title: Structural basis by which a recessive mutation in the alpha-subunit of the insulin receptor affects insulin binding.
abstract: Recently, a mutation substituting Leu for Ser323 in the alpha-subunit of the human insulin receptor has been identified in an insulin-resistant patient. The Leu323 mutation leads to a severe impairment in insulin binding without significantly altering the processing or cell surface expression of the receptor. In order to study how alpha beta half-receptors interact to form the insulin-binding site, we cotransfected NIH-3T3 cells with two insulin receptor cDNA constructs: a truncated insulin receptor lacking the C-terminal 43 amino acids (delta 43) and the full-length Leu323 mutant receptor. A clonal cell line from cotransfected cells expresses a hybrid receptor consisting of a Leu323 half-receptor and a delta 43 half-receptor. We demonstrate that the Leu323-delta 43 hybrid receptor binds insulin with high affinity. Furthermore, by cross-linking 125I-insulin to immobilized hybrid receptors, we show that only the alpha beta delta half of the hybrid receptor binds insulin. Since the isolated half-insulin receptor has low affinity for insulin, this suggests that the addition of even a non-binding alpha-subunit can result in high affinity binding to the holoreceptor (alpha alpha mut beta delta beta). Both beta and beta delta-subunits of the Leu323-delta 43 hybrid receptor are phosphorylated in vivo and in vitro in an insulin-dependent manner, suggesting an intramolecular transphosphorylation mechanism and that the presence of the Leu323 mutant receptor that lacks an intrinsic high affinity binding site does not prevent the associated beta-subunit from functioning either as a tyrosine kinase or as a phosphate acceptor in the hybrid insulin receptor molecule (alpha alpha mut beta delta beta). Furthermore, we show that the hybrid receptor can phosphorylate insulin receptor substrate-1 (IRS-1) in response to insulin and can be coimmunoprecipitated together with IRS-1 by anti-IRS-1 antibody.
authors.affiliation: Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: M
authors.initials: M
authors.lastname: Taouis
authors.forename: R
authors.initials: R
authors.lastname: Levy-Toledano
authors.forename: P
authors.initials: P
authors.lastname: Roach
authors.forename: S I
authors.initials: SI
authors.lastname: Taylor
authors.forename: P
authors.initials: P
authors.lastname: Gorden
mesh_terms: D016475:3T3 Cells; D000818:Animals; D005808:Genes, Recessive; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D007930:Leucine / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
chemical_list: C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D010750:Phosphoproteins; D012694:Serine; D011972:Receptor, Insulin; D007930:Leucine