Role of arginine 86 of the insulin receptor in insulin binding and activation of glucose transport

Abstract

Mutations in the insulin receptor gene cause the inherited insulin resistant syndrome leprechaunism. Patient Atl-1 with leprechaunism was homozygous for the substitution of Arg-86 with Pro (R86P) in the α subunit of the insulin receptor. Fibroblasts homozygous for the mutant receptor had defective insulin binding, but increased glucose transport and receptor kinase activity. The R86P mutation is located in a putative β turn N-terminal to a proposed insulin binding domain of the receptor [P. DeMeyts, J.-L. Gu, R.M. Shymko, B.E. Kaplan, G.I. Bell, J. Whittaker, Mol. Endocrinol. 4 (1990) 409–416]. To get further insight into the mechanism of the paradoxical activation of receptor signalling by the R86P mutation, the codons for proline, alanine, and glycine were substituted in the R86 position of the insulin receptor cDNA by PCR-mediated mutagenesis and stably transfected into Chinese hamster ovary (CHO) cells. Insulin binding increased 10–20 fold in CHO cells transfected with the wild type, the R86A, and the R86G insulin receptor cDNA, but did not increase in cells expressing the R86P mutation. The R86P mutation caused a constitutive activation of insulin receptor phosphorylation in CHO cells, but did not increase basal glucose transport or its sensitivity to insulin stimulation. By contrast, transfection with the wild type and the R86A receptors increased 20–30 fold the sensitivity of glucose transport to stimulation by insulin. The R86G insulin receptor bound insulin normally, but was four times less efficient than the wild type or R86A insulin receptor in increasing the sensitivity for insulin stimulation of glucose transport. These results indicate that position 86 of the insulin receptor α subunit is tolerant to substitution by alanine, but not by proline. Substitution with glycine allows insulin binding, but does not activate normally glucose transport, further supporting an essential role of this position in the initiation of insulin receptor signalling of glucose transport.