Agonist exposure of many G protein-coupled receptors induces a rapid receptor phosphorylation and uncoupling from G proteins. Resensitization of these desensitized receptors requires endocytosis and subsequent dephosphorylation. Using a yeast two-hybrid screen, the rat -opioid receptor (MOR1, also termed MOP) was found to be associated with phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane, which has been implicated in the formation of endocytotic vesicles. Coimmunoprecipitation experiments in HEK293 cells coexpressing MOR1 and PLD2 confirmed that MOR1 constitutively interacts with PLD2. ]enkephalin) led to an increase in PLD2 activity, whereas morphine, which does not induce MOR1 receptor internalization, failed to induce PLD2 activation. The DAMGO-mediated PLD2 activation was inhibited by brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) but not by the protein kinase C (PKC) inhibitor calphostin C indicating that opioid receptor-mediated activation of PLD2 is ARF-but not PKC-dependent. Furthermore, heterologous stimulation of PLD2 by phorbol ester led to an accelerated internalization of the -opioid receptor after both DAMGO and morphine exposure. Conversely the inhibition of PLD2-mediated phosphatidic acid formation by 1-butanol or overexpression of a negative mutant of PLD2 prevented agonist-mediated endocytosis of MOR1. Together, these data suggest that PLD2 play a key role in the regulation of agonist-induced endocytosis of the -opioid receptor. Phospholipase D (PLD)1 is a widely distributed phospholipid-specific diesterase that hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) and choline and is assumed to play an important function in cell regulation (1, 2). Signal-dependent activation of PLD was demonstrated in numerous cell types stimulated by various hormones, growth factors, cytokines, neurotransmitters, adhesion molecules, drugs, and physical stimuli (reviewed in Ref. 3). Pathways leading to PLD activation include protein serine/threonine kinases, e.g. protein kinase C, small GTPases, e.g. ADP-ribosylation factor (ARF), RhoA and Ral, phosphatidylinositol 4,5-bisphosphate (PIP2), and tyrosine kinases (4 -6). Recently two mammalian PLDs (PLD1 and PLD2) have been identified (7-10). Subcellular fractionation studies have demonstrated the presence of PLD1 in intracellular membranes, e.g. ER, Golgi, and vesicular compartment (9, 11), whereas PLD2 was largely associated with the plasma membrane (9). After stimulation with serum, redistribution of PLD2 from the plasma membrane into submembraneous endocytotic vesicles (early endosomes) was observed (9). Another study revealed that PLD2 is associated with the EGF receptor (12). Interestingly, EGF receptor endocytosis is impaired when PLD activity is inhibited (13) suggesting a role for PLD2 in receptor trafficking.Using the yeast two-hybrid system to identify proteins that interact with the -opioid receptor, we isolated a rat cDNA encoding for the NH 2 terminus of PLD2. We therefore investig...

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) ...

Receptor phosphorylation, arrestin binding, uncoupling from G protein and subsequent endocytosis have been implicated in G protein-coupled receptor desensitization after chronic agonist exposure. In search of proteins regulating the l-opioid receptor endocytosis, we have recently established that activation of phospholipase D (PLD)2 is required for agonistinduced l-opioid receptor endocytosis. In this study, we determined the effect of PLD2 activity on the desensitization and resensitization rate of the l-opioid receptor. We clearly demonstrated that inhibition of PLD2-mediated phosphatidic acid formation by alcohol (1-butanol or ethanol) or overexpression of a dominant negative mutant of PLD2 prevented agonist-mediated endocytosis and resulted in a faster desensitization rate of the l-opioid receptor after chronic (DAla 2 , Me Phe 4 , Glyol 5 )enkephalin treatment in human embryonic kidney 293 cells. Moreover, inhibition of PLD2 activity led to an impairment of the resensitization rate of the l-opioid receptor. In summary, our data strongly suggest that PLD2 is a modulator of agonist-induced endocytosis, desensitization and resensitization of the l-opioid receptor.

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.