INTERACTION OF INSULIN-RECEPTOR SUBSTRATE-1 WITH THE SIGMA-3A SUBUNITOF THE ADAPTER PROTEIN COMPLEX-3 IN CULTURED ADIPOCYTES

Signaling through the insulin receptor tyrosine kinase involves its au tophosphorylation in response to insulin and the subsequent tyrosine p hosphorylation of substrate proteins such as insulin receptor substrat e-1 (IRS-1). In basal 3T3-L1 adipocytes, IRS-1 is predominantly membra ne-bound, and this localization may be important in targeting downstre am signaling elements that mediate insulin action. Since IRS-1 localiz ation to membranes may occur through its association with specific mem brane proteins, a 3T3-F442A adipocyte cDNA expression library was scre ened with non-tyrosine-phosphorylated, baculovirus-expressed IRS-1 in order to identify potential IRS-1 receptors. A cDNA clone that encodes sigma 3A, a small subunit of the AP-3 adaptor protein complex, was de monstrated to bind IRS-1 utilizing this cloning strategy. The specific interaction between IRS-1 and sigma 3A was further verified by in vit ro binding studies employing baculovirus-expressed IRS-1 and a glutath ione S-transferase (GST)-sigma 3A fusion protein. IRS-1 and sigma 3A w ere found to co-fractionate in a detergent-resistant population of low density membranes isolated from basal 3T3-L1 adipocytes. Importantly, the addition of exogenous purified GST-sigma 3A to low density membra nes caused the release of virtually all of the IRS-1 bound to these me mbranes, while GST alone had no effect. These results are consistent w ith the hypothesis that sigma 3A serves as an IRS-1 receptor that may dictate the subcellular localization and the signaling functions of IR S-1.