Insulin binding leads to the formation of covalent (-S-S-) hormone receptor complexes.

Abstract

The recent finding that the lymphocyte insulin receptor is covalently labeled after '261-insulin binding (Saviolaskis, G., Harrison, L. c., and Roth, J. (1981) J. BioL Chem 256,4924-4928) led us to directly test a previous hypothesis that insulin binds via disulfide bonds. "% Insulin was bound to isolated rat adipocytes at 37 "C, the cells were washed extensively at 37 "C to remove nondissociable radioactivity, and then extracted in Triton X-lOO/Dulbecco's buffer containing protease inhibitors. The solubilized radioactivity obtained after centrifugation at 100,000 X g was chromatographed on Sephadex G-50 and resolved as a major high molecular weight (HMW) peak and two smaller peaks representing intact and degraded "%insulin, respectively. The formation of the HMW component was dependent on the concentration of 1261-insulin, although the total formed did not exceed 10% of the specifically bound radioactivity. Its formation was inhibited in a dosedependent manner by sulfhydryl-blocking agents in concentrations that did not alter specific binding. The HMW component was also formed in paraformaldehyde-treated cells and in adipocyte membranes. It eluted at the position of the native Triton-solubilized insulin receptor on Sepharose CL6B chromatography and was immunoprecipitated by antireceptor antibodies. Radioactivity bound to the receptor in the HMW complex was dissociated by the reducing agent dithiothreitol and eluted off Sepharose CL-GB at the position of reduced '261-insulin. Oxidative sulfitolysis revealed that the radioactivity in the HMW complex was present in both A and B chains of insulin, in the same proportions as in intact '261-insulin. We conciude that a fraction of bound insuLin becomes covalently linked to its receptor via a disulfide-sulfhydry1 exchange on the cell surface. The significance of this modification in initiating insulin action deserves further study.

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@article{Clark1982InsulinBL, title={Insulin binding leads to the formation of covalent (-S-S-) hormone receptor complexes.}, author={Sharon F. Clark and Leonard C. Harrison}, journal={The Journal of biological chemistry}, year={1982}, volume={257 20}, pages={12239-44} }