The -opioid receptor (MOR1) and the substance P receptor (NK1) coexist and functionally interact in nociceptive brain regions; however, a molecular basis for this interaction has not been established. (14). Given the high level of sequence homology between opioid and somatostatin receptors existing in domains proposed to contribute to the dimerization interface (i.e. transmembrane helices) it was not unexpected that these closely related receptors can form heterodimers. However, so far little is known about dimerization of opioid receptors with distantly related GPCRs. A major prerequisite for the physiological assembly of heterodimeric GPCRs in vivo is their coexpression in the same cell. MOR1 and NK1, the principal receptor for substance P (SP), coexist and functionally interact in pain-processing brain regions (15)(16)(17)(18)(19)(20)(21)(22). SP is released from nociceptive primary afferents in the spinal and trigeminal dorsal horn, where it activates spino-thalamic projection neurons. Recently, NK1 and MOR1 have been shown to coexist in trigeminal dorsal horn neurons, suggesting that MOR and NK1 functionally interact in these neurons during nociceptive neurotransmission. Furthermore, MOR1 and NK1 are highly expressed in brain regions implicated in depression, anxiety, and stress, but also in other regions such as the nucleus accumbens, which mediate the motivational properties of drugs of abuse including opioids. Interestingly, the rewarding effects of morphine are absent in mice lacking the NK1 receptor (20,21). Despite these observations, a molecular basis for the interactions between opioid and substance P receptors has not been established.Here, we report that the MOR1 receptor forms stable heterodimers with the NK1 receptor. MOR1-NK1 heterodimerization did not substantially change ligand binding and signaling properties of the MOR1 receptor, but it dramatically altered its trafficking and resensitization profile.

Agonist‐induced internalization of G protein‐coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid‐rafts‐ and clathrin‐coated‐pits‐mediated pathways were involved in agonist‐induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Δ9‐THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212‐2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist‐induced CB1 receptor desensitization. Furthermore, co‐expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co‐expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212‐2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Δ9‐THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212‐2, CP 55940 or 2‐arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid‐mediated effects.

Using a yeast two-hybrid screen, the neuronal membrane glycoprotein M6a, a member of the proteolipid protein family, was identified to be associated with the -opioid receptor (MOPr). Bioluminescence resonance energy transfer and co-immunoprecipitation experiments confirmed that M6a interacts agonist-independently with MOPr in human embryonic kidney 293 cells co-expressing MOPr and M6a. Co-expression of MOPr with M6a, but not with M6b or DM20, exists in many brain regions, further supporting a specific interaction between MOPr and M6a. After opioid treatment M6a co-internalizes and then co-recycles with MOPr to cell surface in transfected human embryonic kidney 293 cells. Moreover, the interaction of M6a and MOPr augments constitutive and agonist-dependent internalization as well as the recycling rate of -opioid receptors. On the other hand, overexpression of a M6a-negative mutant prevents -opioid receptor endocytosis, demonstrating an essential role of M6a in receptor internalization. In addition, we demonstrated the interaction of M6a with a number of other G protein-coupled receptors (GPCRs) such as the ␦-opioid receptor, cannabinoid receptor CB1, and somatostatin receptor sst2A, suggesting that M6a might play a general role in the regulation of certain GPCRs. Taken together, these data provide evidence that M6a may act as a scaffolding molecule in the regulation of GPCR endocytosis and intracellular trafficking.The clinical utility of opiates is greatly limited by adaptive changes in the nervous system causing tolerance, dependence, and addiction. These adaptive changes are initiated by binding of opiate drugs to opioid receptors that are also activated by endogenous opioid peptides (1). The -opioid receptor mediates the analgesic effects of opioids but is also important for initiating adaptive changes in the nervous system causing opioid tolerance, dependence, and addiction (2-4). On the cellular level, the repeated opioid exposure induces a rapid phosphorylation of intracellular receptor domains, leading to receptor desensitization followed by receptor internalization (5). Internalized receptors can be sorted either to a degradative pathway for down-regulation or recycling pathway for reactivation (6). There is increasing evidence that the trafficking and signaling of opioid receptors are regulated by direct interaction with membranal and/or cytosolic proteins (7).In the course of identifying new -opioid receptor-interacting proteins using a yeast two-hybrid method, we isolated a cDNA encoding for the membrane glycoprotein M6a. M6a is a member of the proteolipid protein (PLP) 2 family of tetraspan membrane proteins and mainly expressed in neurons (8 -10). M6a shares 40% homology with DM20, the smaller splice isoform of the major central nervous system myelin proteolipid PLP, which is mainly expressed in myelinating glial cells (11). M6a is 55% homologous to proteolipid M6b, which is expressed in neurons and oligodendrocytes (8,9,12). M6a is suggested to play a role as a modulator for neurite outgrowth (13) ...

Stromal-cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) play a well-established role during embryonic development of dentate gyrus granule cells. However, little is known about the regulation and function of CXCR4 in the postnatal dentate gyrus. Here, we identify a striking mismatch between intense CXCR4 mRNA and limited CXCR4 protein expression in adult rat subgranular layer (SGL) neurons. We demonstrate that CXCR4 protein expression in SGL neurons is progressively lost during postnatal day 15 (P15) to P21. This loss of CXCR4 protein expression was paralleled by a reduction in the number of SDF-1-responsive SGL neurons and a massive upregulation of SDF-1 mRNA in granule cells. Intraventricular infusion of the CXCR4-antagonist AMD3100 dramatically increased CXCR4 protein expression in SGL neurons, suggesting that CXCR4 is tonically activated and downregulated by endogenous SDF-1. Infusion of AMD3100 also facilitated detection of CXCR4 protein in bromodeoxyuridine-, nestin-, and doublecortin-labeled cells and showed that the vast majority of adult-born granule cells transiently expressed CXCR4. Chronic AMD3100 administration impaired formation of new granule cells as well as neurogenesis-dependent long-term recognition of novel objects. Therefore, our findings suggest that tonic activation of CXCR4 in newly formed granule cells by endogenous SDF-1 is essential for neurogenesis-dependent long-term memory in the adult hippocampus.

The protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in stroke models is poorly understood. We studied patterns of PACAP, vasoactive intestinal peptide, and the PACAP-selective receptor PAC1 after middle cerebral artery occlusion and neuroprotection by PACAP in cortical cultures exposed to oxygen/glucose deprivation (OGD). Within hours, focal ischemia caused a massive, NMDA receptor (NMDAR)-dependent up-regulation of PACAP in cortical pyramidal cells. PACAP expression dropped below the control level after 2 days and was normalized after 4 days. Vasoactive intestinal peptide expression was regulated oppositely to that of PACAP. PAC1 mRNA showed ubiquitous expression in neurons and astrocytes with minor changes after ischemia. In cultured cortical neurons PACAP27 strongly activated Erk1/2 at low and p38 MAP kinase at higher nanomolar concentrations via PAC1. In astrocyte cultures, effects of PACAP27 on Erk1/2 and p38 were weak. During OGD, neurons showed severely reduced Erk1/2 activity and dephosphorylation of Erk1/2-regulated Ser112 of pro-apoptotic Bad. PACAP27 stimulation counteracted Erk1/2 inactivation and Bad dephosphorylation during short-term OGD but was ineffective after expanded OGD. Consistently, PACAP27 caused MEK-dependent neuroprotection during mild but not severe hypoxic/ischemic stress. While PACAP27 protected neurons at 1-5 nmol/L, full PAC1 activation by 100 nmol/L PACAP exaggerated hypoxic/ischemic damage. PACAP27 stimulation of astrocytes increased the production of Aktactivating factors and conferred ischemic tolerance to neurons. Thus, ischemia-induced PACAP may act via neuronal and astroglial PAC1. PACAP confers protection to ischemic neurons by maintaining Erk1/2 signaling via neuronal PAC1 and by increasing neuroprotective factor production via astroglial PAC1.

We have recently shown that the l-opioid receptor [MOR1, also termed l-opioid peptide (MOP) receptor] is associated with the phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane. We further demonstrated that, in human embryonic kidney (HEK) 293 cells co-expressing MOR1 and PLD2, treatment with (D-Ala2, Me Phe4, Glyol5)enkephalin (DAMGO) led to an increase in PLD2 activity and an induction of receptor endocytosis, whereas morphine, which does not induce opioid receptor endocytosis, failed to activate PLD2. In contrast, a C-terminal splice variant of the l-opioid receptor (MOR1D, also termed MOP 1D ) exhibited robust endocytosis in response to both DAMGO and morphine treatment. We report here that MOR1D also mediates an agonist-independent (constitutive) PLD2-activation facilitating agonist-induced and constitutive receptor endocytosis. Inhibition of PLD2 activity by over-expression of a dominant negative PLD2 (nPLD2) blocked the constitutive PLD2 activation and impaired the endocytosis of MOR1D receptors. Moreover, we provide evidence that the endocytotic trafficking of the d-opioid receptor [DOR, also termed d-opioid peptide (DOP) receptor] and cannabinoid receptor isoform 1 (CB1) is also mediated by a PLD2-dependent pathway. These data indicate the generally important role for PLD2 in the regulation of agonist-dependent and agonist-independent G protein-coupled receptor (GPCR) endocytosis. Keywords: cannabinoid receptor 1, endocytosis, d-opioid receptor, l-opioid receptor, phospholipase D2. Address correspondence and reprint requests to Thomas Koch, Department of Pharmacology and Toxicology, Otto-von-Guericke University, 39120 Magdeburg, Leipziger Str. 44, Germany. E-mail: Thomas.Koch@Medizin.Uni-Magdeburg.deAbbreviations used: AM281, N-(morpholin-4-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AP, adapter protein 2; ARF, ADP-ribosylation factor; BFA, brefeldin A; CB1, cannabinoid receptor isoform 1; DAMGO, (D-Ala2, Me Phe4, Glyol5)enkephalin; DOR, d-opioid receptor; DPDPE, [D-Pen(2,5)]-enkephalin; GPCR, G protein-coupled receptor; HAMOR1, HA epitopetagged rat l-opioid receptor isoform 1; HEK293, human embryonic kidney 293; MOR1, rat l-opioid receptor isoform 1; MOR1D, rat lopioid receptor splice variant 1D; PA, phosphatidic acid; PC, phosphatidylcholine; PKC, protein kinase C; PLD, phospholipase D; PMA, phorbol-12-myristate-13-acetate; PtdEtOH, phosphatidylethanol.

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The interaction of μ-opioid receptor (MOPr) with the neuronal membrane glycoprotein M6a is known to facilitate MOPr endocytosis in human embryonic kidney 293 (HEK293) cells. To further study the role of M6a in the post-endocytotic sorting of MOPr, we investigated the agonist-induced co-internalization of MOPr and M6a and protein targeting after internalization in HEK293 cells that co-expressed HA-tagged MOPr and Myc-tagged M6a. We found that M6a, MOPr, and Rab 11, a marker for recycling endosomes, co-localized in endocytotic vesicles, indicating that MOPr and M6a are primarily targeted to recycling endosomes after endocytosis. Furthermore, co-expression of M6a augmented the post-endocytotic sorting of δ-opioid receptors into the recycling pathway, indicating that M6a might have a more general role in opioid receptor post-endocytotic sorting. The enhanced post-endocytotic sorting of MOPr into the recycling pathway was accompanied by a decrease in agonist-induced receptor down-regulation of M6a in co-expressing cells. We tested the physiological relevance of these findings in primary cultures of cortical neurons and found that co-expression of M6a markedly increased the translocation of MOPrs from the plasma membrane to intracellular vesicles at steady state and significantly enhanced both constitutive and agonist-induced receptor endocytosis. In conclusion, our results strongly indicate that M6a modulates MOPr endocytosis and post-endocytotic sorting and has an important role in receptor regulation.