Different Pathways of Postreceptor Desensitization following Chronic Insulin Treatment and in Cells Overexpressing Constitutively Active Insulin Receptors

Gen Inoue, Bentley Cheatham, C. Ronald Kahn
1996 Journal of Biological Chemistry  
We have reported previously that substitution of the transmembrane domain of the insulin receptor with that of the erbB-2 oncogene (IR erbV3 E ) results in constitutive activation of the insulin receptor kinase. Compared to NIH3T3 cells overexpressing wild-type insulin receptors (IR wt ), cells overexpressing IR erbV3 E displayed a decrease in IRS-1 protein content by 55%, but basal tyrosine phosphorylation of IRS-1 was increased. This resulted in an increased association of IRS-1 with the p85
more » ... IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase, increased phosphatidylinositol 3-kinase activity in anti-IRS-1 immunoprecipitates, constitutive activation of p70 S6 protein kinase, and an increased association of Grb2 with Shc in the absence of ligand. However, Grb2 association with IRS-1 could not be detected in the basal or insulin-stimulated states, and mitogen-activated protein kinase (MAPK) activity could not be stimulated by insulin, epidermal growth factor, or platelet-derived growth factor. In contrast to IR erbV3 E , the insulin receptor content and its tyrosine phosphorylation were significantly decreased in IR wt cells chronically stimulated (>24 h) with insulin. With decreased IRS-1 content, tyrosine phosphorylation of IRS-1 was decreased by over 75%, leading to decreased IRS-1-associated PI 3-kinase and Grb2. In addition, Grb2 association with Shc and activation of MAPK and the p70 S6 kinase were insensitive to insulin stimulation. By contrast, association of Grb2 with Shc and activation of MAPK, but not the p70 S6 kinase, could be stimulated by epidermal growth factor or plateletderived growth factor. These data suggest that there are multiple levels of postreceptor desensitization to insulin action. These are used somewhat differently in these two different models, probably due in part to the difference in receptor down-regulation. The insulin receptor (IR) 1 is an integral membrane protein comprised of two extracellular ␣-subunits, which bind insulin, and two transmembrane ␤-subunits, which contain a tyrosine kinase activity that is activated following insulin binding (1). Activation of the receptor tyrosine kinase appears to be essential for most, if not all, insulin actions (1). In addition to insulin binding, the receptor-associated tyrosine kinase activity can be activated by tryptic cleavage of the ␣-subunit or removal of the ␣-subunit by in vitro mutagenesis, suggesting that the ␣-subunit acts to repress kinase activity (1). Recently, we have shown that substitution of the insulin receptor transmembrane domain with that from the erbB-2 oncogene (IR erbV3 E ) also results in a constitutively active insulin receptor kinase (2, 3). Both in intact animals and in cultured cells, chronic stimulation by insulin induces a desensitization and down-regulation of insulin signaling. This occurs in part through internalization and degradation of the insulin receptor (4, 5), as well as downregulation of IRS-1 (3). In hyperinsulinemic conditions, tyrosine phosphorylation of IRS-1 and association with PI 3-kinase are also decreased, consistent with the insulin resistance that is present (6, 7). In acute insulin signaling, there may be other levels of feedback inhibition, including serine phosphorylation of the insulin receptor and IRS-1, feedback inhibition of MAPK at the level of Ras and Raf-1 association (8), and dissociation of SOS from Grb2 (9 -11). In this study, we have examined the mechanism of down-regulation of insulin signaling and the relationship to the changes of insulin actions using two different cell culture models. The first is NIH3T3 cells overexpressing wild-type human IR (IR wt ) and chronically treated by insulin, in which desensitization presumably occurs at the level of the insulin receptor. The second is NIH3T3 cells overexpressing the constitutively active insulin receptor (IR erbV3 E ), in which desensitization may occur at postreceptor levels, but the level of the activated insulin receptors remains high. EXPERIMENTAL PROCEDURES Materials and Cell Lines-Insulin was purchased from Boehringer Mannheim, [␥-32 P]ATP was from DuPont NEN; polyclonal anti-insulin receptor antibodies, anti-IRS-1 antibodies, anti-p70 S6 kinase antibodies, and anti-MAPK antibodies were prepared as described previously (12). Monoclonal anti-phosphotyrosine antibody (4G10), monoclonal anti-Grb2 antibody, polyclonal anti-Shc antibodies, and polyclonal anti-85 kDa subunit of PI 3-kinase antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). The parental mouse NIH3T3 fibroblasts were transfected with the wild-type or mutant insulin receptor expression plasmids as described previously (3) . Stable transfectants were selected after selection in the presence of G418 (500 g/ml) for 8 weeks. Resistant clones were selected by limiting dilution, expanded, and screened further by immunoblotting for insulin receptor expression. Preparation of Total Cell Lysates and Immunoblotting-Cells were serum-starved for 24 h before experiments. For chronic treatment with insulin, IR wt cells were incubated with 10 Ϫ6 M insulin for 24 h. Cells were acutely treated with ligands, washed twice with ice-cold phosphate-buffered saline, and harvested in lysis buffer (50 mM HEPES, 10
doi:10.1074/jbc.271.45.28206 pmid:8910437 fatcat:am75dm244va7lgudiibc6eqls4