Functional properties of a heterozygous mutation (Arg¹¹⁷⁴ → Gln) in the tyrosine kinase domain of the insulin receptor from a Type A insulin resistant patient

Abstract

We analysed the biochemical properties of insulin receptors of a Type A insulin resistant patient with a single heterozygous point mutation substituting Gln for Arg 1174. Insulin binding capacity and affinty to Epstein-Barr virus transformed lymphocytes was normal. Quantitative analysis of autophosphorylation and substrate phosphorylation of soluble insulin receptors isolated from patient cells revealed no differences in the basal state whereas in the presence of insulin autophosphorylation activity was only 30% of control receptors. The stimulation of substrate phosphorylation and down-regulation of receptors on patient cells after chronic exposure to insulin was diminished when compared to controls. We conclude that the heterozygous Arg 1174 mutation does not perturb basal kinase activity but specifically interferes with the kinase activation by insulin and that the mutation has a dominant negative effect on the wild type kinase.

Partial rescue of in vivo insulin signalling in skeletal muscle by impaired insulin clearance in heterozygous carriers of a mutation in the insulin receptor gene

“…In agreement with a study of the Arg1174Gln mutation in patient lymphocytes transformed by Epstein-Barr virus [5], the number of insulin receptors in muscle tended to be lower (36%) in Arg1174Gln carriers compared with control subjects. Studies of CHO cells cotransfected with wild-type and mutant INSR cDNA have shown that the Arg1174Gln mutation is more susceptible to proteasomal degradation and impairs the formation of mt/wt hybrids, causing an increased fraction (∼55%) of fully functional (wt/wt) receptors at the cell surface [32].…”

“…All ten family members affected by hypoglycaemia carried a heterozygous mutation in the IRTK domain of INSR (Arg1174Gln). This mutation has previously been found in three females with the type A syndrome of insulin resistance, and in an apparently healthy male [4][5][6][7]. Complete cosegregation (logarithm of the odds score 3.21) with the disease phenotype supported the proposition that the Arg1174Gln mutation was the cause of hypoglycaemia [3].…”

“…The signalling properties of the naturally occurring Arg1174Gln mutation in INSR have been studied previous- [7,19] and in patient lymphocytes transformed by Epstein-Barr virus [5]. In transfected cells, only receptors formed by two mutant (mt) alleles-mt/mt receptors-are synthesised, and the mutation is present in a homozygous state, which facilitates the interpretation of receptor function studies.…”

“…In transfected cells, only receptors formed by two mutant (mt) alleles-mt/mt receptors-are synthesised, and the mutation is present in a homozygous state, which facilitates the interpretation of receptor function studies. In contrast, patient cells transformed by Epstein-Barr virus provide the possibility of studying the mutation in a heterozygous state similar to the situation in vivo, in which all three possible receptor species-wt/wt, wt/mt hybrid and mt/mt receptors-are formed in an expected ratio of 1:2:1 [1,5]. In the present case-control study, we had the opportunity to investigate the functional consequences of a heterozygous mutation in INSR in skeletal muscle in vivo.…”

“…Studies of the functional properties of the Arg1174Gln mutation have shown normal insulin binding and affinity to the receptor, but a 70-75% reduction in insulin-stimulated IRTK activity [5][6][7]. Furthermore, in CHO cell lines homozygous for the mutant INSR, stimulation with insulin in the physiological range (1,000 pmol/l) showed a pronounced inability to increase tyrosine phosphorylation of IRS-1, PI3K activity, SLC2A4 translocation and glycogen synthesis [7,19].…”