Involvement of mitogen-activated protein kinase in agonist-induced phosphorylation of the mu-opioid receptor in HEK 293 cells

Agonist exposure of many G protein-coupled receptors stimulates an activati on of extracellular signal-regulated protein kinases (ERKs) 1 and 2, member s 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 mu-opioid receptor (MOR1) with [D-Ala(2),MePhe(4),Gly(5)-ol ]enkephalin (DAMGO) stimulated a rapid and transient (3-5-min) activation a nd nuclear translocation of MAPK. Exposure of these cells to the MAPK kinas e 1 inhibitor PD98059 not only prevented MAPK activation but also inhibited homologous desensitization of the mu-opioid receptor. We have therefore de termined the effect of PD98059 on agonist-induced mu-receptor phosphorylati on. 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 mu-receptor phosphorylation w ith an IC50 of 20 mu M. DAMGO also induced MOR1 internalization that peaked at 30 min. Confocal microscopy revealed that DAMGO-induced MOR1 internaliz ation was also largely inhibited in the presence of PD98059, U0126, another chemically unrelated inhibitor of the MAPK cascade, mimicked the effect of PD98059 on ct-receptor phosphorylation and desensitization. MOR1 itself, h owever, appears to be a poor substrate for MAPK because mu-receptors immuno precipitated from stably transfected HEK 293 cells were not phosphorylated by exogenous ERK 2 in vitro. The fact that morphine also triggered MAPK act ivation but did not induce MOR1 internalization indicates that receptor int ernalization was not required for MOR1-mediated mitogenic signaling. We con clude that MOR1 stimulates a rapid and internalization-independent MAPK act ivation. Activation of the MAPK cascade in turn may not only relay mitogeni c signals to the nucleus but also trigger initial events leading to phospho rylation and desensitization of the mu-opioid receptor.