title: Localization of the insulin-binding site to the cysteine-rich region of the insulin receptor alpha-subunit.
abstract: Affinity-purified insulin receptor was photoaffinity labeled with a cleavable radioactive insulin photoprobe. Exhaustive digestion of the labeled alpha-subunit with endoproteinase Glu-C produced a major radioactive fragment of 23 kDa as a part of the putative insulin-binding domain. This fragment could contain either residues 205-316 or 518-633 of the alpha-subunit. Rat hepatoma cells and Chinese hamster ovary cells were transfected with cDNA encoding a human insulin receptor mutant with a deletion of the cysteine-rich region spanning amino acid residues 124-319. Insulin binding by these cells was not increased in spite of high numbers of the mutant insulin receptors being expressed. A panel of monoclonal antibodies which was specific for the receptor alpha-subunit and inhibited insulin binding immunoprecipitated the photolabeled 23-kDa receptor fragment but not the receptor mutant. A synthetic peptide containing residues 243-251 was specifically bound by agarose-insulin beads. We therefore suggest that the 23-kDa fragment contains residues 205-316, and that insulin binding occurs, in part, in the cysteine-rich region of the alpha-subunit.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Ont., Canada.
authors.forename: C C
authors.initials: CC
authors.lastname: Yip
authors.forename: H
authors.initials: H
authors.lastname: Hsu
authors.forename: R G
authors.initials: RG
authors.lastname: Patel
authors.forename: D M
authors.initials: DM
authors.lastname: Hawley
authors.forename: B A
authors.initials: BA
authors.lastname: Maddux
authors.forename: I D
authors.initials: ID
authors.lastname: Goldfine
mesh_terms: D000345:Affinity Labels; D001665:Binding Sites; D003545:Cysteine; D004252:DNA Mutational Analysis; D007328:Insulin / Q000378:metabolism*; D015394:Molecular Structure; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010777:Photochemistry; D011233:Precipitin Tests; D011972:Receptor, Insulin / Q000378:metabolism*; D014162:Transfection
chemical_list: D000345:Affinity Labels; D007328:Insulin; D009842:Oligopeptides; D011972:Receptor, Insulin; D003545:Cysteine