Insulin-induced protein tyrosine phosphorylation cascade and signalling molecules are localized in a caveolin-enriched cell membrane domain

Abstract

The cellular localisation of time-and temperature-dependant 125I-insulin binding, insulin-sensitive signalling proteins and the insulin-induced protein tyrosine phosphorylation cascade were assessed in subcellular fractions isolated on Iodixanol gradients from control and insulin-treated H35 hepatoma cells. Western blot analysis demonstrated that the concentrations of IRS-1, Shc, GRB-2,SOS, Syp, PI 3-kinase, MAP kinase and G10 were at least 10-fold higher in cell surface-derived, caveolin-enriched fraction than in a cell surface-derived, caveolin-poor fraction (i.e., the plasma membranes). Insulin treatment caused a 15-fold increase in tyrosine phosphorylation of IRS-1 in the caveolin-enriched fraction in 5 min at 37°C compared with a 3-fold increase in plasma membranes and a 6-fold increases in the cytosol and endosomes. Insulin also increased tyrosine phosphorylation of both a 72-kDA protein and the 46-kDA Shc isoform only in the caveolin-enriched fraction. Insulin treatment did not change the concentrations of insulin receptors or Shc but increased the concentrations of insulin receptors and proteins activated with them. Electron microscopic analysis, with the use of a combination of colloidal gold labelled insulin to label the insulin receptor and immunolabelling to detect caveolin or IRS-1, demonstrated the co-localisation of insulin receptors in caveolin-and IRS-1 containing vesicular structures. Differences in the insulin-induced protein tyrosine phosphorylation and concentrations of these proximal signalling proteins in the caveolin-enriched fraction, plasma membranes, and cytosol suggest that insulin receptors in the caveolae play a major role in initiating insulin's signal transduction processes.