Mitogenic Signaling via Endogenous κ-Opioid Receptors in C6 Glioma Cells

Evidence for the Involvement of Protein Kinase C and the Mitogen-Activated Protein Kinase Signaling Cascade

Authors

  • Laura M. Bohn,

    1. E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, U.S.A.
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  • Mariana M. Belcheva,

    1. E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, U.S.A.
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  • Carmine J. Coscia

    1. E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, U.S.A.
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  • International Society for Neurochemistry

  • Abbreviations used: Ab, antibody; βARK, β-adrenergic receptor kinase; CS, calf serum; DMEM, Dulbecco’s modified Eagle’s medium; DMI, desipramine; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GFX, bisindolylmaleimide I; GPCR, G protein-coupled receptors; HEK, human embryonic kidney; IP, inositol phosphate; MAP, mitogen-activated protein; MEK, extracellular signal-regulated kinase kinase; MEM, minimal essential medium; nor-BNI, nor-binalthorphimine; OR, opioid receptor; PBS, phosphate-buffered saline; PI, phosphoinositide; PLC, phospholipase C; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PTX, pertussis toxin.

Address correspondence and reprint requests to Dr. C. J. Coscia at Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104, U.S.A. E-mail: cosciacc@slu.edu

Abstract

Abstract: As reports on G protein-coupled receptor signal transduction mechanisms continue to emphasize potential differences in signaling due to relative receptor levels and cell type specificities, the need to study endogenously expressed receptors in appropriate model systems becomes increasingly important. Here we examine signal transduction mechanisms mediated by endogenous κ-opioid receptors in C6 glioma cells, an astrocytic model system. We find that the κ-opioid receptor-selective agonist U69,593 stimulates phospholipase C activity, extracellular signal-regulated kinase 1/2 phosphorylation, PYK2 phosphorylation, and DNA synthesis. U69,593-stimulated extracellular signal-regulated kinase 1/2 phosphorylation is shown to be upstream of DNA synthesis as inhibition of signaling components such as pertussis toxin-sensitive G proteins, L-type Ca2+ channels, phospholipase C, intracellular Ca2+ release, protein kinase C, and mitogen-activated protein or extracellular signal-regulated kinase kinase blocks both of these downstream events. In addition, by overexpressing dominant-negative or sequestering mutants, we provide evidence that extracellular signal-regulated kinase 1/2 phosphorylation is Ras-dependent and transduced by Gβγ subunits. In summary, we have delineated major features of the mechanism of the mitogenic action of an agonist of the endogenous κ-opioid receptor in C6 glioma cells.

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