The -opioid receptor mediates the analgesic and addictive properties of morphine. Despite the clinical importance of this G-protein-coupled receptor and many years of pharmacological research, few intracellular signaling mechanisms triggered by morphine and other -opioid agonists have been described. We report that -opioid agonists stimulate three different effectors of a phosphoinositide 3-kinase (PI3K)-dependent signaling cascade. By using a cell line stably transfected with the -opioid receptor cDNA, we show that the specific agonist [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]enkephalin (DAMGO) stimulates the activity of Akt, a serine/threonine protein kinase implicated in protecting neurons from apoptosis. Activation of Akt by DAMGO correlates with its phosphorylation at serine 473. The selective PI3K inhibitors wortmannin and LY294002 blocked phosphorylation of this site, previously shown to be necessary for Akt enzymatic activity. DAMGO also stimulates the phosphorylation of two other downstream effectors of PI3K, the p70 S6 kinase and the repressors of mRNA translation, 4E-BP1 and 4E-BP2. Upon -opioid receptor stimulation, p70 S6 kinase is activated and phosphorylated at threonine 389 and at threonine 421/serine 424. Phosphorylation of p70 S6 kinase and 4E-BP1 is also repressed by PI3K inhibitors as well as by rapamycin, the selective inhibitor of FRAP/mTOR. Consistent with these findings, DAMGO-stimulated phosphorylation of 4E-BP1 impairs its ability to bind the translation initiation factor eIF-4E. These results demonstrate that the -opioid receptor activates signaling pathways associated with neuronal survival and translational control, two processes implicated in neuronal development and synaptic plasticity.The -opioid receptor mediates the effects of morphine as well as the actions of endogenous opioid ligands involved in diverse functions in the central and peripheral nervous system (1, 2). Activation of the -opioid receptor modulates neuronal excitability as it inhibits neuronal firing and neurotransmitter release in different regions of the nervous system (1). In addition to antinociception, the -opioid receptor has been also implicated in many other functions such as hippocampal plasticity, gastrointestinal motility, and modulation of the immune response (1, 3). Recent evidence using mice lacking the -opioid receptor underscored its role in hematopoiesis and in reproductive physiology (4). Although the behavioral and pharmacological properties of this receptor have been extensively studied, analysis of intracellular signaling has focused almost entirely on inhibition of the cAMP system. Opioid agonists inhibit adenylyl cyclase through coupling of the receptor to a G o/i pertussis-sensitive protein (1, 5). This activity is mediated by the G␣ subunit of the G-protein. Studies on other G-protein-coupled receptors have shown that, upon agonist binding, the GTPbound G␣ subunit dissociates from the G␤␥ subunit. The G␤␥ subunit then serves as an independent activator of different effector pathways that require a p...

The -opioid receptor mediates not only the beneficial painkilling effects of opiates like morphine but also the detrimental effects of chronic exposure such as tolerance and dependence. Different studies have linked tolerance to opioid receptor desensitization. Agonist activation of the -opioid receptor stimulates a mitogenactivated protein kinase (MAPK) activity, but the functional significance of this pathway remains unclear. We have focused on the MAPK signaling cascade to study -opioid receptor desensitization. We report that inhibition of the MAPK pathway blocks desensitization of -opioid receptor signaling as well as the loss of receptor density due to internalization. Our results suggest that a feedback signal emanating from the MAPK cascade is required for -opioid receptor desensitization.Agonists for the -opioid receptor are the therapeutic choice in the management of severe acute and chronic pain despite the potential development of tolerance, dependence, and addiction. The mechanisms of tolerance, defined as the diminishing effect of the drug in response to chronic exposure, are not fully understood. It has been postulated that receptor desensitization and down-regulation could be associated with aspects of tolerance in vivo (1). Opioid receptors undergo homologous desensitization upon continuous or repeated agonist exposure in a fashion similar to other G protein-coupled receptors (GPCR) 1 (2-4). Desensitization of the -opioid receptor involves agonistdependent phosphorylation of the receptor, most likely by a member of the family of G-coupled receptor protein kinases (GRKs) (4, 5). According to this model, upon phosphorylation and uncoupling from the G protein, the receptor is bound to ␤-arrestins and internalized into endosomes, reducing the number of receptors available at the cell surface for further agonist binding (6, 7).Desensitization of the -opioid receptor has been mainly described as the attenuated reduction of forskolin-stimulated cAMP levels in response to the agonist (2-4). However, other signaling pathways might also be desensitized upon prolonged agonist exposure. GPCRs can trigger a G␤␥-mediated activation of a phosphoinositide 3-kinase (PI3K)/Ras-dependent MAPK signaling pathway (8, 9). Opioid receptors stimulate MAPK activity as well, although the components of this signaling cascade have not been fully described (10 -12). In this report we have examined the role of the MAPK (Erk1/2) signaling pathway on -opioid receptor desensitization. This pathway, which involves the activation of a phosphatidylinositol 3-kinase activity as well as Raf and MEK1/2, is essential for -opioid receptor desensitization. EXPERIMENTAL PROCEDURESReagents-[D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]enkephalin (DAMGO), dermorphin, morphine, Met-enkephalin, naloxone, D-Pen 2 ,D-Pen 5 -enkephalin (DPDPE), lysophosphatidic acid (LPA), pertussis toxin, cycloheximide, forskolin, and isobutylmethylxanthine (IBMX) were from Sigma. Wortmannin and LY294002 were from Calbiochem. Antibodies that recognize only the phosphorylated...