Insulin stimulation of 3T3-L1 adipocytes results in rapid activation of the insulin receptor tyrosine kinase followed by autophosphorylation of the receptor and phosphorylation of insulin receptor substrate 1 (IRS-1), its major substrate. The insulin receptor resides mostly at the cell surface of 3T3-L1 adipocytes under basal conditions, while about two-thirds of IRS-1 fractionates with intracellular membranes and one-third fractionates with cytosol. To test whether insulin receptor internalization is required for optimal tyrosine phosphorylation of IRS-1, 3T3-L1 adipocytes and CHO-T cells were incubated at 4 °C which inhibits receptor endocytosis but not its tyrosine kinase activity. Under these conditions, tyrosine phosphorylation of IRS-1 in the low density microsome fraction in response to insulin was as intense as that observed at 37 °C, indicating that endocytosis of insulin receptors is not necessary for tyrosine phosphorylation of IRS-1 to occur. Surprisingly, at 37 °C, insulin action on 3T3-L1 adipocytes progressively decreased the amount of IRS-1 protein associated with the low density microsome fraction and increased that in the cytosol. This redistribution of IRS-1 from the low density microsome fraction to the cytosol in response to insulin was accompanied by decreased electrophoretic mobility of IRS-1 on SDS-polyacrylamide gel electrophoresis. Incubation of adipocytes at 4 °C blocked the appearance of tyrosine-phosphorylated IRS-1 in the cytosol. Taken together, these data indicate that insulin receptors phosphorylate IRS-1 at the cell surface, perhaps in coated pits which are included in the low density microsome fraction. The results also suggest a desensitization mechanism in which the tyrosine-phosphorylated membrane-bound IRS-1, associated with signaling molecules such as phosphatidylinositol 3-kinase, is released into the cytoplasm in concert with its serine/threonine phosphorylation.
CITATION STYLE
Heller-Harrison, R. A., Morin, M., & Czech, M. P. (1995). Insulin regulation of membrane-associated insulin receptor substrate 1. Journal of Biological Chemistry, 270(41), 24442–24450. https://doi.org/10.1074/jbc.270.41.24442
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