Biochemistry zyxwvu 1983, 22, zyxwvut 2519-2586 2579 Kretsinger, R. H. (1980a) CRC Crit. Rev. Biochem., 119. Kretsinger, R. H. (1980b) Ann. N.Y. Acad. Sci. 356, 14. Levine, B. A., Mercola, D., Coffman, D., zyxwvuts & Thornton, J. M. (1977) zyxwvutsrqpon J. Mol. Biol. 115, 743. Malencik, D. A,, Anderson, zyxwvutsrq S. R., Shalitin, Y., & Schimerlik, M. I. (1980) Biochem. Biophys. Res. Commun. 101,390. Moews, P. C., & Kretsinger, R. H. (1975) J. Mol. Biol. 91, 201. Richman, P. G., & Klee, C. B. (1978) Biochemistry 17, 928. Seamon, K. B. (1979) Biochem. Biophys. Res. Commun. 86, Seamon, K. B. (1980) Biochemistry 19, 207. Seamon, K. B., & Moore, B. W. (1980) J. Biol. Chem. 255, Sin, J. L., Fernandes, R., & Marcola, D. (1978) Biochem. 1256. 11644. Biophys. Res. Commun. 82, 1132. Teo, T. S., Wang, T. H., & Wang, J. H. (1973) J. Biol. Chem. 248, 588. Wallace, R. W., Tallant, E. W., Dockter, M. E., & Cheung, W. Y. (1982) J. Biol. Chem. 275, 1845. Wang, C. A., Aquaron, R. R., Leavis, P. C., & Gergely, J. (1982) Eur. J. Biochem. 124, 7. Wolff, D. J., Poirier, P. G., Brostrom, C. O., & Brostrom, M. A. (1977) J. Biol. Chem. 252, 4108. Yagi, K., Matsuda, S., Nagamoto, H., Mikuni, T., & Yazawa, M. (1982) in Calmodulin and Intracellular zyx Ca++ Receptors (Kakiuchi, S., Hidaka, H., & Means, A. R., Eds.) p 75, Plenum Press, New York. Yazawa, M., Kuwayama, H., & Yagi, K. (1978) J. Biochem. (Tokyo) 84, 1253. Yazawa, M., Sakuma, M., & Yagi, K. (1980) J. Biochem. (Tokyo) 87, 1313. Insulin Receptor: Insulin-Modulated Interconversion between Distinct Molecular Forms Involving Disulfide-Sulfhydryl Exchanget Joseph M. Maturo III,* Morley D. Hollenberg, and Linda S. Aglio* ABSTRACT: When insulin receptor was isolated from human placenta membranes by a sequential chromatographic pro- cedure (method I) that did not expose the receptor to insulin, the purified receptor (about 2000-fold purification) was eluted on columns of Sepharose 6B in a volume corresponding to a K,, of 0.31 (R, form of the receptor, with an apparent Stokes radius of about 7.2 nm). In contrast, the placenta receptor isolated by insulin-agarose affinity chromatography (method 11) was eluted on Sepharose 6B columns with a K,, of 0.53 (RII form of the receptor, with an apparent Stokes radius of about 3.8 nm). When receptor prepared by method I was exposed to insulin, the RI form of the receptor was converted to the RII form; this insulin-mediated change in the receptor elution behavior was mimicked by dithiothreitol (0.1 mM) treatment. In concert with the insulin-mediated conversion of the RI to the RII form of the receptor, we observed insu- lin-stimulated incorporation of 3H-labeled N-ethylmaleimide (3H-NEM) into the receptor preparation. Both the insulin- mediated interconversion of the receptor from the RI to the RII form and the insulin-stimulated incorporation of 3H-NEM were dependent on insulin concentration; the concentration dependence indicated that a half-maximal effect occurred at about 1 nM insulin. Both 1251-labeled receptor, converted to the RII form in the presence of insulin, and the RII form of the receptor labeled with 3H-NEM in the presence of insulin T e r e has recently been considerable progress in under- standing the molecular structure of the receptor for insulin + From the Biology Department, C. W. Post College, Greenvale, New York 11548 (J.M.M. and L.S.A.), and the Endocrine Research Group, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Canada T2N 4N1 (M.D.H.). Receiued August 27, 1982. This work was supported in large part by grants-in-aid from C. W. Post College and in some part by a grant (to M.D.H.) from the Canadian Medical Research Council. L.S.A. was supported by a Student National Institutes of Health fellowship granted to the Albert Einstein College of Medicine, Bronx, New York, NY. were bound by anti-insulin receptor immunoglobulin, obtained from an individual with severe insulin resistance. Exposure of the RI form of the receptor to insulin-agarose also led concurrently to the conversion of the receptor from the RI to the RII form and to the incorporation of 3H-NEM. The R,, form of the receptor obtained by method I1 from insulin- agarose could be converted back to the RI form by treatment with oxidized glutathione. The experiments with purified receptor preparations were complemented by studies with particulate membrane preparations cross-link labeled with '251-labeled insulin both at low (0.5 ng/mL) and comparatively high (25 ng/mL) insulin concentrations. Solubilization and immunoaffinity purification of receptor, cross-link labeled in the membranes with 0.5 ng/mL IZ5I-labeled insulin, yielded material that on Sepharose 5B columns behaved like the RI form of the receptor. In contrast, immunoaffinity-isolated receptor, cross-link labeled in the membranes with 25 ng/mL '251-labeled insulin, behaved on Sepharose 6B columns like the RII form of the receptor; receptor cross-link labeled at the high (25 ng/mL) insulin concentration (but not at 0.5 ng/mL insulin) simultaneously incorporated 3H-NEM. Our results indicate that both in membranes and in purified soluble re- ceptor preparations, insulin causes an interconversion of its receptor from one hydrodynamic form to another by a process involving a disulfide-sulfhydryl exchange. [current views summarized in volume edited by Andreani et al. (1981)l. One salient feature of the heterodimeric structure proposed for the insulin receptor (Jacobs et al., 1980; Massagut et al., 1980) relates to the presence of a number of disulfide bonds that stabilize the proposed oligomeric (aP)2 structure. It is further evident that the entire receptor structure, as it exists in the cell membrane and in crude detergent extracts of cell membranes, may comprise not only the recog- nition oligomer that has been the species of intensive inves- tigation but may also include other closely associated poly- peptide chains. Evidence for the presence of such receptor- 0006-2%0/83/0422-2519$01.50/0 @ 1983 American Chemical Society