Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction.

Opiate analgesia, tolerance, and addiction are mediated by drug-induced activation of the mu opioid receptor. A fundamental question in addiction biology is why exogenous opiate drugs have a high liability for inducing tolerance and addiction while native ligands do not. Studies indicate that highly addictive opiate drugs such as morphine are deficient in their ability to induce the desensitization and endocytosis of receptors. Here, we demonstrate that this regulatory mechanism reveals an independent functional property of opiate drugs that can be distinguished from previously established agonist properties. Moreover, this property correlates with agonist propensity to promote physiological tolerance, suggesting a fundamental revision of our understanding of the role of receptor endocytosis in the biology of opiate drug action and addiction.     

Insights into mu opioid pharmacology the role of mu opioid receptor subtypes

Although mu opioids share many pharmacological characteristics, they also reveal many differences. Many approaches over the years have suggested the existence of multiple mu opioid receptors. The unique selectivities of naloxonazine, for example, provided a way of distinguishing mu, from mu2 actions. Studies of morphine-6beta-gluruconide suggested that its actions involved yet another mu opioid receptor subtype. The cloning of a mu opioid receptor, MOR-1, provided a way of exploring this possibility at the molecular level. Recent studies have now identified a number of splice variants of this gene that appear to be important in the production of mu opioid analgesia.     

The role of mu opioid receptor desensitization and endocytosis in morphine tolerance and dependence

Following activation, most G protein coupled receptors undergo regulation by a cascade of events that promote receptor desensitization and endocytosis. Following endocytosis, receptors can then be recycled to the plasma membrane, retained in an intracellular compartment, or targeted for degradation. For receptors that are recycled, like the mu opioid receptor (MOR), endocytosis serves as the first step toward resensitizing receptors. For receptors that are degraded, endocytosis serves as the first step toward receptor downregulation. Thus, for receptors like the MOR, the desensitization-endocytosis-resensitization cycle serves as a rapid and dynamic means to titrate signaling through the receptor. However, not all agonist ligands at the MOR promote the same degree of receptor desensitization and endocytosis. For example, the endogenous peptide ligands at the MOR induce rapid desensitization, endocytosis, and recycling. By contrast, morphine induces only weak or partial desensitization and little to no endocytosis. As a consequence, signal transduction promoted by morphine is less dynamic than that induced by endogenous ligands as well as other opioid agonists that promote endocytosis. The resulting imbalance of desensitization-endocytosis-resensitization has at least two consequences: (1) in cell types where morphine induces desensitization but not endocytosis and/or resensitization, desensitization is protracted; (2) in cell types where morphine induces neither desensitization nor endocytosis, prolonged signaling through the receptor leads to multiple cellular adaptations downstream of receptor-G protein coupling. Both protracted desensitization and adaptive cellular changes probably contribute to the pronounced in vivo tolerance and dependence that occur with chronic morphine treatment. As a consequence, facilitating receptor endocytosis, using either genetic or pharmacological approaches, can restore the balance of signaling through the receptor and affect the development of tolerance and dependence.     

Chronic ethanol consumption in rats produces opioid antinociceptive tolerance through inhibition of mu opioid receptor endocytosis

It is well known that the mu-opioid receptor (MOR) plays an important role in the rewarding properties of ethanol. However, it is less clear how chronic ethanol consumption affects MOR signaling. Here, we demonstrate that rats with prolonged voluntary ethanol consumption develop antinociceptive tolerance to opioids. Signaling through the MOR is controlled at many levels, including via the process of endocytosis. Importantly, agonists at the MOR that promote receptor endocytosis, such as the endogenous peptides enkephalin and β-endorphin, show a reduced propensity to promote antinociceptive tolerance than do agonists, like morphine, which do not promote receptor endocytosis. These observations led us to examine whether chronic ethanol consumption produced opioid tolerance by interfering with MOR endocytosis. Indeed, here we show that chronic ethanol consumption inhibits the endocytosis of MOR in response to opioid peptide. This loss of endocytosis was accompanied by a dramatic decrease in G protein coupled receptor kinase 2 (GRK2) protein levels after chronic drinking, suggesting that loss of this component of the trafficking machinery could be a mechanism by which endocytosis is lost. We also found that MOR coupling to G-protein was decreased in ethanol-drinking rats, providing a functional explanation for loss of opioid antinociception. Together, these results suggest that chronic ethanol drinking alters the ability of MOR to endocytose in response to opioid peptides, and consequently, promotes tolerance to the effects of opioids.     

High-affinity naloxone binding to filamin a prevents mu opioid receptor-Gs coupling underlying opioid tolerance and dependence

Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [(3)H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [(3)H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A(2561-2565) as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A(2561-2565) abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor-Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence.     

An opiate cocktail that reduces morphine tolerance and dependence

Morphine is an exceptionally effective analgesic whose utility is compromised by the development of tolerance and dependence to the drug. Morphine analgesia and dependence are mediated by its activity at the mu opioid peptide (MOP) receptor [1]. The MOP receptor is activated not only by morphine, but also by other opiate drugs such as methadone and endogenous opioids such as endorphins. Morphine, however, is a unique opioid agonist ligand because it fails to induce endocytic trafficking of the MOP receptor [2], whereas the endogenous ligands and methadone do facilitate endocytosis [3]. Using the unique pharmacology of the MOP receptor and its proposed existence as an oligomeric structure [4], we designed a pharmacological cocktail that facilitates endocytosis of the MOP receptor in response to morphine. This cocktail consists of morphine and a small dose of methadone. Importantly, this cocktail, while retaining full analgesic potency, does not promote morphine dependence. We further demonstrate that dependence is reduced, at least in part, because endocytosis of the MOP receptor in response to morphine prevents the upregulation of N-methyl-D-aspartate (NMDA) receptors.     

Downregulation of mu opioid receptor by RNA interference in the ventral tegmental area reduces ethanol consumption in mice

Pharmacological and genetic studies have implicated the mu opioid receptor (MOR) in the regulation of ethanol intake in animal models and humans. Non-specific antagonists of opioid receptors have been shown to affect ethanol consumption when infused directly into the ventral tegmental area (VTA) of rats. However, administration of MOR-selective antagonists into the VTA has yielded mixed results. We used RNA interference (RNAi) to specifically decrease levels of MOR messenger RNA in the VTA of mice and examined the effect on ethanol consumption in a two-bottle choice paradigm. Mice were injected in the VTA with lentivirus expressing either a small hairpin RNA (shRNA) targeting MOR or a control shRNA. One week after virus injection, mice were examined for ethanol consumption in a two-bottle choice experiment with increasing concentrations of ethanol over the course of 1 month. Expression of an shRNA targeting MOR in the VTA led to a significant reduction in ethanol consumption. These results strengthen the hypothesis that MOR in the VTA is one of the key brain substrates mediating alcohol consumption. The RNAi combined with lentiviral delivery can be used successfully in brain to effect a sustained reduction in expression of specific genes for behavioral analysis.     

Impedance-based analysis of mu opioid receptor signaling and underlying mechanisms

The mu opioid receptor is a G-protein coupled receptor able to signal through the Gα_i/o class of G-protein and β-arrestin pathways, stimulating down-stream effector pathways. Signaling bias occurs when different receptor agonists lead to different signaling outcomes. Traditionally these have been studied using end-point assays. Real-time cellular analysis platforms allow for the analysis of the holistic effects of receptor activation as an integrated output. While this allows for different ligands to be compared rapidly, the cellular mechanisms underlying the signal are not well described. Using an impedance based system, the impedance responses for two opioid ligands, morphine and DAMGO were examined.The impedance responses for these two agonists, while showing similar features, were distinct from each other. Some of the mechanisms underlying the mu opioid receptor coupled impedance changes were investigated. It was found that the response is a result of discrete cellular processes, including G-protein signaling and protein kinase phosphorylation.     

Molecular identification and functional expression of mu 3, a novel alternatively spliced variant of the human mu opiate receptor gene

Studies from our laboratory have revealed a novel mu opiate receptor, mu 3, which is expressed in both vascular tissues and leukocytes. The mu 3 receptor is selective for opiate alkaloids and is insensitive to opioid peptides. We now identify the mu 3 receptor at the molecular level using a 441-bp conserved region of the mu 1 receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the mu opiate receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the mu 3 receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the mu 3 receptor cDNA exhibited dose-dependent release of NO following treatment with morphine, but not opioid peptides (i.e., Met-enkephalin). Naloxone was able to block the effect of morphine on COS-1 transfected cells. Nontransfected COS-1 cells did not produce NO in the presence of morphine or the opioid peptides at similar concentrations. Receptor binding analysis with [(3)H]dihydromorphine further supports the opiate alkaloid selectivity and opioid peptide insensitivity of this receptor. These data suggest that this new mu opiate receptor cDNA encodes the mu 3 opiate receptor, since it exhibits biochemical characteristics known to be unique to this receptor (opiate alkaloid selective and opioid peptide insensitive). Furthermore, using Northern blot, RT-PCR, and sequence analysis, we have demonstrated the expression of this new mu variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.     

Etonitazene: An opioid selective for the mu receptor types

Specific radioligand binding protocols were utilized to compare the affinity of morphine and the high-potency opioid etonitazene at mu₁, mu₂, delta, kappa₁ and sigma receptors. Both etonitazene and morphine displayed a mu₁-selective binding profile; however, etonitazene had a 2500-fold higher affinity at this receptor type. The latter result is consistent with the relative potencies of morphine and etonitazene in various behavioral tests.     

Multiple actions of spinophilin regulate mu opioid receptor function

Spinophilin, a dendritic spine-enriched scaffold protein, modulates synaptic transmission via multiple functions mediated by distinct domains of the protein. Here, we show that spinophilin is a key modulator of opiate action. Knockout of the spinophilin gene causes reduced sensitivity to the analgesic effects of morphine and early development of tolerance but a higher degree of physical dependence and increased sensitivity to the rewarding actions of the drug. At the cellular level, spinophilin associates with the mu opioid receptor (MOR) in striatum and modulates MOR signaling and endocytosis. Activation of MOR by opiate agonists such as fentanyl and morphine promotes these events, which feedback to suppress MOR responsiveness. Our findings support a potent physiological role of spinophilin in regulating MOR function and provide a potential new target for the treatment of opiate addiction.     

Evidence for a mu-delta opioid receptor complex in CHO cells co-expressing mu and delta opioid peptide receptors

Based on non-competitive binding interactions we suggested that mu and delta receptors associate as a mu/delta receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express mu and delta receptors, but not in cells that express just mu or delta receptors. We used CHO cells expressing the cloned human mu receptor, cloned human delta receptor, or cloned mouse delta/human mu ("dimer cell"). Cell membranes were prepared from intact cells pretreated with 100nM SUPERFIT. [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to delta receptors by approximately 75% and to mu receptors by approximately 50% in dimer cells. SUPERFIT treatment did not decrease [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to mu cells. The IC(50) values observed in SUPERFIT-treated dimer cells were: [d-Pen(2),d-Pen(5)]enkephalin (1820nM) and morphine (171nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen(2),d-Pen(5)]enkephalin (5000nM) was a competitive inhibitor. In contrast, morphine (1000nM) lowered the B(max) from 1944fmol/mg to 1276fmol/mg protein (35% decrease). Both [d-Pen(2),d-Pen(5)]enkephalin and morphine competitively inhibited [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to SUPERFIT-treated mu cells. The results indicate that the mu-delta opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of mu-delta heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of mu-delta heterodimers.     

Analgesic effect of interferon-alpha via mu opioid receptor in the rat

Using the tail-flick induced by electro-stimulation as a pain marker, it was found that pain threshold (PT) was significantly increased after injecting interferon-alpha (IFN alpha) into the lateral ventricle of rats. This effect was dosage-dependent and abolished by monoclonal antibody (McAb) to IFN alpha. Naloxone could inhibit the analgesic effect of IFN alpha, suggesting that the analgesic effect of IFN alpha be related to the opioid receptors. Beta-funaltrexamine (beta-FNA), the mu specific receptor antagonist could completely block the analgesic effect of IFN alpha. The selective delta-opioid receptor antagonist, ICI174,864 and the kappa-opioid receptor antagonist, nor-BNI both failed to prevent the analgesic effect of IFN alpha. IFN alpha could significantly inhibit the production of the cAMP stimulated by forskolin in SK-N-SH cells expressing the mu-opioid receptor, not in NG108-15 cells expressing the delta-opioid receptor uniformly. The results obtained provide further evidence for opioid activity of IFN alpha and suggest that this effect is mediated by central opioid receptors of the mu subtype. The evidence is consistent with the hypothesis that multiple actions of cytokines, such as immunoregulatory and neuroregulatory effects, might be mediated by distinct domains of cytokines interacting with different receptors.     

Identification of an opioid receptor subunit carrying the mu binding site

The enkephalin affinity reagent [3H]Tyr-D-Ala-Gly-Phe-Leu-CH2Cl [( 3H]DALECK) was synthesized. It exhibited high-affinity reversible binding, at pH 7.4, to both mu and delta opioid receptor sites in rat brain membranes. At pH 8.1, nanomolar levels of [3H]DALECK produced an irreversible labeling in synaptic membranes, essentially only in one subunit of 58 000 daltons. The irreversible phase of the reaction reduced the subsequent binding of a mu-selective enkephalin derivative but not that of a delta-selective one. It is concluded that a mu subunit of the opioid receptor exists, can be alkylated specifically, and is of Mr 58 000.     

Agonist-induced, G protein-dependent and -independent down-regulation of the mu opioid receptor. The receptor is a direct substrate for protein-tyrosine kinase.

The mu opioid receptor (MOR) has been shown to desensitize after 1 h of exposure to the opioid peptide, [D-Ala(2), N-MePhe(4), Gly-ol(5)]enkephalin (DAMGO), largely by the loss of receptors from the cell surface and receptor down-regulation. We have previously shown that the Thr(394) in the carboxyl tail is essential for agonist-induced early desensitization, presumably by serving as a primary phosphorylation site for G protein-coupled receptor kinase. Using a T394A mutant receptor, we determined that Thr(394) was also responsible for mu opioid receptor down-regulation. The T394A mutant receptor displayed 50% reduction of receptor down-regulation (14.8%) compared with wild type receptor (34%) upon 1 h of exposure to DAMGO. Agonist-induced T394A receptor down-regulation was unaffected by pertussis toxin treatment, indicating involvement of a mechanism independent of G protein function. Interestingly, pertussis toxin-insensitive T394A receptor down-regulation was completely inhibited by a tyrosine kinase inhibitor, genistein. Tyrosine kinase inhibition blocked wild type MOR down-regulation by 50%, and the genistein-resistant wild type MOR down-regulation was completely pertussis toxin-sensitive. Following DAMGO stimulation, MOR was shown to be phosphorylated at tyrosine residue(s), indicating that the receptor was a direct substrate for tyrosine kinase action. Mutagenesis of the four intracellular tyrosine residues resulted in complete inhibition of the G protein-insensitive MOR internalization. Therefore, agonist-induced MOR down-regulation appears to be mediated by two distinct cellular signal transduction pathways. One is G protein-dependent and GRK-dependent, which can be abolished by pertussis toxin treatment of wild type MOR or by mutagenesis of Thr(394). The other novel pathway is G protein-independent but tyrosine kinase-dependent, blocked by genistein treatment, and one in which Thr(394) has no regulatory role but phosphorylation of tyrosine residues appears essential.     

Delta and mu opiate receptor probes: fluorescent enkephalins with high receptor affinity and specificity

The fluorescent amino acid, L-1-pyrenylalanine (Pya) was incorporated into [D-Ala2,Leu5]enkephalin and its methyl ester at position 4 or 5. Pya-enkephalins showed strong fluorescent intensity and displayed high binding affinity for opiate receptors. Pya4-enkephalins showed high specificity for the mu receptors, while Pya5-enkephalins showed high specificity and selectivity for the delta receptors. Particularly, [D-Ala2,Pya5]enkephalin was as potent as the most utilized delta-specific ligand of [D-Ala2,D-Leu5]enkephalin (DADLE), and yet its delta-selectivity was about 5-times greater than that of DADLE. Thus, Pya-enkephalins per se can be utilized as a fluorescent probe or tracer for the opiate receptor-binding assays.     

Molecular and cellular mechanisms of the age-dependency of opioid analgesia and tolerance

ABSTRACT: The age-dependency of opioid analgesia and tolerance has been noticed in both clinical observation and laboratory studies. Evidence shows that many molecular and cellular events that play essential roles in opioid analgesia and tolerance are actually age-dependent. For example, the expression and functions of endogenous opioid peptides, multiple types of opioid receptors, G protein subunits that couple to opioid receptors, and regulators of G protein signaling (RGS proteins) change with development and age. Other signaling systems that are critical to opioid tolerance development, such as N-methyl-D-aspartic acid (NMDA) receptors, also undergo age-related changes. It is plausible that the age-dependent expression and functions of molecules within and related to the opioid signaling pathways, as well as age-dependent cellular activity such as agonist-induced opioid receptor internalization and desensitization, eventually lead to significant age-dependent changes in opioid analgesia and tolerance development.     

Role for the C-terminus in agonist-induced mu opioid receptor phosphorylation and desensitization

Determining which domains and amino acid residues of the mu opioid receptor are phosphorylated is critical for understanding the mechanism of mu opioid receptor phosphorylation. The role of the C-terminus of the receptor was investigated by examining the C-terminally truncated or point-mutated mu opioid receptors in receptor phosphorylation and desensitization. Both wild-type and mutated receptors were stably expressed in Chinese hamster ovary (CHO) cells. The receptor expression was confirmed by receptor radioligand binding and immunoblottting. After exposure to 5 microM of DAMGO, phosphorylation of the C-terminally truncated receptor and the mutant receptor T394A was reduced to 40 and 10% of that of the wild-type receptor, respectively. Mutation effects on agonist-induced desensitization were studied using adenylyl cyclase inhibition assays. The C-terminally truncated receptor and mutant receptor T394A both showed complete loss of DAMGO-induced desensitization, while the mutant T/S-7A receptor only lost part of its ability to desensitize. Taken together, these results suggest that the C-terminus of the mu opioid receptor participates in receptor phosphorylation and desensitization with threonine 394, a crucial residue for both features. DAMGO-induced mu opioid receptor phosphorylation and desensitization are associated and appear to involve both the mu opioid receptor C-terminus and other domains of the receptor.