Insulin-mimetic anti-insulin receptor monoclonal antibodies stimulate receptor kinase activity in intact cells.
In the present studies, nine different monoclonal antibodies to the extracellular domain of the insulin receptor were tested in three different cell types for their ability to stimulate the intrinsic tyrosine kinase activity of the receptor. Previous studies had suggested that several of these monoclonal antibodies stimulate biological responses without stimulating the intrinsic tyrosine kinase activity of the receptor (Hawley, D. M., Maddux, B. A., Patel, R. G., Wong, K. Y., Manula, P. W., Firestone, G. L., Brunetti, A., Verspohl, E., and Goldfine, I. D. (1989) J. Biol. Chem. 264, 2438-2444 and Soos, M. A., O'Brien, R. M., Brindle, N. P. J., Stigter, J. M., Okamoto, A. K., Whittaker, J., and Siddle, K. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5217-5221). In the present study, a more sensitive assay was utilized, and these same monoclonal antibodies, when added to intact cells, were found to stimulate the phosphotransferase activity of the receptor. This increase in activity was reversed by phosphatase treatment of the receptor. In contrast, monoclonal antibodies which had no insulin-mimetic activities did not stimulate the receptor's kinase activity. In addition, Western blot analyses of lysates with anti-phosphotyrosine antibodies showed that insulin-mimetic, but not non-insulin-mimetic antibodies, stimulated tyrosine phosphorylation of the receptor as well as an endogenous substrate (phosphoprotein Mr = 160,000). Finally, these antibodies were found to stimulate the tyrosine phosphorylation of another endogenous substrate of the insulin receptor kinase, the type I phosphatidylinositol kinase. These studies support the hypothesis that monoclonal antibodies, like insulin, stimulate biological responses via their ability to stimulate the tyrosine kinase activity of the receptor.
Steele-Perkins
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Department of Pharmacology, Stanford University School of Medicine, California 94305-5332.
Roth
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D019870:1-Phosphatidylinositol 4-Kinase; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000276:immunology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007328:Insulin; D019000:Phosphotyrosine; D014443:Tyrosine; D004247:DNA; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D010744:Phosphoric Monoester Hydrolases