DYNAMICS OF INSULIN SIGNALING IN 3T3-L1 ADIPOCYTES - DIFFERENTIAL COMPARTMENTALIZATION AND TRAFFICKING OF INSULIN-RECEPTOR SUBSTRATE (IRS)-1 AND IRS-2

The ability of the insulin receptor to phosphorylate multiple substrat es and their subcellular localization are two of the determinants that contribute to diversity of signaling, We find that insulin receptor s ubstrate (IRS)-1 is 2-fold more concentrated in the intracellular memb rane (IM) compartment than in cytosol, whereas IRS-2 is 2-fold more co ncentrated in cytosol than in IM. Insulin stimulation induces rapid ty rosine phosphorylation of both IRS-1 and IRS-2. This occurs mainly in the IM compartment, even though IRS-S is located predominantly in cyto sol, Furthermore, after insulin stimulation, both IRS-1 and IRS-2 tran slocate from IRI to cytosol with a t(1/2) of 3.5 min. Using an in vitr o reconstitution assay, we have demonstrated an association between IR S-1 and internal membranes and have shown that the dissociation of IRS -1 from IM is dependent on serine/ threonine phosphorylation of IM. By comparison, within 1 min after insulin stimulation, 40% of the total pool of the 85-kDa subunit of phosphatidylinositol 3-kinase (p85) is r ecruited from cytosol to IM, the greater part of which can be accounte d for by binding to IRS-1 present in the IM. The p85 binding and phosp hatidylinositol 3-kinase activity associated with IRS-2 rapidly decrea se in both IM and cytosol, whereas those associated with IRS-1 stay at a relatively high level in IM and increase with time in cytosol despi te a return of p85 to the cytosol and decreasing tyrosine phosphorylat ion of cytosolic IRS-1. These data indicate that IRS-1 and IRS-2 are d ifferentially distributed in the cell and move from IRI to cytosol fol lowing insulin stimulation. Insulin-stimulated IRS-1 and IRS-S signali ng occurs mainly in the IM and shows different kinetics; IRS-l-mediate d signaling is more stable, whereas IRS-a-mediated signaling is more t ransient. These differences in substrate utilization and compartmental ization may contribute to the complexity and diversity of the insulin signaling network.