title: Two naturally occurring mutant insulin receptors phosphorylate insulin receptor substrate-1 (IRS-1) but fail to mediate the biological effects of insulin. Evidence that IRS-1 phosphorylation is not sufficient for normal insulin action.
abstract: Two naturally occurring mutant insulin receptors, Arg-1174 --> Gln and Leu-1178 --> Pro, found in patients with dominantly inherited Type A insulin resistance, showed unusual signaling properties when stably expressed in Chinese hamster ovary (CHO) cells. Both mutant receptors were expressed on the cell surface and bound insulin normally, but showed markedly impaired autophosphorylation in response to insulin. In addition, the in vitro tyrosine kinase activity of both mutant receptors toward an artificial substrate was also severely impaired. Despite these defects of kinase activity, anti-phosphotyrosine immunoblotting of whole cell lysates and anti-phosphotyrosine immunoprecipitation of 32P-labeled cells showed insulin-stimulated tyrosine phosphorylation of a protein of approximately 185 kDa to an extent comparable to that seen in CHO cells expressing wild-type human insulin receptors. Anti-insulin receptor substrate-1 (IRS-1) immunoprecipitation followed by anti-phosphotyrosine immunoblotting confirmed that this tyrosine-phosphorylated protein was IRS-1. In contrast, CHO cells expressing an insulin receptor mutated at the ATP binding site (Lys-1030 --> Arg) showed no insulin-stimulated autophosphorylation or phosphorylation of IRS-1. Despite exhibiting apparently normal insulin stimulation of IRS-1 tyrosine-phosphorylation, cells expressing the Arg-1174 --> Gln or Pro-1178 --> Leu receptors showed marked impairment in insulin stimulation of glycogen synthesis, thymidine incorporation, and activation of MAP kinase. The inability of these mutant receptors to signal normally to metabolic and mitogenic responses suggests that insulin-stimulated tyrosine phosphorylation of IRS-1 alone is insufficient to fully mediate insulin action.
authors.affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR, United Kingdom. Harvard Medical School, Boston, Massachusetts.
authors.forename: A
authors.initials: A
authors.lastname: Krook
authors.forename: D E
authors.initials: DE
authors.lastname: Moller
authors.forename: K
authors.initials: K
authors.lastname: Dib
authors.forename: S
authors.initials: S
authors.lastname: O'Rahilly
mesh_terms: D000293:Adolescent; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011494:Protein Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D017931:DNA Primers; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D014443:Tyrosine; D011494:Protein Kinases; D011972:Receptor, Insulin