ROLE OF ARGININE-86 OF THE INSULIN-RECEPTOR IN INSULIN BINDING AND ACTIVATION OF GLUCOSE-TRANSPORT

Mutations in the insulin receptor gene cause the inherited insulin res istant syndrome leprechaunism. Patient Atl-1 with leprechaunism was ho mozygous for the substitution of Arg-86 with Pro (R86P) in the alpha s ubunit of the insulin receptor. Fibroblasts homozygous for the mutant receptor had defective insulin binding, but increased glucose transpor t and receptor kinase activity. The R86P mutation is located in a puta tive beta 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, Mel. Endocrinol. 4 (1990) 409-416]. To get further insigh t into the mechanism of the paradoxical activation of receptor signall ing 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 o vary (CHO) cells. Insulin binding increased 10-20 fold in CHO cells tr ansfected 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 pho sphorylation in CHO cells, but did not increase basal glucose transpor t or its sensitivity to insulin stimulation. By contrast, transfection with the wild type and the R86A receptors increased 20-30 fold the se nsitivity of glucose transport to stimulation by insulin. The R86G ins ulin receptor bound insulin normally, but was four times less efficien t than the wild type or R86A insulin receptor in increasing the sensit ivity for insulin stimulation of glucose transport. These results indi cate that position 86 of the insulin receptor cu subunit is tolerant t o substitution by alanine, but not by proline. Substitution with glyci ne allows insulin binding, but does not activate normally glucose tran sport, further supporting an essential role of this position in the in itiation of insulin receptor signalling of glucose transport. (C) 1998 Elsevier Science B.V.