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. 1987 Feb;84(3):704–708. doi: 10.1073/pnas.84.3.704

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.

Y Ebina, E Araki, M Taira, F Shimada, M Mori, C S Craik, K Siddle, S B Pierce, R A Roth, W J Rutter
PMCID: PMC304284  PMID: 3101064

Abstract

To test whether the tyrosine kinase activity of the insulin receptor is crucial for insulin action, we have constructed mutations of the human insulin receptor at Lys-1030, which is in the presumed ATP-binding region. By using oligonucleotide-directed mutagenesis, this lysine residue was replaced with either methionine, arginine, or alanine. Chinese hamster ovary cells were transfected by mutant cDNAs and the expressed insulin receptors were characterized. We show here that none of these mutants exhibited insulin-activated autophosphorylation and kinase activity in vitro. They also do not mediate insulin- and antibody-stimulated uptake of 2-deoxyglucose. The tyrosine kinase activity is thus required for a key physiological response of insulin.

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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., Alexander M. C., Palmer J. L., Pierce M. W., Nemenoff R. A., Blackshear P. J., Tipper J. P., Witters L. A. Role of insulin-stimulated protein phosphorylation in insulin action. Fed Proc. 1982 Aug;41(10):2629–2633. [PubMed] [Google Scholar]
  2. Craik C. S., Largman C., Fletcher T., Roczniak S., Barr P. J., Fletterick R., Rutter W. J. Redesigning trypsin: alteration of substrate specificity. Science. 1985 Apr 19;228(4697):291–297. doi: 10.1126/science.3838593. [DOI] [PubMed] [Google Scholar]
  3. Czech M. P. The nature and regulation of the insulin receptor: structure and function. Annu Rev Physiol. 1985;47:357–381. doi: 10.1146/annurev.ph.47.030185.002041. [DOI] [PubMed] [Google Scholar]
  4. Ebina Y., Edery M., Ellis L., Standring D., Beaudoin J., Roth R. A., Rutter W. J. Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8014–8018. doi: 10.1073/pnas.82.23.8014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ebina Y., Ellis L., Jarnagin K., Edery M., Graf L., Clauser E., Ou J. H., Masiarz F., Kan Y. W., Goldfine I. D. The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling. Cell. 1985 Apr;40(4):747–758. doi: 10.1016/0092-8674(85)90334-4. [DOI] [PubMed] [Google Scholar]
  6. Ellis L., Clauser E., Morgan D. O., Edery M., Roth R. A., Rutter W. J. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell. 1986 Jun 6;45(5):721–732. doi: 10.1016/0092-8674(86)90786-5. [DOI] [PubMed] [Google Scholar]
  7. Gardell S. J., Craik C. S., Hilvert D., Urdea M. S., Rutter W. J. Site-directed mutagenesis shows that tyrosine 248 of carboxypeptidase A does not play a crucial role in catalysis. Nature. 1985 Oct 10;317(6037):551–555. doi: 10.1038/317551a0. [DOI] [PubMed] [Google Scholar]
  8. Grigorescu F., Flier J. S., Kahn C. R. Defect in insulin receptor phosphorylation in erythrocytes and fibroblasts associated with severe insulin resistance. J Biol Chem. 1984 Dec 25;259(24):15003–15006. [PubMed] [Google Scholar]
  9. 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]
  10. Hunter T., Cooper J. A. Protein-tyrosine kinases. Annu Rev Biochem. 1985;54:897–930. doi: 10.1146/annurev.bi.54.070185.004341. [DOI] [PubMed] [Google Scholar]
  11. Kahn C. R. The molecular mechanism of insulin action. Annu Rev Med. 1985;36:429–451. doi: 10.1146/annurev.me.36.020185.002241. [DOI] [PubMed] [Google Scholar]
  12. Kamps M. P., Sefton B. M. Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src. Mol Cell Biol. 1986 Mar;6(3):751–757. doi: 10.1128/mcb.6.3.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kamps M. P., Taylor S. S., Sefton B. M. Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites. Nature. 1984 Aug 16;310(5978):589–592. doi: 10.1038/310589a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Morgan D. O., Ho L., Korn L. J., Roth R. A. Insulin action is blocked by a monoclonal antibody that inhibits the insulin receptor kinase. Proc Natl Acad Sci U S A. 1986 Jan;83(2):328–332. doi: 10.1073/pnas.83.2.328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Roth R. A., Cassell D. J., Maddux B. A., Goldfine I. D. Regulation of insulin receptor kinase activity by insulin mimickers and an insulin antagonist. Biochem Biophys Res Commun. 1983 Aug 30;115(1):245–252. doi: 10.1016/0006-291x(83)90996-8. [DOI] [PubMed] [Google Scholar]
  18. Simpson I. A., Hedo J. A. Insulin receptor phosphorylation may not be a prerequisite for acute insulin action. Science. 1984 Mar 23;223(4642):1301–1304. doi: 10.1126/science.6367041. [DOI] [PubMed] [Google Scholar]
  19. Snyder M. A., Bishop J. M., McGrath J. P., Levinson A. D. A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity. Mol Cell Biol. 1985 Jul;5(7):1772–1779. doi: 10.1128/mcb.5.7.1772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Soos M. A., Siddle K., Baron M. D., Heward J. M., Luzio J. P., Bellatin J., Lennox E. S. Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor. Biochem J. 1986 Apr 1;235(1):199–208. doi: 10.1042/bj2350199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  22. Takayama S., White M. F., Lauris V., Kahn C. R. Phorbol esters modulate insulin receptor phosphorylation and insulin action in cultured hepatoma cells. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7797–7801. doi: 10.1073/pnas.81.24.7797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tamura S., Brown T. A., Whipple J. H., Fujita-Yamaguchi Y., Dubler R. E., Cheng K., Larner J. A novel mechanism for the insulin-like effect of vanadate on glycogen synthase in rat adipocytes. J Biol Chem. 1984 May 25;259(10):6650–6658. [PubMed] [Google Scholar]
  24. Tamura S., Fujita-Yamaguchi Y., Larner J. Insulin-like effect of trypsin on the phosphorylation of rat adipocyte insulin receptor. J Biol Chem. 1983 Dec 25;258(24):14749–14752. [PubMed] [Google Scholar]
  25. 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]
  26. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  27. Zick Y., Rees-Jones R. W., Taylor S. I., Gorden P., Roth J. The role of antireceptor antibodies in stimulating phosphorylation of the insulin receptor. J Biol Chem. 1984 Apr 10;259(7):4396–4400. [PubMed] [Google Scholar]
  28. Zoller M. J., Nelson N. C., Taylor S. S. Affinity labeling of cAMP-dependent protein kinase with p-fluorosulfonylbenzoyl adenosine. Covalent modification of lysine 71. J Biol Chem. 1981 Nov 10;256(21):10837–10842. [PubMed] [Google Scholar]
  29. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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