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

Abstract

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 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 decr...

Increase in hepatocyte growth factor receptor tyrosine kinase activity in renal carcinoma cells is associated with increased motility partly through phosphoinositide 3-kinase activation

“…The amino-truncated variant form of EGFR, denoted EGFRvIII, is constitutively active and is found in human brain, breast, lung and ovarian tumors (Voldborg et al, 1997). Downstream of this receptor, the Ras-MAPK pathway was downregulated (Moscatello et al, 1996), and PI3-kinase and c-Jun N-terminal kinase were chronically activated (Inoue et al, 1996;Moscatello et al, 1998) in NIH3T3 cells. Constitutive activation of the insulin receptor in NIH3T3 cells induced constitutive activation of PI3-kinase and unresponsiveness of MAPK activation by insulin, EGF, and platelet-derived growth factor (Antonyak et al, 1998).…”

Constitutively active mitogen-activated protein kinase kinase increases GLUT1 expression and recruits both GLUT1 and GLUT4 at the cell surface in 3T3-L1 adipocytes.

“…MAPK assay. MAPK activity was measured with an immune complex assay as previously described (10). The incorporation of radioactivity to myelin basic protein was analyzed with the Bio-Imaging Analyzer BAS-2000 (Fuji Photo Film) after separating with SDS-PA G E .…”

Insulin-Like Growth Factor I-Induced Degradation of Insulin Receptor Substrate 1 Is Mediated by the 26S Proteasome and Blocked by Phosphatidylinositol 3′-Kinase Inhibition

“…We measured the half-life of IRS-1 in the presence and in the absence of IGF-I by [ 35 S]methionine- Increased insulin concentrations result in decreased IRS-1 expression. It has previously been shown that insulin at high (micromolar) concentrations causes a decrease in IRS-1 expression (23,47). We therefore tested the ability of insulin, at low (nanomolar) and at high (micromolar) concentrations, to decrease IRS-1 expression in MCF-7 cells (Fig.…”

“…Conversely, decreased IRS-1 expression, achieved by antiestrogen treatment, inhibited IGF-I signaling through IRS-1. In a number of cell systems it has been shown that high concentrations of insulin can cause posttranscriptional downregulation of IRS-1 expression (23,47), a potential mechanism being proteolytic degradation of IRS-1 protein by the calpain pathway (54). IRS-1 phosphorylation is regulated by receptor tyrosine kinases, protein tyrosine phosphatases, and serine/threonine kinases.…”