Ligand-dependent intersubunit association within the insulin receptor complex activates its intrinsic kinase activity.

Insulin receptor halves (alpha beta) were obtained upon selective reduction of the holoreceptor (alpha 2 beta 2) and were isolated in concentrated form. Autophosphorylation of concentrated alpha beta receptor halves can be stimulated by insulin an average of 4.0-fold, whereas nonreduced holoreceptor can be stimulated 5.4-fold. If alpha beta half-receptors are immobilized on wheat germ agglutinin-agarose, no insulin-stimulated autophosphorylation is observed, whereas immobilized holoreceptor retains insulin responsiveness. Treatment of alpha beta half-receptors with glutathione in the presence of insulin results in reoxidation to the holoreceptor form (alpha 2 beta 2) with an efficiency of 60-70% as visualized by immunoblotting, thus providing evidence that two alpha beta halves are in close physical proximity. This reoxidation reaction, which is evident prior to autophosphorylation, is rapid and strictly dependent on the presence of insulin, consistent with the hypothesis that insulin promotes the association of two alpha beta halves. Furthermore, the insulin-induced reoxidation reaction and the insulin-induced autophosphorylation show the same dose dependence ED50 3-4 x 10(-8) M insulin), suggesting that the noncovalent association of alpha beta half-receptors upon insulin binding is a prerequisite for insulin-stimulated autophosphorylation in concentrated alpha beta half-receptor preparations. If the alpha beta half-receptor forms are phosphorylated in the presence of an anti-phosphotyrosine antibody and separated from nonphosphorylated alpha beta receptors, we observed that the phosphorylated alpha beta receptor halves contain bound insulin. This excludes the possibility that alpha beta half-receptors that bind insulin, preferentially phosphorylate alpha beta halves that have no insulin bound.