Abstract
The intracellular mechanisms used by insulin and insulin-like growth factors to block programmed cell death are unknown. To identify receptor structures and signaling pathways essential for anti-apoptotic effects on cells, we have created a chimeric receptor (colony-stimulating factor-1 receptor/insulin receptor chimera (CSF1R/IR)) connecting the extracellular, ligand-binding domain of the colony-stimulating factor-1 (CSF-1) receptor to the transmembrane and cytoplasmic domains of the insulin receptor. Upon activation with CSF-1, the CSF1R/IR phosphorylates itself and intracellular substrates in a manner characteristic of normal insulin receptors. CSF-1 treatment protected cells expressing the CSF1R/IR from staurosporine-induced apoptosis. A chimeric receptor (CSF1R/IRDelta960) with a deletion of 12 amino acids from its juxtamembrane domain was constructed and expressed. CSF-1-treated cells expressing the CSF1R/IRDelta960 are unable to phosphorylate IRS-1 and Shc (Chaika, O. V., Chaika, N., Volle, D. J., Wilden, P. A. , Pirrucello, S. J., and Lewis, R. E. (1997) J. Biol. Chem. 272, 11968-11974). CSF-1 stimulated glucose uptake, mitogen-activated protein kinases, and IRS-1-associated phosphatidylinositol 3' kinase in cells expressing the CSF1R/IR but not in cells expressing the CSF1R/IRDelta960. Surprisingly, the CSF1R/IRDelta960 was as effective as the CSF1R/IR in mediating CSF-1 protection of cells from staurosporine-induced apoptosis. These observations indicate that the anti-apoptotic effects of the insulin receptor cytoplasmic domain can be mediated by signaling pathways distinct from those requiring IRS-1 and Shc.
Highlights
The intracellular mechanisms used by insulin and insulin-like growth factors to block programmed cell death are unknown
The tyrosine-phosphorylated CSF1R/IR and CSF1R/IR⌬960 were immunoprecipitated with the monoclonal antibody CT-1, which is specific for the carboxyl terminus of the insulin receptor
The amounts of CSF1R/IR and CSF1R/IR⌬960 in each cell line were equal as determined by a Western blot using the CT-1 antibody (Fig. 1), showing that the difference in tyrosine phosphorylation between the CSF1R/IR and the CSF1R/IR⌬960 is due to the 12-amino acid deletion of the juxtamembrane domain that is present in the CSF1R/IR⌬960 and not to clonal variation
Summary
The insulin receptor cytoplasmic domain has generated insulin receptors with altered biological properties [21,22,23,24], suggesting that different regions of the insulin receptor cytoplasmic domain may modulate the distinct biological effects of insulin. Analysis of the ability of mutated insulin receptors to mediate signaling in cells is obscured by the activity of endogenous insulin receptors. We have avoided this dilemma by constructing a chimeric receptor (CSF1R/IR) consisting of the extracellular ligand binding domain of the human CSF-1 receptor [25] coupled to the transmembrane and cytoplasmic domains of the human insulin receptor (26 –28). Deletions within the cytoplasmic domain of the CSF1R/IR that disrupt the ability of the insulin receptor cytoplasmic domain to activate MAP kinase and IRS1-associated PI 3Ј kinase activity do not disrupt its anti-apoptotic activity These observations indicate that the phosphorylation of IRS-1 and its subsequent association with and direct activation of PI 3Ј kinase are not requisite components of antiapoptotic signaling by the insulin receptor kinase
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