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.
Esposito
Diana L
DL
Department of Oncology and Neurosciences, Section of Molecular Pathology, University Gabriele D'Annunzio, 66013 Chieti, Italy.
Li
Yunhua
Y
Vanni
Cinzia
C
Mammarella
Sandra
S
Veschi
Serena
S
Della Loggia
Fulvio
F
Mariani-Costantini
Renato
R
Battista
Pasquale
P
Quon
Michael J
MJ
Cama
Alessandro
A
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D051275:Glucose Transporter Type 4; D006801:Humans; D007163:Immunosorbent Techniques; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007333:Insulin Resistance / Q000235:genetics; D008297:Male; D051379:Mice; D008875:Middle Aged; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D020125:Mutation, Missense; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D051381:Rats; D015398:Signal Transduction; D014162:Transfection
D051275:Glucose Transporter Type 4; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D019869:Phosphatidylinositol 3-Kinases