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 formation of hybrid dimers between normal and mutant receptors may explain the dominant negative effect of these mutations. To investigate this mechanism, we expressed two types of human insulin receptors in NIH-3T3 cells; wild type and the tyrosine kinase-deficient Ile1153 mutant. To distinguish the two types of receptors, 43 amino acids were deleted 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. Nevertheless, insulin stimulated the kinase competent delta 43 receptors to transphosphorylate the kinase-deficient Ile1153 mutant receptor in co-transfected cells via an intermolecular mechanism. Furthermore, transphosphorylation of the Ile1153 mutant receptor is sufficient to trigger insulin-stimulated endocytosis. Despite the absence of alpha 2 beta beta mut hybrids, expression of the Ile1153 mutant receptor inhibited the ability of the delta 43 truncated receptor to mediate insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1). Evidence is presented to support the hypothesis that the Ile1153 mutant receptor retains the ability to bind IRS-1, and that sequestration of substrate may explain the dominant negative effect of the mutant receptor to inhibit phosphorylation of IRS-1.