title: Recombinant human insulin receptor substrate-1 protein. Tyrosine phosphorylation and in vitro binding of insulin receptor kinase.
abstract: Insulin receptor substrate-1 (IRS-1) is a major endogenous substrate of the insulin receptor. To study the interaction of the insulin receptor with IRS-1 in vitro, we expressed in Escherichia coli the amino acids 516-777 of human IRS-1 (hIRS-p30) covering five potential tyrosine phosphorylation sites within YXXM motifs. Kinetic data for tyrosine phosphorylation of hIRS-p30 by partially purified insulin receptor and insulin-like growth factor I receptor and by baculovirus-expressed insulin receptor kinase domain were determined. Native insulin receptor demonstrated the highest affinity to hIRS-p30 (Km = 6.8 +/- 0.6 microM), followed by the insulin-like growth factor I receptor (Km = 9.9 +/- 1.0 microM). We used the soluble recombinant insulin receptor kinase domain, which phosphorylated hIRS-p30 with high affinity (Km = 11.9 +/- 0.8 microM), and affinity columns prepared by coupling hIRS-p30 to NHS-activated Sepharose for binding assays. The insulin receptor kinase domain phosphorylated the hIRS-p30 on the column, was bound by the immobilized hIRS-p30, and was eluted with high salt buffer. Autophosphorylated and EDTA-inactivated insulin receptor kinase domain was bound only by immobilized hIRS-p30 protein that has been prephosphorylated. Our results indicate that the recombinant hIRS-p30 protein is a high affinity substrate for insulin receptor and insulin-like growth factor I receptor in vitro. Moreover, we show that only tyrosine-phosphorylated hIRS-p30 is able to bind to the insulin receptor.
authors.affiliation: Klinik II und Poliklinik für Innere Medizin, University of Cologne, Federal Republic of Germany.
authors.forename: G
authors.initials: G
authors.lastname: Siemeister
authors.forename: H
authors.initials: H
authors.lastname: al-Hasani
authors.forename: H W
authors.initials: HW
authors.lastname: Klein
authors.forename: S
authors.initials: S
authors.lastname: Kellner
authors.forename: R
authors.initials: R
authors.lastname: Streicher
authors.forename: W
authors.initials: W
authors.lastname: Krone
authors.forename: D
authors.initials: D
authors.lastname: Müller-Wieland
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011972:Receptor, Insulin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D017931:DNA Primers; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014443:Tyrosine; D011972:Receptor, Insulin