INVESTIGATION OF THE MECHANISM OF THE DOMINANT-NEGATIVE EFFECT OF MUTATIONS IN THE TYROSINE KINASE DOMAIN OF THE INSULIN-RECEPTOR

Mutations in the tyrosine kinase domain of the insulin receptor cause insulin resistance in a dominant fashion. It has been proposed that fo rmation of hybrid dimers between normal and mutant receptors may expla in the dominant negative effect of these mutations. To investigate thi s mechanism, we expressed two types of human insulin receptors in NIH- 3T3 cells; wild type and the tyrosine kinase-deficient Ile(1153) mutan t. To distinguish the two types of receptors, 43 amino acids were dele ted from the C-terminus of the wild type receptor (Delta 43 truncation ). If mutant and wild type receptors assemble in a random fashion, 50% of the receptors would be hybrid oligomers (alpha(2) beta beta(mut)). However, alpha(2) beta beta(mut) hybrids were undetectable. Neverthel ess, insulin stimulated the kinase competent Delta 43 receptors to tra nsphosphorylate the kinase-deficient Ile(1153) mutant receptor in co-t ransfected cells via an intermolecular mechanism. Furthermore, transph osphorylation of the Ile(1153) mutant receptor is sufficient to trigge r insulin-stimulated endocytosis. Despite the absence of alpha(2) beta beta(mut) hybrids, expression of the Ile(1153) mutant receptor inhibi ted the ability of the Delta 43 truncated receptor to mediate insulin- stimulated phosphorylation of insulin receptor substrate-1 (IRS-1). Ev idence is presented to support the hypothesis that the Ile(1153) mutan t receptor retains the ability to bind IRS-1, and that sequestration o f substrate may explain the dominant negative effect of the mutant rec eptor to inhibit phosphorylation of IRS-1.