Interaction of insulin receptor substrate-1 with the sigma3A subunit of the adaptor protein complex-3 in cultured adipocytes.
Signaling through the insulin receptor tyrosine kinase involves its autophosphorylation in response to insulin and the subsequent tyrosine phosphorylation of substrate proteins such as insulin receptor substrate-1 (IRS-1). In basal 3T3-L1 adipocytes, IRS-1 is predominantly membrane-bound, and this localization may be important in targeting downstream signaling elements that mediate insulin action. Since IRS-1 localization to membranes may occur through its association with specific membrane proteins, a 3T3-F442A adipocyte cDNA expression library was screened with non-tyrosine-phosphorylated, baculovirus-expressed IRS-1 in order to identify potential IRS-1 receptors. A cDNA clone that encodes sigma3A, a small subunit of the AP-3 adaptor protein complex, was demonstrated to bind IRS-1 utilizing this cloning strategy. The specific interaction between IRS-1 and sigma3A was further verified by in vitro binding studies employing baculovirus-expressed IRS-1 and a glutathione S-transferase (GST)-sigma3A fusion protein. IRS-1 and sigma3A were 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-sigma3A to low density membranes caused the release of virtually all of the IRS-1 bound to these membranes, while GST alone had no effect. These results are consistent with the hypothesis that sigma3A serves as an IRS-1 receptor that may dictate the subcellular localization and the signaling functions of IRS-1.
VanRenterghem
B
B
Program in Molecular Medicine and the Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, USA.
Morin
M
M
Czech
M P
MP
Heller-Harrison
R A
RA
D016475:3T3 Cells; D033963:Adaptor Protein Complex 3; D034002:Adaptor Protein Complex sigma Subunits; D017667:Adipocytes / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D007328:Insulin / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
C115637:AP3S1 protein, human; D033963:Adaptor Protein Complex 3; D034002:Adaptor Protein Complex sigma Subunits; C485460:Ap3s1 protein, mouse; D002352:Carrier Proteins; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins