The main analgesic effects of the opioid alkaloid morphine are mediated by the -opioid receptor. In contrast to endogenous opioid peptides, morphine activates the -opioid receptor without causing its rapid endocytosis. Recently, three novel C-terminal splice variants (MOR1C, MOR1D, and MOR1E) of the mouse -opioid receptor (MOR1) have been identified. In the present study, we show that these receptors differ substantially in their agonist-selective membrane trafficking. MOR1 and MOR1C stably expressed in human embryonic kidney 293 cells exhibited phosphorylation, internalization, and down-regulation in the presence of the opioid peptide [D-Ala 2 ,Me-Phe 4 ,Gly 5 -ol]enkephalin (DAMGO) but not in response to morphine. In contrast, MOR1D and MOR1E exhibited robust phosphorylation, internalization, and down-regulation in response to both DAMGO and morphine. DAMGO elicited a similar desensitization (during an 8-h exposure) and resensitization (during a 50-min drug-free interval) of all four -receptor splice variants. After morphine treatment, however, MOR1 and MOR1C showed a faster desensitization and no resensitization as compared with MOR1D and MOR1E. These results strongly reinforce the hypothesis that receptor phosphorylation and internalization are required for opioid receptor reactivation thus counteracting agonist-induced desensitization. Our findings also suggest a mechanism by which cell-and tissuespecific C-terminal splicing of the -opioid receptor may significantly modulate the development of tolerance to the various effects of morphine.Opioid receptors couple via G proteins to a variety of downstream effectors including adenylate cyclase (1) and mitogenactivated protein kinases (2-5). During repeated or continuous agonist stimulation these responses undergo rapid desensitization. An important mechanism of desensitization of G proteincoupled receptors is the phosphorylation of intracellular receptor domains by G protein-coupled receptor kinases or second messenger-regulated protein kinases such as Ca 2ϩ /calmodulindependent protein kinase II, cAMP-dependent protein kinase, or protein kinase C. After phosphorylation of the receptor, arrestins are frequently recruited to the plasma membrane, at which they facilitate endocytosis by serving as scaffolding proteins that bind to clathrin (6, 7). For some time it has been assumed that the rapid removal of ligand-activated receptors from the cell surface plays a major role in the receptor degradation, thus enhancing functional desensitization. Recent studies have suggested that endocytozed receptors are predominantly recycled to the cell surface in a reactivated state (8, 9).We have shown previously that two C-terminal splice variants (rMOR1 and rMOR1B) 1 of the rat -opioid receptor differ in their DAMGO-mediated internalization and resensitization rates (10, 11). The rapid internalizing variant rMOR1B revealed a faster resensitization and consequently a slower desensitization as compared with rMOR1. These results clearly show that receptor recycling after interna...

In this study we report that human phosphatidylethanolamine-binding protein (hPBP) facilitates heterotrimeric G protein-coupled signaling. In Xenopus laevis oocytes, coexpression of hPBP with human opioid receptor, human ␦ opioid receptor, or human somatostatin receptor 2 evoked an agonist-induced increase in potassium conductance of G protein-activated inwardly rectifying potassium channels. This activation of heterotrimeric G protein signaling in oocytes could also be elicited by injection of bacterially overexpressed and purified hPBP. Stimulatory effect was pertussis toxinsensitive and present even in the absence of coexpressed receptors. Additionally, an increase in G protein-mediated inhibition of adenylate cyclase activity, measured by the inhibition of forskolin-mediated cAMP accumulation, could be detected in HEK293 and NIH3T3 cells after expression of hPBP and in Xenopus oocytes after injection of hPBP. As

The relief from an aversive event is rewarding. Since organisms are able to learn which environmental cues can cease an aversive event, relief learning helps to better cope with future aversive events. Literature data suggest that relief learning is affected in various psychopathological conditions, such as anxiety disorders. Here, we investigated the role of the mesolimbic dopamine system in relief learning. Using a relief learning procedure in Sprague Dawley rats, we applied a combination of behavioral experiments with anatomical tracing, c-Fos immunohistochemistry, and local chemogenetic and pharmacological interventions to broadly characterize the role of the mesolimbic dopamine system. The present study shows that a specific part of the mesolimbic dopamine system, the projection from the posterior medial ventral tegmental area (pmVTA) to the nucleus accumbens shell (AcbSh), is activated by aversive electric stimuli. 6-OHDA lesions of the pmVTA blocked relief learning but fear learning and safety learning were not affected. Chemogenetic silencing of the pmVTA-AcbSh projection using the DREADD approach, as well as intra-AcbSh injections of the dopamine D2/3 receptor antagonist raclopride inhibited relief learning. Taken together, the present data demonstrate that the dopaminergic pmVTA-AcbSh projection is critical for relief learning but not for similar learning phenomena. This novel finding may have clinical implications since the processing of signals predicting relief and safety is often impaired in patients suffering from anxiety disorders. Furthermore, it may help to better understand psychological conditions like non-suicidal self-injury, which are associated with pain offset relief.

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