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 α₂β₂ heterotetrameric complexes that are assembled from two identical αβ heterodimeric half-receptor precursors. Recent evidence suggests that insulin and IGF-I half-receptors can heterologously assemble to form α₂β₂ 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 (αβ_INS.WT) or IGF-I (αβIGF.WT) half-receptors assembled with a kinase-defective insulin half-receptor mutant (αβ_INS.A/K). In vitro assembly of either (αβ)_IGF.WT/(αβ)_INS.A/K or (αβ)_INS.WT/(αβ)_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 (αβ)_INS.WT/(αβ)_INS.A/K hybrid receptor complex was completely unaffected in comparison to the wild-type (αβ)_INS.WT/(αβ)_INS.WT receptor. To assess a molecular basis for this difference, autophosphorylation of a hybrid receptor composed of a truncated β-subunit insulin half-receptor with the kinase-defective half-receptor, (αβ)_INS.ΔCT/ (αβ)_INS.A/K, demonstrated the exclusive autophosphorylation of the (αβ)_INS.A/K half-receptor β subunit. These results demonstrate that ligand-dependent substrate phosphorylation by insulin and IGF-I holoreceptors requires interactions between two functional β subunits within the α₂β₂ heterotetrameric complex and occurs through an intramolecular trans-phosphorylation reaction.