Role of disulfides in the subunit structure of the insulin receptor. Reduction of class I disulfides does not impair transmembrane signalling.

Abstract

The native insulin receptor affinity-labeled by covalent cross-linking to 125I-insulin has been proposed to consist of two alpha receptor subunits and two beta-receptor subunits all disulfide-linked as a Mr = 350,000 (beta-S-S-alpha)-S-S-(alpha-S-S-beta) receptor complex (Massague, J., Pilch, P. F., and Czech, M. P. (1981) J. Biol. Chem. 256, 3181-3190). We denote the disulfide bonds linking the two symmetrical (alpha-S-S-beta) receptor halves as class I insulin receptor disulfides, whereas the disulfide bonds linking one alpha receptor subunit to one beta receptor subunit are termed class II disulfides. In the present studies, dithiothreitol added directly to intact rat fat cells or to membranes prepared from rat fat cell, rat liver, or human placenta, reduced the class I disulfides, but not the class II disulfides, of the insulin receptor. Reduction of class I insulin receptor disulfides did not prevent binding of insulin to the insulin receptor. After treatment with dithiothreitol, the number of apparent "high affinity" insulin binding sites was increased in rat fat cells or fat cell membranes but was decreased in rat liver and human placenta membranes. Reduction of class I insulin receptor disulfides did not alter the sensitivity to insulin of glucose oxidation in intact rat fat cells. The resistance of class II disulfides to reduction by dithiothreitol was not affected by solubilization of the membrane structure by Triton X-100. Class II insulin receptor disulfides were fully reduced by dithiothreitol only after denaturation of the insulin receptor by sodium dodecyl sulfate. Class I insulin receptor disulfides were partially reoxidized by an incubation mixture consisting of reduced and oxidized glutathione. When solubilized insulin receptor containing reduced class I disulfides was exposed to ethylene glycol bis(succinimidyl succinate), the receptor subunits were internally cross-linked and migrated like intact receptor complexes on nonreduced dodecyl sulfate gels. We conclude that the (alpha-S-S-beta) receptor halves in the partially reduced receptor remain associated by noncovalent interactions and that this receptor structure without class I disulfides remains biologically competent.