An arginine to cysteine 252 mutation in insulin receptors from a patient with severe insulin resistance inhibits receptor internalisation but preserves signalling events

Abstract

Aims/hypothesis. We examined the properties of a mutant insulin receptor (IR) with an Arg 252 to Cys (IR R252C) substitution in the α-subunit originally identified in a patient with extreme insulin resistance and acanthosis nigricans. Methods. We studied IR cell biology and signalling pathways in Chinese Hamster Ovary cells overexpressing this IR R252C. Results. Our investigation showed an impairment in insulin binding to IR R252C related mostly to a reduced affinity of the receptor for insulin and to a reduced rate of IR R252C maturation; an inhibition of IR R252C-mediated endocytosis resulting in a decreased insulin degradation and insulin-induced receptor down-regulation; a maintenance of IR R252C on microvilli even in the presence of insulin; a similar autophosphorylation of mutant IR R252C followed by IRS 1/IRS 2 phosphorylation, p85 association with IRS 1 and IRS 2 and Akt phosphorylation similar to those observed in cells expressing wild type IR (IRwt); and finally, a reduced insulin-induced Shc phosphorylation accompanied by decreased ERK1/2 phosphorylation and activity and of thymidine incorporation into DNA in cells expressing IR R252C as compared to cells expressing IRwt. Conclusion/interpretation. These observations suggest that: parameters other than tyrosine kinase activation participate in or control the first steps of IR internalisation or both; IR-mediated IRS 1/2 phosphorylation can be achieved from the cell surface and microvilli in particular; Shc phosphorylation and its subsequent signalling pathway might require IR internalisation; defective IR endocytosis correlates with an enhancement of some biological responses to insulin and attenuation of others.