Insulin-mimetic anti-insulin receptor monoclonal antibodies stimulate receptor kinase activity in intact cells
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1Department of Pharmacology, Stanford University School of Medicine, California 94305-5332.
Abstract:
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.
Insights
Monoclonal antibodies targeting the insulin receptor activate its tyrosine kinase activity, stimulating biological responses. This research clarifies the mechanism by which these antibodies mimic insulin signaling.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Previous studies suggested some monoclonal antibodies (mAbs) to the insulin receptor (IR) elicit biological responses without activating IR's intrinsic tyrosine kinase activity.
- This raised questions about the signaling mechanisms of IR-targeting mAbs.
Purpose of the Study:
- To investigate the ability of nine different monoclonal antibodies to the extracellular domain of the insulin receptor to stimulate its intrinsic tyrosine kinase activity.
- To clarify the role of tyrosine kinase activation in mediating the biological responses induced by these monoclonal antibodies.
Main Methods:
- Testing nine monoclonal antibodies against the insulin receptor in three cell types.
- Utilizing a sensitive assay to measure receptor phosphotransferase activity in intact cells.
- Performing Western blot analyses with anti-phosphotyrosine antibodies.
- Assessing the effect of phosphatase treatment on receptor activity.
Main Results:
- All tested monoclonal antibodies, when added to intact cells, stimulated the phosphotransferase activity of the insulin receptor.
- This stimulation was reversed by phosphatase treatment.
- Insulin-mimetic monoclonal antibodies, unlike non-insulin-mimetic ones, stimulated tyrosine phosphorylation of the receptor and its substrates, including a 160,000 Mr phosphoprotein and type I phosphatidylinositol kinase.
- Western blot analysis confirmed increased tyrosine phosphorylation.
Conclusions:
- Monoclonal antibodies to the insulin receptor stimulate biological responses by activating the receptor's tyrosine kinase activity.
- This provides evidence supporting the hypothesis that insulin-mimetic antibodies, similar to insulin, signal through IR tyrosine kinase activation.