CYSTEINE-647 IN THE INSULIN-RECEPTOR IS REQUIRED FOR NORMAL COVALENT INTERACTION BETWEEN ALPHA-SUBUNIT AND BETA-SUBUNIT AND SIGNAL TRANSDUCTION

We have investigated the structural and functional properties of two m utant insulin receptors in which Cys647 and Cys682,683,685 have been r eplaced with Ser (IR(S647) and IR(S682,683,685), respectively). Compar ed with the wild-type receptor (IR(WT)), both mutant receptors display ed altered sensitivities to dithiothreitol with respect to insulin bin ding and reduction of oligomeric forms. Subunit composition of the oli gomeric forms of the receptors as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of I-125-labeled r eceptors indicated that Cys682,683,685 are required for normal heterot etrameric structure and that Cys647 plays a major role in the normal c ovalent association of the alpha- and beta-subunits. Under nonreducing conditions, the affinity-labeled IR(S647) migrated, almost exclusivel y, as a 230-kDa species which appeared to represent an alpha(2) form o f the receptor. Furthermore, Chinese hamster ovary cells expressing IR (S647) did not exhibit basal or insulin-stimulated autophosphorylation , suggesting that Cys647 is also required for signal transduction.