Abstract
We have recently characterized a mutant insulin receptor (Y/F2) in which the two tyrosines in the carboxyl terminus (Tyr1316, Tyr1322) were mutated to phenylalanine. Compared with wild type receptors, the Y/F2 receptor exhibited markedly enhanced sensitivity to insulin-stimulated DNA synthesis with normal insulin-stimulated glucose uptake (Takata, Y., Webster, N. J. G., and Olefsky, J. M. (1991) J. Biol. Chem. 266, 9135-9139). In this paper, we present further evidence for the divergence of the metabolic and mitogenic signaling pathways utilized by the insulin receptor. The mutant receptor showed normal sensitivity and responsiveness for insulin-stimulated glucose incorporation into glycogen. The insulin sensitivity for phosphorylation of two substrates (pp180 and pp220) was the same in both Y/F2 cells and HIRc cells. Phosphotyrosine content, however, was greater in Y/F2 cells than in HIRc cells, especially in the basal state. Insulin stimulated S6 kinase activity 2-6-fold, with an ED50 of -10 nM in Rat 1 cells and 0.5 nM in HIRc cells. The sensitivity to insulin was enhanced in Y/F2 cells with an ED50 of 0.1 nM. These effects were insulin-specific, since insulin-like growth factor (IGF)-I-stimulated mitogenesis was normal. In summary: 1) Y/F2 receptors exhibit normal metabolic and enhanced mitogenic signaling; 2) the enhanced mitogenic signaling is specific for the insulin receptor in the Y/F2 cells, since IGF-I-stimulated mitogenesis is normal; 3) Y/F2 cells display increased endogenous substrate phosphorylation and augmented insulin-stimulated S6 kinase activity placing these responses among insulin's mitogenic effects; and 4) these results are consistent with the concept that the COOH-terminal tyrosine residues of the insulin receptor are normally inhibitory to mitogenic signaling.
Highlights
We have recentlycharacterized a mutantinsulin as other potential downstream coupling, or signaling, molereceptor (Y/F2) in which the two tyrosinesin the car- cules
Within the COOH terminusare two tyrosine residues (Tyr1316,Tyr13”)which are autophosphorylated in response to insulin [11].To explore the role of these two tyrosines in the function of the COOH terminus, we constructed a mutant insulin receptor in which both residues were changed to phenylalanine by site-directed mutagenesis (Y/F2 receptor)
Cell Preparation and Thymidine Incorporation Assay-Cells were starved in DME medium containing 0.05% fetal calf serum and 40 mM Hepes(pH 7.4) for 24 h priortohormonestimulation.With the ED5, for insulin stimulation of DNA synthesis was 0.1nM in Y/F2 cells compared with 0.6 nM in HIRc cells expressing comparable numbers of wild type receptors [8].We show serum starvation, rat fibroblasts are known to secrete IGF-I into thethat this augmentation is restricted to the insulin signaling medium [15]
Summary
We have recentlycharacterized a mutantinsulin as other potential downstream coupling, or signaling, molereceptor (Y/F2) in which the two tyrosinesin the car- cules. Cell Preparation and Thymidine Incorporation Assay-Cells were starved in DME medium containing 0.05% fetal calf serum and 40 mM Hepes(pH 7.4) for 24 h priortohormonestimulation.With the ED5,, for insulin stimulation of DNA synthesis was 0.1nM in Y/F2 cells compared with 0.6 nM in HIRc cells expressing comparable numbers of wild type receptors [8].We show serum starvation, rat fibroblasts are known to secrete IGF-I into thethat this augmentation is restricted to the insulin signaling medium [15].
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