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.