Defective insulin receptor function in down-regulated HepG2 cells.
Insulin receptors on the surface of down-regulated HepG2 cells were studied, and multiple defects of receptor function were demonstrated. Insulin treatment led to a 58% decrease in cell surface receptor number, and the remaining receptors exhibited a 50% decrease in insulin internalization and degradation on a per receptor basis. Down-regulated cells internalized 36% of receptors photolabeled with 125I-NAPA-insulin vs. 56% of labeled receptors in control cells, indicating a 40% decrease in ligand-mediated receptor internalization. Total cellular receptors purified from down-regulated cells by wheat germ affinity chromatography demonstrated a 20% decrease in maximal autophosphorylation. Assessment of autophosphorylation of cell surface receptors in intact cells by restimulation of cells with insulin at 12 C (a temperature nonpermissive for receptor recycling), followed by immunoblotting with antiphosphotyrosine antibodies revealed a decrease of approximately 70% in insulin-stimulated autophosphorylation compared with controls. This method also revealed a comparable decrease in insulin-stimulable tyrosine phosphorylation of pp185, a putative endogenous substrate. When receptors were stimulated to undergo autophosphorylation with 125I-NAPA-insulin in intact cells and then solubilized, only 11% of 125I-NAPA-insulin receptor complexes from down-regulated cells were immunoprecipitated with antiphosphotyrosine antibodies compared with 35% of labeled control receptors. These results indicate that treatment with high concentrations of insulin results in the accumulation on the cell surface of a population of receptors that display multiple functional abnormalities. This probably results from preferential internalization and degradation of kinase-competent insulin receptors causing an accumulation of kinase-incompetent receptors on the cell surface. These receptors may in part be responsible for the postbinding defects in insulin action observed in down-regulated cells.
Williams
J F
JF
Department of Medicine, University of California, La Jolla 92037.
Olefsky
J M
JM
D000345:Affinity Labels; D001386:Azides / Q000378:metabolism; D001692:Biological Transport; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D015536:Down-Regulation; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism*; D010766:Phosphorylation; D010777:Photochemistry; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism*; D011973:Receptors, LDL / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D001386:Azides; D007328:Insulin; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; C036767:insulin, (2-nitro-4-azidophenyl)(A1)-; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin