A novel T608R missense mutation in insulin receptor substrate-1 identified in a subject with type 2 diabetes impairs metabolic insulin signaling.

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.

Novel insulin receptor substrate 1 and 2 variants in breast and colorectal cancer.

The insulin/insulin-like growth factor pathway is involved in breast and colorectal cancer (CRC) development. In the present study, we analyzed the coding region and short intron-exon borders of the insulin receptor substrate 1 and 2 (IRS­1 and IRS­2) genes in 12 cell lines derived from breast cancer (BC), 14 cell lines derived from CRC and 33 primary CRCs. The nucleotide variants identified in BC were 3 in IRS­1, 1 of which (p.Arg267Cys) was novel and with a pathogenic potential as predicted by in silico analysis and 6 in IRS­2. Twenty­one variants in IRS­1 and 18 in IRS­2 were identified in the CRC samples. These included 11 novel IRS­1 variants detected exclusively in CRCs, which included 5 missense (p.Pro559Leu, p.Gln655His, p.Asp1014Gly, p.Asp1181His and pPro1203Ser) with a pathogenic potential as predicted by in silico analysis, 2 frameshifts predicted to generate a truncated protein, 1 splice-site mutation and 3 silent variants. In the CRC samples we also identified 7 novel IRS­2 variants, including 4 missense variants, which included 2 (p.Asp782Asn and p.Gly1230Ser) with a pathogenic potential as predicted by in silico analysis, 2 frame insertion mutations and 1 silent variant. Most of the novel IRS­1 and IRS­2 variants may be involved in the modulation of IRS-1 or IRS­2 functions and could be relevant to breast and colorectal tumorigenesis.