TRANSDOMINANT INHIBITION OF TYROSINE KINASE-ACTIVITY IN MUTANT INSULIN/INSULIN-LIKE GROWTH FACTOR-I HYBRID RECEPTORS

Classical insulin and insulin-like growth factor I (IGF-I) receptors exist as well defined alpha-2-beta-2 heterotetrameric complexes that are assembled from two identical alpha-beta heterodimeric half-receptor precursors. Recent evidence suggests that insulin and IGF-I half-receptors can heterologously assemble to form alpha-2-beta-2 insulin/IGF-I hybrid receptor complexes in vivo and in vitro. We have utilized hybrid receptor complexes to examine ligand-stimulated transmembrane signaling of wild-type insulin (alpha-beta(INS.WT) or IGF-I (alpha-beta(IGF.WT)) half-receptors or (alpha-beta)INS.WT/(alpha-beta)INS.A/K hybrid receptors resulted in decreased substrate protein kinase activity. The degree of protein kinase inactivation directly correlated with the amount of immunologically cross-reactive hybrid receptors formed. In contrast to substrate kinase activity, insulin-stimulated autophosphorylation of the (alpha-beta)INS.WT/(alpha-beta)INS.A/K hybrid receptor complex was completely unaffected in comparison to the wild type (alpha-beta)INS.WT/(alpha-beta)INS/WT receptor. To assess a molecular basis for this difference, autophosphorylation of a hybrid receptor composed of a truncated beta-subunit insulin half-receptor with the kinase-defective half-receptor, (alpha-beta)INS.DELTA-CT/(alpha-beta)INS.A/K, demonstrated the exclusive autophosphorylation of the (alpha-beta)INS.A/K half-receptor beta-subunit. These results demonstrrate that ligand-dependent substrate phosphorylation by insulin and IGF-I holoreceptors requires interactions between two functional beta-subunits within the alpha-2-beta-2 heterotetrameric complex and occurs through an intramolecular trans-phosphorylation reaction.