Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Dec;85(24):9489–9493. doi: 10.1073/pnas.85.24.9489

ATP sensitizes the insulin receptor to insulin.

K D Ridge 1, K Hofmann 1, F M Finn 1
PMCID: PMC282778  PMID: 2849108

Abstract

Insulin receptor with high insulin binding and tyrosine kinase activities has been prepared from human placenta. Based on a molecular mass of 306 kDa for the receptor (the value obtained from the sum of the amino acid residues), this preparation is capable of binding 1.48 mol of insulin per mol of receptor. The receptor is free from phosphatase and ATPase activity and is not stimulated by sodium vanadate. Autophosphorylation is linear with respect to receptor concentration, and the 32P incorporated is stable even in the presence of a 100-fold excess of unlabeled ATP. The Km for ATP is 208 microM. N-Ethylmaleimide inhibits autophosphorylation. Alkylation with 3H-labeled N-ethylmaleimide results in the incorporation of 1.13 +/- 0.37 mol of N-ethylmaleimide per mol of insulin binding activity exclusively into the beta subunit of the receptor. The nonhydrolyzable ATP analog adenosine 5'-[beta,gamma-imido]triphosphate stimulates autophosphorylation of the receptor, an effect that is evident at ATP concentrations below 1 mM. The stimulatory effect of adenosine 5'-[beta,gamma-imido]triphosphate is the result of increasing the binding of insulin to the alpha subunit, and this reflects itself in a shift to the left of the insulin dose-response curve for autophosphorylation. The same is true for ATP. As a consequence, it is now possible to reconcile the concentration of insulin necessary for stimulating the autophosphorylation reaction with physiological levels and with the levels of insulin required for its classical biological effects.

Full text

PDF
9489

Images in this article

9490Image
on p.9490

Selected References

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

  1. AVRON M. Photophosphorylation by swiss-chard chloroplasts. Biochim Biophys Acta. 1960 May 20;40:257–272. doi: 10.1016/0006-3002(60)91350-0. [DOI] [PubMed] [Google Scholar]
  2. Brooker R. J., Slayman C. W. Inhibition of the plasma membrane [H+]-ATPase of Neurospora crassa by N-ethylmaleimide. Protection by nucleotides. J Biol Chem. 1982 Oct 25;257(20):12051–12055. [PubMed] [Google Scholar]
  3. Cantley L. C., Jr, Cantley L. G., Josephson L. A characterization of vanadate interactions with the (Na,K)-ATPase. Mechanistic and regulatory implications. J Biol Chem. 1978 Oct 25;253(20):7361–7368. [PubMed] [Google Scholar]
  4. Cantley L. C., Jr, Josephson L., Warner R., Yanagisawa M., Lechene C., Guidotti G. Vanadate is a potent (Na,K)-ATPase inhibitor found in ATP derived from muscle. J Biol Chem. 1977 Nov 10;252(21):7421–7423. [PubMed] [Google Scholar]
  5. Cooper J. A., Hunter T. Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells. Mol Cell Biol. 1981 Feb;1(2):165–178. doi: 10.1128/mcb.1.2.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cuatrecasas P. Isolation of the insulin receptor of liver and fat-cell membranes (detergent-solubilized-( 125 I)insulin-polyethylene glycol precipitation-sephadex). Proc Natl Acad Sci U S A. 1972 Feb;69(2):318–322. doi: 10.1073/pnas.69.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Finn F. M., Titus G., Horstman D., Hofmann K. Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7328–7332. doi: 10.1073/pnas.81.23.7328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fujita-Yamaguchi Y., Choi S., Sakamoto Y., Itakura K. Purification of insulin receptor with full binding activity. J Biol Chem. 1983 Apr 25;258(8):5045–5049. [PubMed] [Google Scholar]
  10. Fujita-Yamaguchi Y., Kathuria S. The monomeric alpha beta form of the insulin receptor exhibits much higher insulin-dependent tyrosine-specific protein kinase activity than the intact alpha 2 beta 2 form of the receptor. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6095–6099. doi: 10.1073/pnas.82.18.6095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goodno C. C. Inhibition of myosin ATPase by vanadate ion. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2620–2624. doi: 10.1073/pnas.76.6.2620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hofmann K., Romovacek H., Titus G., Ridge K., Raffensperger J. A., Finn F. M. The rat liver insulin receptor. Biochemistry. 1987 Nov 17;26(23):7384–7390. doi: 10.1021/bi00397a028. [DOI] [PubMed] [Google Scholar]
  13. Josephson L., Cantley L. C., Jr Isolation of a potent (Na-K)ATPase inhibitor from striated muscle. Biochemistry. 1977 Oct 18;16(21):4572–4578. doi: 10.1021/bi00640a006. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Machicao F., Urumow T., Wieland O. H. Evidence for phosphorylation of actin by the insulin receptor-associated protein kinase from human placenta. FEBS Lett. 1983 Oct 31;163(1):76–80. doi: 10.1016/0014-5793(83)81167-3. [DOI] [PubMed] [Google Scholar]
  17. O'Connell P. B., Brady C. J. Polyacrylamide gels with modified cross-linkages. Anal Biochem. 1976 Nov;76(50):63–73. doi: 10.1016/0003-2697(76)90264-5. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Schuurmans Stekhoven F., Bonting S. L. Transport adenosine triphosphatases: properties and functions. Physiol Rev. 1981 Jan;61(1):1–76. doi: 10.1152/physrev.1981.61.1.1. [DOI] [PubMed] [Google Scholar]
  21. Shia M. A., Rubin J. B., Pilch P. F. The insulin receptor protein kinase. Physicochemical requirements for activity. J Biol Chem. 1983 Dec 10;258(23):14450–14455. [PubMed] [Google Scholar]
  22. 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]
  23. Udenfriend S., Stein S., Böhlen P., Dairman W., Leimgruber W., Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972 Nov 24;178(4063):871–872. doi: 10.1126/science.178.4063.871. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Van Obberghen E., Rossi B., Kowalski A., Gazzano H., Ponzio G. Receptor-mediated phosphorylation of the hepatic insulin receptor: evidence that the Mr 95,000 receptor subunit is its own kinase. Proc Natl Acad Sci U S A. 1983 Feb;80(4):945–949. doi: 10.1073/pnas.80.4.945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wessel D., Flügge U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. doi: 10.1016/0003-2697(84)90782-6. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Wilden P. A., Boyle T. R., Swanson M. L., Sweet L. J., Pessin J. E. Alteration of intramolecular disulfides in insulin receptor/kinase by insulin and dithiothreitol: insulin potentiates the apparent dithiothreitol-dependent subunit reduction of insulin receptor. Biochemistry. 1986 Jul 29;25(15):4381–4388. doi: 10.1021/bi00363a031. [DOI] [PubMed] [Google Scholar]
  29. Wilden P. A., Pessin J. E. Differential sensitivity of the insulin-receptor kinase to thiol and oxidizing agents in the absence and presence of insulin. Biochem J. 1987 Jul 15;245(2):325–331. doi: 10.1042/bj2450325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Williams P. F., Turtle J. R. Purification of the insulin receptor from human placental membranes. Biochim Biophys Acta. 1979 Aug 28;579(2):367–374. doi: 10.1016/0005-2795(79)90064-3. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES