Molecular Endocrinology, Vol 9, 435-442, Copyright © 1995 by Endocrine Society

ARTICLES

Role of p85 subunit of phosphatidylinositol-3-kinase as an adaptor molecule linking the insulin receptor to insulin receptor substrate 1

V Sanchez-Margalet, ID Goldfine, K Truitt, J Imboden and CK Sung
Department of Medicine, Mount Zion Medical Center/University of California, San Francisco 94115, USA.

After insulin stimulation of cells, signaling complexes are formed, containing the insulin receptor (IR), insulin receptor substrate-1 (IRS- 1), and phosphatidylinositol-3-kinase. To study the nature of these complexes, we employed purified IR, recombinant IRS-1, antibodies to IR and IRS-1, and fusion proteins containing the two SH2 domains of p85. In intact cells, insulin increased tyrosine phosphorylation of both the IR and IRS-1. Both of these proteins were immunoprecipitated with antibodies to p85. Also, fusion proteins containing the two SH2 domains of p85 directly precipitated both the IR and IRS-1. Next, these signaling complexes were reconstituted in vitro with purified IR, recombinant IRS-1, and the two SH2 domains of p85. In the presence of both SH2 domains of p85, the IR associated with IRS-1. Other data, both in intact cells and in vitro, demonstrated that N- and C-terminal SH2 domains of p85 had preferential binding affinities for the IR and IRS- 1, respectively. Studies with an IR mutant truncated in the C terminus indicated 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 p85 in directly linking the IR to IRS-1 via its SH2 domains. The formation of these complexes, therefore, may provide a mechanism for the translocation to the plasma membrane of phosphatidylinositol-3-kinase and other molecules that are involved in IR signaling.

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