ROLE OF P85 SUBUNIT OF PHOSPHATIDYLINOSITOL-3-KINASE AS AN ADAPTER MOLECULE LINKING THE INSULIN-RECEPTOR TO INSULIN-RECEPTOR SUBSTRATE-1

After insulin stimulation of cells, signaling complexes are formed, co ntaining the insulin receptor (IR), insulin receptor substrate-1 (IRS- 1), and phosphatidylinositol-3-kinase. To study the nature of these co mplexes, we employed purified IR, recombinant IRS-1, antibodies to IR and lRS-1, and fusion proteins containing the two SH2 domains of p85. In intact cells, insulin increased tyrosine phosphorylation of both th e IR and IRS-1. Both of these proteins were immunoprecipitated with an tibodies to p85. Also, fusion proteins containing the two SH2 domains of p85 directly precipitated both the IR and IRS-1. Next, these signal ing complexes were reconstituted in vitro with purified IR, recombinan t IRS-1, and the two SH2 domains of p85. In the presence of both SH2 d omains of p85, the IR associated with IRS-1. Other data, both in intac t cells and in vitro, demonstrated that N- and C-terminal SH2 domains of p85 had preferential binding affinities for the IR and IRS-1, respe ctively. Studies with an IR mutant truncated in the C terminus indicat ed that the C-terminal phosphotyrosines of the IR play a major role in interacting with the SH2 domains of p85. In conclusion, both in vivo and in vitro data support a role for 985 in directly linking the IR to IRS-1 via its SH2 domains. The formation of these complexes, therefor e, may provide a mechanism for the translocation to the plasma membran e of phosphatidylinositol-3-kinase and other molecules that are involv ed in IR signaling.