LOCALIZATION OF THE INSULIN-RECEPTOR BINDING-SITES FOR THE SH2 DOMAINPROTEINS P85, SYP, AND GAP

The insulin receptor is known to interact with the SH2 domain proteins p85 (the regulatory subunit of phosphatidylinositol 3-kinase), Syp (a tyrosine phosphatase), and GAP (GTPase-activating protein). In this s tudy, we mapped the insulin receptor binding sites for each of these p roteins by examining the ability of phosphopeptides, corresponding to insulin receptor phosphorylation sites, and mutant insulin receptors t o inhibit an insulin receptor-SH2 domain interaction. Precipitation of partially purified insulin receptors by glutathione S-transferase fus ion proteins containing the N-terminal SH2 domains of p85 and GAP and both SH2 domains of Syp was demonstrated. The effect of the addition o f each phosphopeptide on insulin receptor precipitation was tested. pY 1322, the C-terminal insulin receptor peptide, inhibited insulin recep tor precipitation by both p85- and Syp GST. The NPXY internalization d omain peptide inhibited insulin receptor precipitation by GAP GST. The se data were confirmed by mutant insulin receptor experiments. The ins ulin receptor C-terminal mutants, Delta CT and Y/F2, were not precipit ated by p85 or Syp GST and the NPXY mutant insulin receptors, Delta Ex 16 and HI Delta NPEY, were not precipitated by GAP-GST. Therefore, we conclude that p85 and Syp bind to the insulin receptor C terminus at t yrosine 1322 and GAP binds to the insulin receptor NPXY domain at tyro sine 960.