title: Down-regulation of c-Cbl by morphine accounts for persistent ERK1/2 signaling in delta-opioid receptor-expressing HEK293 cells.
abstract: Opioids display ligand-specific differences in the time course of ERK1/2 signaling. Whereas full agonists, like etorphine, induce only transient activation of ERK1/2, the partial agonist morphine mediates persistent stimulation of mitogenic signaling. Here we report that in stably delta-opioid receptor (DOR)-expressing HEK293 (HEK/DOR) cells, the transient nature of etorphine-induced ERK1/2 signaling is due to desensitization of epidermal growth factor (EGF) receptor-mediated activation of the Ras/Raf-1/ERK1/2 cascade. Desensitization of ERK1/2 activity by etorphine is associated with down-regulation of EGF receptors, an effect mediated by the ubiquitin ligase c-Cbl. In contrast, chronic morphine treatment failed to desensitize EGF receptors, resulting in unimpeded ERK1/2 signaling. The failure of morphine to desensitize ERK1/2 signaling is mediated by persistent activation of c-Src, which induces degradation of c-Cbl. The role of c-Src in opioid-specific ERK1/2 signaling is further demonstrated by pretreatment of the cells with PP2 and SKI-I as well as overexpression of a dominant negative c-Src mutant (c-Src(dn)) or a c-Src-resistant c-Cbl mutant (CblY3F), both of which facilitate desensitization of ERK1/2 signaling by morphine. Conversely, overexpression of c-Src as well as down-regulation of c-Cbl by small interfering RNA results in persistent etorphine-induced stimulation of ERK1/2 activity. Subcellular fractionation experiments finally attributed the ability of morphine to persistently activate c-Src to its redistribution from Triton X-100-insensitive membrane rafts to DOR and EGF receptor containing high density membrane compartments implicated in ERK1/2 signaling. These results demonstrate that agonist-specific differences in the temporal and spatial pattern of c-Src activation determine the kinetics of DOR-mediated regulation of ERK1/2 signaling.
authors.affiliation: Institute of Pharmacology, Toxicology and Pharmacy, Ludwig-Maximilians-University Munich, 80539 Muenchen, Germany. eisinger@lrz.uni-muenchen.de
authors.forename: Daniela A
authors.initials: DA
authors.lastname: Eisinger
authors.forename: Hermann
authors.initials: H
authors.lastname: Ammer
mesh_terms: D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects*; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism; D005048:Etorphine / Q000494:pharmacology; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009020:Morphine / Q000494:pharmacology*; D009294:Narcotics / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D019061:src-Family Kinases
chemical_list: D009294:Narcotics; D034741:RNA, Small Interfering; D017465:Receptors, Opioid, delta; D005048:Etorphine; D009020:Morphine; D050721:Proto-Oncogene Proteins c-cbl; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3