Insulin-induced protein tyrosine phosphorylation cascade and signalling molecules are localized in a caveolin-enriched cell membrane domain.
The cellular localisation of time- and temperature-dependent 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 Gi alpha 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 degrees 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 IRS-1 in the caveolin-enriched fraction, possibly recruited from the cytosolic pool. Insulin also increased the concentrations of insulin receptors, IRS-1 and Shc in endosomes, suggesting insulin-induced internalization of the 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.
Smith
R M
RM
Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
Harada
S
S
Smith
J A
JA
Zhang
S
S
Jarett
L
L
D001665:Binding Sites; D051242:Caveolin 1; D022461:Caveolins; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D012967:Sodium Dodecyl Sulfate; D014443:Tyrosine; D011972:Receptor, Insulin