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. 1985 Dec;82(23):7899–7903. doi: 10.1073/pnas.82.23.7899

An antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity.

R Herrera, L Petruzzelli, N Thomas, H N Bramson, E T Kaiser, O M Rosen
PMCID: PMC390877  PMID: 2415975

Abstract

Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the beta subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the beta subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor; it was not detected when the receptor was autophosphorylated prior to addition of AbP4. AbP4 did not inhibit activity of the related epidermal growth factor (EGF)-receptor tyrosine protein kinase nor did it inhibit the activity of cAMP-dependent kinase or protein kinase C. The observation that an antibody directed to residues 952-967 of the proreceptor neutralizes the protein kinase activity of the beta subunit suggests that this region may play a critical role in the function of the hormone-dependent, protein tyrosine-specific kinase activity of the insulin receptor.

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Selected References

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

  1. 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]
  2. Bishop J. M. Cellular oncogenes and retroviruses. Annu Rev Biochem. 1983;52:301–354. doi: 10.1146/annurev.bi.52.070183.001505. [DOI] [PubMed] [Google Scholar]
  3. Blake J., Li C. H. The synthesis and biological activity of (165, 182, 189-S-carbamidomethylcysteine)-human growth hormone-(140-191). Int J Pept Protein Res. 1975;7(6):495–501. doi: 10.1111/j.1399-3011.1975.tb02471.x. [DOI] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Cohen S., Carpenter G., King L., Jr Epidermal growth factor-receptor-protein kinase interactions. Co-purification of receptor and epidermal growth factor-enhanced phosphorylation activity. J Biol Chem. 1980 May 25;255(10):4834–4842. [PubMed] [Google Scholar]
  6. Deutsch P. J., Wan C. F., Rosen O. M., Rubin C. S. Latent insulin receptors and possible receptor precursors in 3T3-L1 adipocytes. Proc Natl Acad Sci U S A. 1983 Jan;80(1):133–136. doi: 10.1073/pnas.80.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Flier J. S., Kahn C. R., Roth J., Bar R. S. Antibodies that impair insulin receptor binding in an unusual diabetic syndrome with severe insulin resistance. Science. 1975 Oct 3;190(4209):63–65. doi: 10.1126/science.170678. [DOI] [PubMed] [Google Scholar]
  8. Grunberger G., Zick Y., Gorden P. Defect in phosphorylation of insulin receptors in cells from an insulin-resistant patient with normal insulin binding. Science. 1984 Mar 2;223(4639):932–934. doi: 10.1126/science.6141638. [DOI] [PubMed] [Google Scholar]
  9. Hedo J. A., Kahn C. R., Hayashi M., Yamada K. M., Kasuga M. Biosynthesis and glycosylation of the insulin receptor. Evidence for a single polypeptide precursor of the two major subunits. J Biol Chem. 1983 Aug 25;258(16):10020–10026. [PubMed] [Google Scholar]
  10. Jacobs S., Chang K. J., Cuatrecasas P. Antibodies to purified insulin receptor have insulin-like activity. Science. 1978 Jun 16;200(4347):1283–1284. doi: 10.1126/science.663609. [DOI] [PubMed] [Google Scholar]
  11. Jacobs S., Hazum E., Shechter Y., Cuatrecasas P. Insulin receptor: covalent labeling and identification of subunits. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4918–4921. doi: 10.1073/pnas.76.10.4918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacobs S., Kull F. C., Jr, Cuatrecasas P. Monensin blocks the maturation of receptors for insulin and somatomedin C: identification of receptor precursors. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1228–1231. doi: 10.1073/pnas.80.5.1228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jarrett D. B., Roth J., Kahn C. R., Flier J. S. Direct method for detection and characterization of cell surface receptors for insulin by means of 125I-labeled autoantibodies against the insulin receptor. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4115–4119. doi: 10.1073/pnas.73.11.4115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kahn C. R., Baird K. L., Jarrett D. B., Flier J. S. Direct demonstration that receptor crosslinking or aggregation is important in insulin action. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4209–4213. doi: 10.1073/pnas.75.9.4209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kahn C. R., Baird K., Filier J. S., Jarrett D. B. Effects of autoantibodies to the insulin receptor on isolated adipocytes. Studies of insulin binding and insulin action. J Clin Invest. 1977 Nov;60(5):1094–1106. doi: 10.1172/JCI108861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Kull F. C., Jr, Jacobs S., Su Y. F., Svoboda M. E., Van Wyk J. J., Cuatrecasas P. Monoclonal antibodies to receptors for insulin and somatomedin-C. J Biol Chem. 1983 May 25;258(10):6561–6566. [PubMed] [Google Scholar]
  19. Langone J. J. 125I-Labeled protein A: reactivity with IgG and use as a tracer in radioimmunoassay. Methods Enzymol. 1980;70(A):356–375. doi: 10.1016/s0076-6879(80)70064-2. [DOI] [PubMed] [Google Scholar]
  20. Le Peuch C. J., Ballester R., Rosen O. M. Purified rat brain calcium- and phospholipid-dependent protein kinase phosphorylates ribosomal protein S6. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6858–6862. doi: 10.1073/pnas.80.22.6858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lerner R. A., Green N., Alexander H., Liu F. T., Sutcliffe J. G., Shinnick T. M. Chemically synthesized peptides predicted from the nucleotide sequence of the hepatitis B virus genome elicit antibodies reactive with the native envelope protein of Dane particles. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3403–3407. doi: 10.1073/pnas.78.6.3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Massague J., Pilch P. F., Czech M. P. A unique proteolytic cleavage site on the beta subunit of the insulin receptor. J Biol Chem. 1981 Apr 10;256(7):3182–3190. [PubMed] [Google Scholar]
  23. Massague J., Pilch P. F., Czech M. P. Electrophoretic resolution of three major insulin receptor structures with unique subunit stoichiometries. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7137–7141. doi: 10.1073/pnas.77.12.7137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Naito S., Ueda T. Affinity-purified anti-protein I antibody. Specific inhibitor of phosphorylation of protein I, a synaptic protein. J Biol Chem. 1981 Oct 25;256(20):10657–10663. [PubMed] [Google Scholar]
  25. 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]
  26. Petruzzelli L., Herrera R., Rosen O. M. Insulin receptor is an insulin-dependent tyrosine protein kinase: copurification of insulin-binding activity and protein kinase activity to homogeneity from human placenta. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3327–3331. doi: 10.1073/pnas.81.11.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pilch P. F., Czech M. P. Interaction of cross-linking agents with the insulin effector system of isolated fat cells. Covalent linkage of 125I-insulin to a plasma membrane receptor protein of 140,000 daltons. J Biol Chem. 1979 May 10;254(9):3375–3381. [PubMed] [Google Scholar]
  28. Ronnett G. V., Knutson V. P., Kohanski R. A., Simpson T. L., Lane M. D. Role of glycosylation in the processing of newly translated insulin proreceptor in 3T3-L1 adipocytes. J Biol Chem. 1984 Apr 10;259(7):4566–4575. [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Rubin C. S., Erlichman J., Rosen O. M. Cyclic adenosine 3',5'-monophosphate-dependent protein kinase of human erythrocyte membranes. J Biol Chem. 1972 Oct 10;247(19):6135–6139. [PubMed] [Google Scholar]
  32. Siegel T. W., Ganguly S., Jacobs S., Rosen O. M., Rubin C. S. Purification and properties of the human placental insulin receptor. J Biol Chem. 1981 Sep 10;256(17):9266–9273. [PubMed] [Google Scholar]
  33. Stadtmauer L. A., Rosen O. M. Phosphorylation of exogenous substrates by the insulin receptor-associated protein kinase. J Biol Chem. 1983 Jun 10;258(11):6682–6685. [PubMed] [Google Scholar]
  34. Taylor J. W., Miller R. J., Kaiser E. T. Characterization of an amphiphilic helical structure in beta-endorphin through the design, synthesis, and study of model peptides. J Biol Chem. 1983 Apr 10;258(7):4464–4471. [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. Yip C. C., Yeung C. W., Moule M. L. Photoaffinity labeling of insulin receptor of rat adiopocyte plasma membrane. J Biol Chem. 1978 Mar 25;253(6):1743–1745. [PubMed] [Google Scholar]
  39. Yu K. T., Czech M. P. Tyrosine phosphorylation of the insulin receptor beta subunit activates the receptor-associated tyrosine kinase activity. J Biol Chem. 1984 Apr 25;259(8):5277–5286. [PubMed] [Google Scholar]
  40. 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]
  41. Zick Y., Rees-Jones R. W., Grunberger G., Taylor S. I., Moncada V., Gorden P., Roth J. The insulin-stimulated receptor kinase is a tyrosine-specific casein kinase. Eur J Biochem. 1983 Dec 15;137(3):631–637. doi: 10.1111/j.1432-1033.1983.tb07872.x. [DOI] [PubMed] [Google Scholar]

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