line. Science. 1981 Feb 27;211(4485):947–949. doi: 10.1126/science.7008195. [DOI] [PubMed] [Google Scholar]
  25. LIEBERMAN I., OVE P. Growth factors for mammalian cells in culture. J Biol Chem. 1959 Oct;234:2754–2758. [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. Machicao F., Urumow T., Wieland O. H. Phosphorylation--dephosphorylation of purified insulin receptor from human placenta. Effect of insulin. FEBS Lett. 1982 Nov 22;149(1):96–100. doi: 10.1016/0014-5793(82)81079-x. [DOI] [PubMed] [Google Scholar]
  28. Nishimura J., Huang J. S., Deuel T. F. Platelet-derived growth factor stimulates tyrosine-specific protein kinase activity in Swiss mouse 3T3 cell membranes. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4303–4307. doi: 10.1073/pnas.79.14.4303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Oppermann H., Levinson A. D., Varmus H. E., Levintow L., Bishop J. M. Uninfected vertebrate cells contain a protein that is closely related to the product of the avian sarcoma virus transforming gene (src). Proc Natl Acad Sci U S A. 1979 Apr;76(4):1804–1808. doi: 10.1073/pnas.76.4.1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pawelek J., Sansone M., Koch N., Christie G., Halaban R., Hendee J., Lerner A. B., Varga J. M. Melanoma cells resistant to inhibition of growth by melanocyte stimulating hormone. Proc Natl Acad Sci U S A. 1975 Mar;72(3):951–955. doi: 10.1073/pnas.72.3.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Petruzzelli L. M., Ganguly S., Smith C. J., Cobb M. H., Rubin C. S., Rosen O. M. Insulin activates a tyrosine-specific protein kinase in extracts of 3T3-L1 adipocytes and human placenta. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6792–6796. doi: 10.1073/pnas.79.22.6792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pike L. J., Gallis B., Casnellie J. E., Bornstein P., Krebs E. G. Epidermal growth factor stimulates the phosphorylation of synthetic tyrosine-containing peptides by A431 cell membranes. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1443–1447. doi: 10.1073/pnas.79.5.1443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rechler M. M., Podskalny J. M., Goldfine I. D., Wells C. A. DNA synthesis in human fibroblasts: stimulation by insulin and by nonsuppressible insulin-like activity (NSILA-S). J Clin Endocrinol Metab. 1974 Sep;39(3):512–521. doi: 10.1210/jcem-39-3-512. [DOI] [PubMed] [Google Scholar]
  34. Rosen O. M., Herrera R., Olowe Y., Petruzzelli L. M., Cobb M. H. Phosphorylation activates the insulin receptor tyrosine protein kinase. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3237–3240. doi: 10.1073/pnas.80.11.3237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Roth R. A., Cassell D. J. Insulin receptor: evidence that it is a protein kinase. Science. 1983 Jan 21;219(4582):299–301. doi: 10.1126/science.6849137. [DOI] [PubMed] [Google Scholar]
  36. Smith C. J., Wejksnora P. J., Warner J. R., Rubin C. S., Rosen O. M. Insulin-stimulated protein phosphorylation in 3T3-L1 preadipocytes. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2725–2729. doi: 10.1073/pnas.76.6.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ushiro H., Cohen S. Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A-431 cell membranes. J Biol Chem. 1980 Sep 25;255(18):8363–8365. [PubMed] [Google Scholar]
  38. Van Obberghen E., Kowalski A. Phosphorylation of the hepatic insulin receptor: stimulating effect of insulin on intact cells and in a cell-free system. FEBS Lett. 1982 Jul 5;143(2):179–182. doi: 10.1016/0014-5793(82)80094-x. [DOI] [PubMed] [Google Scholar]
  39. White M. F., Haring H. U., Kasuga M., Kahn C. R. Kinetic properties and sites of autophosphorylation of the partially purified insulin receptor from hepatoma cells. J Biol Chem. 1984 Jan 10;259(1):255–264. [PubMed] [Google Scholar]
  40. Witte O. N., Dasgupta A., Baltimore D. Abelson murine leukaemia virus protein is phosphorylated in vitro to form phosphotyrosine. Nature. 1980 Feb 28;283(5750):826–831. doi: 10.1038/283826a0. [DOI] [PubMed] [Google Scholar]
  41. Zick Y., Kasuga M., Kahn C. R., Roth J. Characterization of insulin-mediated phosphorylation of the insulin receptor in a cell-free system. J Biol Chem. 1983 Jan 10;258(1):75–80. [PubMed] [Google Scholar]
  42. Zick Y., Whittaker J., Roth J. Insulin stimulated phosphorylation of its own receptor. Activation of a tyrosine-specific protein kinase that is tightly associated with the receptor. J Biol Chem. 1983 Mar 25;258(6):3431–3434. [PubMed] [Google Scholar]

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