Transmembrane signaling by the human insulin receptor kinase. Relationship between intramolecular β subunit trans- and cis-autophosphorylation and substrate kinase activation

To examine the role of intramolecular β subunit trans- and cis- autophosphorylation in signal transduction, the vaccinia virus/bacteriophage T7 expression system was used to generate insulin holoreceptors composed of a kinase-defective half-receptor precursor (αβ(A/K) or αβ(A/K.ΔCT)) and a kinase-active half-receptor precursor (αβ(ΔCT) or αβ(WT)). In the αβ(A/K)-αβ(ΔCT) hybrid insulin receptor, insulin stimulated a 20-fold increase in intramolecular β subunit trans-phosphorylation, whereas cis- phosphorylation increased only 3-fold over the basal state. Similarly, in the αβ(WT)-αβ(A/K.ΔCT) hybrid insulin receptor, insulin stimulated trans- phosphorylation approximately 30-fold and cis-phosphorylation only 3-fold over the basal state. Although cis-phosphorylation of the kinase-functional αβ half-receptor was observed within these hybrid receptor species, this was not sufficient to stimulate exogenous substrate kinase activity. These data demonstrate that insulin primarily activates an intramolecular β subunit trans-phosphorylation reaction within the insulin holoreceptor and suggest that this reaction is necessary for activation of the holoreceptors. Furthermore, our results suggest a molecular basis for the dominant-negative phenotype observed in insulin-resistant patients possessing one kinase- defective insulin receptor allele.