Functional Properties of a Naturally Occurring Trp1200→ Ser1200Mutation of the Insulin Receptor

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Abstract
Based on the sequence of cDNA encoding the intracellular domain of the insulin receptor β-subunit, we recently defined a heterozygous point mutation causing a Ser for Trp substitution at position 1200 in the tyrosine kinase domain of a patient (BI-2) with the type A syndrome of insulin resistance. We have now sequenced the remainder of Bl-2's insulin receptor cDNA-coding region and find no additional alterations in the encoded proreceptor protein. The nucleotide sequence of cDNA encoding the portion of the β3-subunit which includes Trp1200 was normal in Bl-2's unaffected mother. Hybridization of a mutant allele-specific oligonucleotide to polymerase chain reaction-amplified cDNA confirmed the presence of the mutant allele in the proband and excluded it in her unaffected sister and mother, 18 normal control subjects, and six other subjects with insulin resistance. To determine whether this mutation had functional consequences for receptor signalling, we reconstructed it into a full-length insulin receptor cDNA expression vector. Chinese hamster ovary cells were transfected with mutant cDNA, and the expressed insulin receptors were compared to receptors expressed by cells transfected with wild-type receptor cDNA. Both mutant and wild-type receptors were properly processed into receptor α- and β- subunits, were expressed on the cell surface, and displayed similar insulin-binding affinity. In contrast, insulin-stimulated autophosphorylation of the mutant receptors was severely impaired, whether assessed in intact cells or with a partially purified receptor preparation. The protein tyrosine kinase activity of the mutant receptor was also deficient, as assessed by both phosphorylation of the endogenous substrate pp185 in intact cells and ability of receptors to phosphorylate an exogenous peptide substrate in vitro. These studies demonstrate an important role for Trp1200 in the normal function of the insulin receptor kinase and suggest a similar role for this amino acid in homologous membrane tyrosine kinases. We have also demonstrated that severe insulin resistance may be caused by a mutation in one allele of the insulin receptor gene.

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