Abstract
Monoclonal antibodies for the human insulin receptor were produced following immunization of mice with IM-9 lymphocytes and/or purified placental receptor. Four separate fusions yielded 28 antibodies, all of which reacted with receptor from human placenta, liver and IM-9 cells. Some antibodies cross-reacted to varying degrees with receptor from rabbit, cow, pig and sheep, but none reacted with rat receptor. At least 10 distinct epitopes were recognized as indicated by species specificity and binding competition experiments. All of these epitopes appeared to be on extracellular domains of the receptor as shown by binding of antibodies to intact cells. In some cases the epitopes were further localized to alpha or beta subunits by immunoblotting. Several antibodies inhibited binding of 125I-insulin to the receptor, some had no effect on binding, and others enhanced the binding of 125I-insulin. It is concluded that these antibodies will be valuable probes of receptor structure and function.
Full text
PDF









Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Baron M. D., Sönksen P. H. Characterization of two insulin-binding components of rat-liver plasma membranes. Biosci Rep. 1982 Oct;2(10):785–793. doi: 10.1007/BF01114938. [DOI] [PubMed] [Google Scholar]
- Beyer C. F. A 'dot-immunobinding assay' on nitrocellulose membrane for the determination of the immunoglobulin class of mouse monoclonal antibodies. J Immunol Methods. 1984 Feb 24;67(1):79–87. doi: 10.1016/0022-1759(84)90087-5. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- Fujita-Yamaguchi Y. Characterization of purified insulin receptor subunits. J Biol Chem. 1984 Jan 25;259(2):1206–1211. [PubMed] [Google Scholar]
- 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]
- Galfrè G., Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 1981;73(Pt B):3–46. doi: 10.1016/0076-6879(81)73054-4. [DOI] [PubMed] [Google Scholar]
- Gazzano H., Kowalski A., Fehlmann M., Van Obberghen E. Two different protein kinase activities are associated with the insulin receptor. Biochem J. 1983 Dec 15;216(3):575–582. doi: 10.1042/bj2160575. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gill G. N., Kawamoto T., Cochet C., Le A., Sato J. D., Masui H., McLeod C., Mendelsohn J. Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem. 1984 Jun 25;259(12):7755–7760. [PubMed] [Google Scholar]
- Grunfeld C. Antibody against the insulin receptor causes disappearance of insulin receptors in 3T3-L1 cells: a possible explanation of antibody-induced insulin resistance. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2508–2511. doi: 10.1073/pnas.81.8.2508. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hales C. N., Woodhead J. S. Labeled antibodies and their use in the immunoradiometric assay. Methods Enzymol. 1980;70(A):334–355. doi: 10.1016/s0076-6879(80)70063-0. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Jacobs S., Cuatrecasas P. Insulin receptor antibodies. Methods Enzymol. 1981;74(Pt 100):471–478. doi: 10.1016/0076-6879(81)74033-3. [DOI] [PubMed] [Google Scholar]
- Jacobs S., Cuatrecasas P. Insulin receptors. Annu Rev Pharmacol Toxicol. 1983;23:461–479. doi: 10.1146/annurev.pa.23.040183.002333. [DOI] [PubMed] [Google Scholar]
- Jacobs S., Hazum E., Cuatrecasas P. The subunit structure of rat liver insulin receptor. Antibodies directed against the insulin-binding subunit. J Biol Chem. 1980 Jul 25;255(14):6937–6940. [PubMed] [Google Scholar]
- Kahn C. R., Baird K. L., Flier J. S., Grunfeld C., Harmon J. T., Harrison L. C., Karlsson F. A., Kasuga M., King G. L., Lang U. C. Insulin receptors, receptor antibodies, and the mechanism of insulin action. Recent Prog Horm Res. 1981;37:477–538. doi: 10.1016/b978-0-12-571137-1.50015-3. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Kemmler W., Renner R., Zynamon A., Hepp K. D. Interactions between insulins and liver membrane receptors of guinea pig, calf and chicken. Exclusion of a species-specific insulin receptor. Biochim Biophys Acta. 1978 Oct 18;543(3):349–356. doi: 10.1016/0304-4165(78)90052-1. [DOI] [PubMed] [Google Scholar]
- Kull F. C., Jr, Jacobs S., Su Y. F., Cuatrecasas P. A monoclonal antibody to human insulin receptor. Biochem Biophys Res Commun. 1982 Jun 15;106(3):1019–1026. doi: 10.1016/0006-291x(82)91813-7. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Linde S., Hansen B., Sonne O., Holst J. J., Gliemann J. Tyrosine A14[125I]monoiodoinsulin: Preparation, Biologic Properties, and long-term stability. Diabetes. 1981 Jan;30(1):1–8. doi: 10.2337/diab.30.1.1. [DOI] [PubMed] [Google Scholar]
- Lindstrom J., Tzartos S., Gullick W., Hochschwender S., Swanson L., Sargent P., Jacob M., Montal M. Use of monoclonal antibodies to study acetylcholine receptors from electric organs, muscle, and brain and the autoimmune response to receptor in myasthenia gravis. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 1):89–99. doi: 10.1101/sqb.1983.048.01.012. [DOI] [PubMed] [Google Scholar]
- Parker P. J., Young S., Gullick W. J., Mayes E. L., Bennett P., Waterfield M. D. Monoclonal antibodies against the human epidermal growth factor receptor from A431 cells. Isolation, characterization, and use in the purification of active epidermal growth factor receptor. J Biol Chem. 1984 Aug 10;259(15):9906–9912. [PubMed] [Google Scholar]
- Pilch P. F., Czech M. P. Hormone binding alters the conformation of the insulin receptor. Science. 1980 Dec 5;210(4474):1152–1153. doi: 10.1126/science.7003712. [DOI] [PubMed] [Google Scholar]
- 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]
- Roth J. Methods for assessing immunologic and biologic properties of iodinated peptide hormones. Methods Enzymol. 1975;37:223–233. doi: 10.1016/s0076-6879(75)37018-3. [DOI] [PubMed] [Google Scholar]
- Roth R. A., Cassell D. J., Wong K. Y., Maddux B. A., Goldfine I. D. Monoclonal antibodies to the human insulin receptor block insulin binding and inhibit insulin action. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7312–7316. doi: 10.1073/pnas.79.23.7312. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roth R. A., Maddux B., Wong K. Y., Styne D. M., Van Vliet G., Humbel R. E., Goldfine I. D. Interactions of a monoclonal antibody to the insulin receptor with receptors for insulin-like growth factors. Endocrinology. 1983 May;112(5):1865–1867. doi: 10.1210/endo-112-5-1865. [DOI] [PubMed] [Google Scholar]
- Soos M., Siddle K. Characterization of monoclonal antibodies directed against human thyroid stimulating hormone. J Immunol Methods. 1982;51(1):57–68. doi: 10.1016/0022-1759(82)90382-9. [DOI] [PubMed] [Google Scholar]
- 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]
- Wisher M. H., Baron M. D., Jones R. H., Sönksen P. H. Photoreactive insulin analogues used to characterise the insulin receptor. Biochem Biophys Res Commun. 1980 Jan 29;92(2):492–498. doi: 10.1016/0006-291x(80)90360-5. [DOI] [PubMed] [Google Scholar]