Insulin bound to the insulin receptor of IM-9 lymphocytes is more accessible to antiinsulin antibodies after treatment with dithiothreitol: bound insulin is deeply buried in the receptor-binding site.

Binding of insulin to its receptor followed by covalent cross-linking with disuccinimidyl suberate (DSS) dramatically impairs the ability of antiinsulin antibodies (both polyclonal and monoclonal) to bind to the insulin moiety. We have used dithiotheitol, which has major effects on the oligomeric structure of the insulin receptor, to determine if this decreased antibody recognition is due to alteration in the conformation of insulin itself or to steric factors. Treatment of the covalently cross-linked insulin-receptor complex with dithiothreitol (DTT) increased the ability of the polyclonal and two monoclonal antiinsulin antibodies to immunoprecipitate the insulin-receptor complex. This treatment decreased immunoprecipitation by antireceptor antibodies. The effect of DTT may have been due to a reversal of either a binding-induced conformational change in the insulin moiety or an alteration in the conformation of the insulin-receptor complex, thereby decreasing steric hindrance. In an effort to choose between these two possible explanations, we prepared a biotinylated derivative of insulin which was cross-linked to the receptor. Since the biotin moiety is relatively rigid, it seemed improbable that binding to the receptor would alter the conformation of the epitope recognized by antibiotin antibodies and that the change would be reversed by DTT. Treatment of the cross-linked biotin-insulin-receptor complex with DTT did increase the ability of both antiinsulin and antibiotin antibodies to immunoprecipitate the cross-linked receptor complex. Identification of the cross-linked receptor on reduced sodium dodecyl sulfate-polyacrylamide gels confirms that the DTT treatment alters the distribution of the oligomeric forms of the receptor. These studies favor the hypothesis that when bound to its receptor, most of the insulin molecule is sequestered within the receptor-binding site such that there is steric hindrance to the approach of antiinsulin antibodies. Moreover, DTT alters the conformation of the cross-linked insulin-receptor complex so as to decrease this steric hindrance.