ALA(1048)-]ASP MUTATION IN THE KINASE DOMAIN OF INSULIN-RECEPTOR CAUSES DEFECTIVE KINASE-ACTIVITY AND INSULIN-RESISTANCE

We identified a heterozygous missense mutation that substituted aspart ic acid (GAC) for alanine (GCC) at codon 1048 of the insulin receptor gene in a patient who displayed typical symptoms of Type A syndrome of insulin resistance. The proband's mother and younger brother were als o found to be heterozygous for the mutation. We constructed the identi fied mutant insulin receptor cDNA by site-directed mutagenesis, transf ected the mutant cDNA into COS 7 cells, and found that kinase activity of the mutant insulin receptors was markedly impaired. Ala1048 is loc ated in the kinase domain of the insulin receptor beta-subunit and is conserved in most of protein-tyrosine kinases. Besides, neighboring Gl u1047 is invariant in all protein kinases and is thought to be involve d in interaction with ATP. Photoaffinity labeling of the mutant insuli n receptor with ATP analogue, 8-azido (alpha-P-32)ATP was not influenc ed by the mutation, suggesting that the mutation did not inhibit ATP b inding but possibly interfered with subsequent phosphoryl transfer. In sulin-stimulated phosphorylation of exogenous substrate by partially p urified insulin receptors prepared from COS 7 cells that were cotransf ected with wild-type and mutant insulin receptor cDNAs was markedly im paired, whereas autophosphorylation was decreased by approximately 50% of wild-type receptors. These results indicated that the identified h eterozygous substitution of Asp for Ala1048 in insulin receptor was re sponsible for insulin resistance of this patient.