Characterization of Drosophila insulin receptor substrate

Insulin receptor substrate (IRS) proteins are phosphorylated by multiple ty rosine kinases, including the insulin receptor. Phosphorylated IRS proteins bind to SH2 domain-containing proteins, thereby triggering downstream sign aling pathways. The Drosophila insulin receptor (dIR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that d IR might not require an IRS protein to accomplish its signaling functions. However, we obtained a cDNA encoding Drosophila IRS (dIRS), and we demonstr ated expression of dIRS in a Drosophila cell line. Like mammalian IRS prote ins, the N-terminal portion of dIRS contains a pleckstrin homology domain a nd a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. When coexpressed with dIRS in COS-7 cells, a chimeric receptor (the extracellular domain of human IR f used to the cytoplasmic domain of dIR) mediated insulin-stimulated tyrosine phosphorylation of dIRS. Mutating the juxtamembrane NPXY motif markedly re duced the ability of the receptor to phosphorylate dIRS. In contrast, the N PXY motifs in the C-terminal extension of dIR were required for stable asso ciation with dIRS. Coimmunoprecipitationexperimentsdemonstratedinsulin-depe ndent binding of dIRS to phosphatidylinositol 3-kinase and SHP2. However, w e did not detect interactions with Grb2, SHC, or phospholipase C-gamma. Tak en together with published genetic studies, these biochemical data support the hypothesis that dIRS functions directly downstream from the insulin rec eptor in Drosophila.