RECOMBINANT HUMAN INSULIN-RECEPTOR SUBSTRATE-1 PROTEIN - TYROSINE PHOSPHORYLATION AND IN-VITRO BINDING OF INSULIN-RECEPTOR KINASE

Insulin receptor substrate-1 (IRS-1) is a major endogenous substrate o f the insulin receptor, To study the interaction of the insulin recept or with IRS-1 in vitro, we expressed in Escherichia coil the amino aci ds 516-777 of human IRS-1 (hIRS-p30) covering five potential tyrosine phosphorylation sites within YXXM motifs, Kinetic data for tyrosine ph osphorylation of hIRS-p30 by partially purified insulin receptor and i nsulin-like growth factor I receptor and by baculovirus-expressed insu lin receptor kinase domain were determined, Native insulin receptor de monstrated the highest affinity to hIRS-p30 (K-m = 6.8 +/- 0.6 mu M), followed by the insulin-like growth factor I receptor (K-m = 9.9 +/- 1 .0 mu M). We used the soluble recombinant insulin receptor kinase doma in, which phosphorylated hIRS-p30 with high affinity (K-m = 11.9 +/- 0 .8 mu M), and affinity columns prepared by coupling hIRS-p30 to NHS-ac tivated Sepharose for binding assays, The insulin receptor kinase doma in phosphorylated the hIRS-p30 on the column, was bound by the immobil ized hIRS-p30, and was eluted with high salt buffer, Autophosphorylate d and EDTA-inactivated insulin receptor kinase domain was bound only b y immobilized hIRS-p30 protein that had been prephosphorylated, Our re sults indicate that the recombinant hIRS-p30 protein is a high affinit y substrate for insulin receptor and insulin-like growth factor I rece ptor in vitro, Moreover, we show that only tyrosine-phosphorylated hIR S-p30 is able to bind to the insulin receptor.