Involvement of Mitogen-Activated Protein Kinase in Agonist-Induced Phosphorylation of the μ-Opioid Receptor in HEK 293 Cells

Authors


  • Abbreviations used: cAMP, cyclic AMP; DAMGO, [D-Ala2, MePhe4,Gly5-ol]enkephalin; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HA, hemagglutinin; HEK, human embryonic kidney; Li, -like immunoreactivity; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MEK, mitogen-activated protein kinase kinase; MOR1, rat μ-opioid receptor; PAGE, polyacrylamide gel electrophoresis; PD98059, 2-(2′-amino-3′-methoxyphenyl)oxanaphthalen-4-one; SDS, sodium dodecyl sulfate; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(aminophenylthio)butadiene.

Address correspondence and reprint requests to Dr. V. Höllt at Department of Pharmacology and Toxicology, Otto-von-Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany. E-mail: Volker.Hoellt@medizin.uni-magdeburg.de

Abstract

Abstract: Agonist exposure of many G protein-coupled receptors stimulates an activation of extracellular signal-regulated protein kinases (ERKs) 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. Here, we show that treatment of human embryonic kidney (HEK) 293 cells stably transfected to express the rat μ-opioid receptor (MOR1) with [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) stimulated a rapid and transient (3-5-min) activation and nuclear translocation of MAPK. Exposure of these cells to the MAPK kinase 1 inhibitor PD98059 not only prevented MAPK activation but also inhibited homologous desensitization of the μ-opioid receptor. We have therefore determined the effect of PD98059 on agonist-induced μ-receptor phosphorylation. DAMGO stimulated a threefold increase in MOR1 phosphorylation within 20 min that could be reversed by the antagonist naloxone. PD98059 produced a dose-dependent inhibition of agonist-promoted μ-receptor phosphorylation with an IC50 of 20 μM. DAMGO also induced MOR1 internalization that peaked at 30 min. Confocal microscopy revealed that DAMGO-induced MOR1 internalization was also largely inhibited in the presence of PD98059. U0126, another chemically unrelated inhibitor of the MAPK cascade, mimicked the effect of PD98059 on μ-receptor phosphorylation and desensitization. MOR1 itself, however, appears to be a poor substrate for MAPK because μ-receptors immunoprecipitated from stably transfected HEK 293 cells were not phosphorylated by exogenous ERK 2 in vitro. The fact that morphine also triggered MAPK activation but did not induce MOR1 internalization indicates that receptor internalization was not required for MOR1-mediated mitogenic signaling. We conclude that MOR1 stimulates a rapid and internalization-independent MAPK activation. Activation of the MAPK cascade in turn may not only relay mitogenic signals to the nucleus but also trigger initial events leading to phosphorylation and desensitization of the μ-opioid receptor.

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