A Novel T608R Missense Mutation in Insulin Receptor Substrate-1 Identified in a Subject with Type 2 Diabetes Impairs Metabolic Insulin Signaling

Abstract

Naturally occurring mutations in insulin receptor substrate-1 (IRS-1) have previously been implicated in impaired insulin action. We now report a novel mutation in IRS-1 with substitution of Arg for Thr(608) that was identified in a patient with type 2 diabetes mellitus. We detected the T608R mutation in 1 of 136 chromosomes from diabetic patients and in 0 of 120 chromosomes from nondiabetic controls, suggesting that this is a rare IRS-1 variant. Conservation of Thr(608) in human, monkey, rat, mouse, and chicken IRS-1 sequences is consistent with a crucial function for this residue. Moreover, Thr(608) is located near the YMXM motif containing Tyr(612) that is important for binding and activation of phosphoinositol 3-kinase (PI 3-kinase). To investigate whether the T608R mutation impairs insulin signaling, we transiently transfected NIH-3T3(IR) cells with hemagglutinin-tagged wild-type or T608R mutant IRS-1 constructs. Recombinant IRS-1 immunoprecipitated from transfected cells treated with or without insulin was subjected to immunoblotting for the p85 regulatory subunit of PI 3-kinase as well as a PI 3-kinase assay. As expected, in control cells transfected with wild-type IRS-1, insulin stimulation caused an increase in p85 coimmunoprecipitated with IRS-1 as well as a 10-fold increase in IRS-1-associated PI 3-kinase activity. Interestingly, when cells transfected with IRS1-T608R were stimulated with insulin, both the amount of p85 coimmunoprecipitated with IRS1-T608R as well as the associated PI 3-kinase activity were approximately 50% less than those observed with wild-type IRS-1. Moreover, in rat adipose cells, overexpression of IRS1-T608R resulted in significantly less translocation of GLUT4 to the cell surface than comparable overexpression of wild-type IRS-1. We conclude that a naturally occurring substitution of Arg for Thr(608) in IRS-1 is a rare human mutation that may contribute to insulin resistance by impairing metabolic signaling through PI 3-kinase-dependent pathways.

Insulin receptor substrate (IRS)‐1 regulates murine embryonic stem (mES) cells self‐renewal

“…We assayed the basal phosphorylation status of IRS-1 on tyrosine residues using an antibody against phospho-tyrosine 608 of IRS-1. This tyrosine residue has been found to be important for the binding of IRS-1 to the p85 regulatory subunit of PI3K (Yamamoto-Honda et al, 1996;Esposito et al, 2003;Valverde et al, 2003). We found that in the presence of LIF, IRS-1 is tyrosine phosphorylated, and the signal decreases after LIF withdrawal and induction of differentiation (Fig.…”

Viral infection and human disease - insights from minimotifs

“…Diabetes mellitus can be caused by rare mutations in IRS-1 that lead to insulin resistance. Mutation of T 608 xxxYxxM 615 → RxxxYxxM in IRS-1 alters a residue near the consensus YxxM motif for binding to the SH2 domain in the p85 subunit of phosphatidylinositol 3-kinase (PI3K) (45). This mutation effects association of IRS-1 with PI3K and insulin-induced PI3K activity, suggesting a role for a residue that is located four residues N-terminal to the critical tyrosine.…”

Modulation of insulin action

“…In fact, PKB-mediated phosphorylation of mouse IRS-1 on serine residues 265, 302, 325 and 358 enhances its action in 293 cells [99] and in vitro a S/T phosphorylation threshold on IRS-1 and 2 must be achieved for full tyrosine phosphorylation by the activated IR to occur [56]. Genetic evidence for a positive role of S/T phosphorylation is provided by the human IRS-1 T608R polymorphism recently identified in a diabetic patient, which impaired metabolic insulin signalling, possibly due to the removal of a potential positive threonine phosphorylation site [100].…”