Opioid Receptor Dimers Research Articles

Opioid Addiction and Opioid Receptor Dimerization: Structural Modeling of the OPRD1 and OPRM1 Heterodimer and Its Signaling Pathways.

Opioid addiction is a complex phenomenon with genetic, social, and other components. Due to such complexity, it is difficult to interpret the outcome of clinical studies, and thus, mutations found in individuals with these addictions are still not indisputably classified as opioid addiction-causing variants. Here, we computationally investigated two such mutations, A6V and N40D, found in the mu opioid receptor gene OPRM1. The mutations are located in the extracellular domain of the corresponding protein, which is important to the hetero-dimerization of OPRM1 with the delta opioid receptor protein (OPRD1). The hetero-dimerization of OPRD1–OPRM1 affects the signaling pathways activated by opioids and natural peptides and, thus, could be considered a factor contributing to addiction. In this study, we built four 3D structures of molecular pathways, including the G-protein signaling pathway and the β-arrestin signaling pathway of the heterodimer of OPRD1–OPRM1. We also analyzed the effect of mutations of A6V and N40D on the stability of individual OPRM1/OPRD1 molecules and the OPRD1–OPRM1 heterodimer with the goal of inferring their plausible linkage with opioid addiction. It was found that both mutations slightly destabilize OPRM1/OPRD1 monomers and weaken their association. Since hetero-dimerization is a key step for signaling processes, it is anticipated that both mutations may be causing increased addiction risk.

Inhibition of endothelin A receptor by a novel, selective receptor antagonist enhances morphine-induced analgesia: Possible functional interaction of dimerized endothelin A and μ-opioid receptors

BackgroundThe misuse of opioids has led to an epidemic in recent times. The endothelin A receptor (ETAR) has recently attracted attention as a novel therapeutic target to enhance opioid analgesia. We hypothesized that endothelin A receptors may affect pain mechanisms by heterodimerization with μ opioid receptors. We examined the mechanisms of ETAR-mediated pain and the potential therapeutic effects of an ETAR antagonist, Compound-E, as an agent for analgesia. MethodsReal-time in vitro effect of Compound-E on morphine response was assessed in HEK293 cells expressing both endothelin A and μ opioid receptors through CellKey™ and cADDis cAMP assays. Endothelin A/μ opioid receptor dimerization was assessed by immunoprecipitation and live cell imaging. The in vivo effect of Compound-E was evaluated using a morphine analgesia mouse model that observed escape response behavior, body temperature, and locomotor activity. ResultsIn CellKey™ and cAMP assays, pretreatment of cells with endothelin-1 attenuated morphine-induced responses. These responses were improved by Compound-E, but not by BQ-123 nor by bosentan, an ETAR and endothelin B receptor antagonist. Dimerization of ETARs and μ opioid receptors was confirmed by Western blot and total internal reflection fluorescence microscopy in live cells. In vivo, Compound-E potentiated and prolonged the analgesic effects of morphine, enhanced hypothermia, and increased locomotor activity compared to morphine alone. ConclusionThe results suggest that attenuation by endothelin-1 of morphine analgesia may be caused by dimerization of Endothelin A/μ opioid receptors. The novel ETAR antagonist Compound-E could be an effective adjunct to reduce opioid use.

Monitoring Opioid Receptor Interaction in Living Cells by Bioluminescence Resonance Energy Transfer (BRET).

Bioluminescence resonance energy transfer (BRET ) is a natural phenomenon that has been successfully applied for the study of protein-protein interactions, including opioid receptor oligomers. The discovery of opioid receptor homomers and heteromers has brought to the discovery of new functions and new way of signaling and trafficking; therefore, opioid receptor oligomers may be considered as novel drug targets. Fusing receptors of interest with Renilla luciferase and with a fluorescent protein (such as EYFP ) it is possible to study opioid receptor dimerization using BRET .

Monitoring opioid receptor dimerization in living cells by bioluminescence resonance energy transfer (BRET).

Bioluminescence resonance energy transfer (BRET) is a natural phenomenon that has been successfully applied for the study of protein-protein interactions, including opioid receptor oligomers. The discovery of opioid receptor homomers and heteromers has brought to the finding of new functions and new way of signaling and trafficking; therefore, opioid receptor oligomers may be considered as novel drug targets. Fusing receptors of interest with Renilla luciferase and with a fluorescent protein (such as EYFP), it is possible to study opioid receptor dimerization using BRET.

Modeling Complexes of Transmembrane Proteins: Systematic Analysis of ProteinProtein Docking Tools.

Proteinprotein docking methodology is frequently used to model complexes of transmembrane proteins, in particular oligomers of G protein-coupled receptors (GPCRs), even if its applicability for these systems has never been fully validated. The aim of this work is to perform a systematic study on the suitability of some widely-used proteinprotein docking software for modeling complexes of transmembrane proteins. In this study we tested the programs ZDOCK, ClusPro, HEX, GRAMM-X, PatchDock, SymmDock, and HADDOCK, using a set of membrane protein oligomers for which the 3D structure has been obtained experimentally, including opsin dimer, the recently published chemokine CXCR4 and kappa opioid receptor dimers. The results show that the docking success depends on the applied docking algorithm and scoring functions, but also on inherent structural features of the transmembrane proteins. Thus, proteins with large interface surfaces, rich in surface cavities, high-order symmetry, and small conformational change upon complex formation are well predicted more often than proteins without these features. The results of this systematic analysis provide guidelines that can be used for obtaining reliable models of transmembrane proteins, including GPCRs. Therefore they can be useful for the application of structure-based methods in drug discovery projects involving these targets.

Spinal interaction between the highly selective μ agonist DAMGO and several δ opioid receptor ligands in naive and morphine-tolerant mice

Since the discovery of opioid receptor dimers their possible roles in opioid actions were intensively investigated. Here we suggest a mechanism that may involve the μ-δ opioid heterodimers. The exact role of δ opioid receptors in antinociception and in the development of opioid tolerance is still unclear. While receptor up-regulation can be observed during the development of opioid tolerance no μ receptor down-regulation could be detected within five days. In our present work we investigated how the selective δ opioid receptor agonists and antagonists influence the antinociceptive effect of the selective μ receptor agonist DAMGO in naïve and morphine-tolerant mice. We treated male NMRI mice with 200 μmol/kg subcutaneous (s.c.) morphine twice daily for three days. On the fourth day we measured the antinociceptive effect of DAMGO alone and combined with delta ligands: DPDPE, deltorphin II (agonists), TIPP and TICPψ (antagonists), respectively, administered intrathecally (i.t.) in mouse tail-flick test. In naive control mice none of the δ ligands caused significant changes in the antinociceptive action of DAMGO. The treatment with s.c. morphine resulted in approximately four-fold tolerance to i.t. DAMGO, i.e. the ED₅₀ value of DAMGO was four times as high as in naive mice. 500 and 1000 pmol/mouse of the δ₁ selective agonist DPDPE enhanced the tolerance to DAMGO while 1000 pmol/mouse of the δ₂ selective agonist deltorphin II did not influence the degree of tolerance. However, both δ antagonists TIPP and TICPψ potentiated the antinociceptive effect of i.t. DAMGO, thus they restored the potency of DAMGO to the control level. The inhibitory action of DPDPE against the antinociceptive effect of DAMGO could be antagonized by TIPP and TICPψ. We hypothesize that during the development of morphine tolerance the formation of μδ heterodimers may contribute to the spinal opioid tolerance. δ ligands may affect the dimer formation differently. Those, like DPDPE may facilitate the dimer formation hence inhibit the antinociceptive effect of DAMGO by causing virtual μ receptor down-regulation. Ligands that do not affect the dimer formation do not influence antinociception either but ligands with the presumed capability of disconnecting the dimers may decrease the spinal tolerance to DAMGO.

Opiate Antagonist Prevents μ- and δ-Opiate Receptor Dimerization to Facilitate Ability of Agonist to Control Ethanol-altered Natural Killer Cell Functions and Mammary Tumor Growth

In the natural killer (NK) cells, δ-opiate receptor (DOR) and μ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.

Mix and Match: Heterodimers and Opioid Tolerance

Heterodimerization of G-protein coupled receptors has become increasingly recognized as a valuable mechanism to increase receptor diversity. Heterodimers have been observed in the opioid receptor family, but one of the most intriguing is that formed between mu and delta opioid receptors. In this issue of Neuron He et al. present evidence further implicating these heterodimers in morphine tolerance.

Modulation of opioid receptor function by protein-protein interactions

Opioid receptors, MORP, DORP and KORP, belong to the family A of G protein coupled receptors (GPCR), and have been found to modulate a large number of physiological functions, including mood, stress, appetite, nociception and immune responses. Exogenously applied opioid alkaloids produce analgesia, hedonia and addiction. Addiction is linked to alterations in function and responsiveness of all three opioid receptors in the brain. Over the last few years, a large number of studies identified protein-protein interactions that play an essential role in opioid receptor function and responsiveness. Here, we summarize interactions shown to affect receptor biogenesis and trafficking, as well as those affecting signal transduction events following receptor activation. This article also examines protein interactions modulating the rate of receptor endocytosis and degradation, events that play a major role in opiate analgesia. Like several other GPCRs, opioid receptors may form homo or heterodimers. The last part of this review summarizes recent knowledge on proteins known to affect opioid receptor dimerization.

In Vivo Characterization of (−)(−)MCL-144 and (+)(−)MCL-193: Isomeric, Bivalent Ligands with Mu/Kappa Agonist Properties

Once opioid receptor dimers were postulated, a goal has been to synthesize and screen novel opioids, with the hope of furthering our knowledge of the structure-activity relationship of opioid ligands with the opioid receptors. The aim of the current study was to address whether two isomeric bivalent ligands would have pharmacological differences after central administration, in vivo. The two compounds, (-) bis(N-cyclobutylmethyl-morphinan-3-yl) sebacoylate dihydrochloride (MCL-144) and 1-((+)N-cyclobutylmethylmorphinan-3-yl)-10-((-) N-cyclobutylmethylmorphinan-3-yl)sebacolyate (MCL-193) are each linked by a 10-carbon chain ester. The active (-) enantiomer for both ligands is 3-hydroxy-N-cyclobutylmethyl morphinan ((-)MCL-101), a N-cyclobutylmethyl analogue of cyclorphan (J Med Chem 43:114-122, 2000). MCL-144 contains two active levo rotatory (-)(-) pharmacophores, while MCL-193 contains one active (-) and one inactive (+) pharmacophore of MCL-101. In vitro analysis demonstrated that all three compounds, (-)(-)MCL-144, (+)(-)MCL-193 and (-)MCL-101 were kappa agonists and mu partial agonists. (-)(-)MCL-144 and (-)MCL-101 had much higher affinity for both the mu and kappa opioid receptors compared to (+)(-)MCL-193. In vivo, (-)(-)MCL-144 and (+)(-)MCL-193 produced full dose-response curves, in the 55 degrees C tail-flick test, with each compound having an ED(50) value of 3.0 nmol after intracerebroventricular (i.c.v.) administration. The analgesic properties of both compounds were antagonized by the mu-selective antagonist, beta-funaltrexamine and the kappa-selective antagonist nor-binaltorphimine. Concomitant, i.c.v., administration of either (-)(-)MCL-144 or (+)(-)MCL-193 with morphine, did not significantly antagonize morphine-induced antinociception at any dose tested. In antinociceptive tests, (-)(-)MCL-144 and (+)(-)MCL-193 had the same pharmacological properties, demonstrating that having two active pharmacophores separated by a 10-carbon spacer group did not increase the antinociceptive efficacy of the compound. Additionally, it was also of interest to compare (-)(-)MCL-145 and (-)(-)MCL-144, as the only difference between these bivalent ligands is the spacer region connecting the two pharmacophores, yet (-)(-)MCL-145 produced an ED(50) value 10-fold lower than (-)(-)MCL-144 (ED(50) values = 0.3 nmol and 3.0 nmol, respectively).

Look before leaping: combined opioids may not be the rave

The use of combinations of potent opioids is a common clinical practice. The addition of one potent opioid to another has been recommended to reduce opioid side effects, improve pain control, and limit dose escalation of the first opioid. The advantages of using combined opioids have been reported to be relative to differences in receptor activation versus endocytosis (RAVE). However, the advantages and detriment to combining opioids are related to naturally occurring opioid receptor dimers. Dimers and oligomers result in a unique opioid pharmacodynamics which influence opioid binding, G protein interactions, desensitization, receptor trafficking, and endocytosis. The pharmacodynamics of dimers may lead to positive or negative cooperativity when two opioids are combined. The use of multiple opioids in practice can lead to increased risk for dosing errors, reduced patient compliance, increased drug interactions and cost. Opioid combinations should not be used until prospective randomized trials clarify the benefits and safety.

Opioid Receptor Homo- and Heterodimerization in Living Cells by Quantitative Bioluminescence Resonance Energy Transfer

Opioid receptors have been shown to dimerize or oligomerize among themselves and each other, affecting their functional properties. This study used bioluminescence resonance energy transfer (BRET) between the mu, delta, and kappa opioid receptors to study opioid receptor aggregation in transfected human embryonic kidney 293 cells. Titration of receptor levels indicated that all three opioid receptors have a similar affinity to form homo- or hetero-oligomers in combination with any other opioid receptor type. In contrast, none of the opioid receptors formed detectable oligomers with the muscarinic M2 receptor, indicating that interactions among opioid receptors are selective. The formation of opioid receptor dimers, rather than higher order oligomers, is supported by binding kinetics in competition experiments between labeled and unlabeled receptors. Opioid receptor dimerization occurred at physiological temperatures upon receptor biosynthesis, before trafficking to the plasma membrane. Moreover, using BRET, coimmunoprecipitation, receptor binding, and G protein coupling, we demonstrate for the first time functional mu opioid receptor-kappa opioid receptor heterodimerization. These combined results demonstrate that opioid receptors can undergo homo- and heterodimerization, a process with potential implications for opioid physiology and pharmacology.

2000 Alfred Burger Award Address in Medicinal Chemistry. From Models to Molecules: Opioid Receptor Dimers, Bivalent Ligands, and Selective Opioid Receptor Probes.

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correction2000 Alfred Burger Award Address in Medicinal Chemistry. From Models to Molecules: Opioid Receptor Dimers, Bivalent Ligands, and Selective Opioid Receptor Probes.Philip S. PortogheseCite this: J. Med. Chem. 2001, 44, 22, 3758Publication Date (Web):September 29, 2001Publication History Published online29 September 2001Published inissue 1 October 2001https://doi.org/10.1021/jm0103514Copyright © 2001 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views389Altmetric-Citations3LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (8 KB) Get e-Alerts Get e-Alerts

From Models to Molecules: Opioid Receptor Dimers, Bivalent Ligands, and Selective Opioid Receptor Probes

From Models to Molecules: Opioid Receptor Dimers, Bivalent Ligands, and Selective Opioid Receptor Probes

Opioids and Their Complicated Receptor Complexes

No field more eagerly awaits a molecular clarification for G-protein coupled receptor (GPCR) dimerization than the opioid receptor field. Extensive evidence of pharmacological and functional interactions between opioid receptor types has primed this field for such a resolution. In retrospect, much of the data collected on synergy between different opioid receptor types may represent the functional correlate for the newly found opioid receptor dimerization. While previous reports of functional synergy have been, for the most part, consistent in demonstrating cross-regulation between two receptor types, the lack of highly receptor-selective ligands allowed skeptics to remain doubtful over the interpretations of these results. Today, two important developments in the opioid receptor field help reinvigorate the hypothesis of functional, cross-modulating opioid receptor complexes: (1) The existence of highly selective ligands which eliminate any possibility of cross-reactivity between receptor types, and (2) the discovery that opioid receptors and a number of other GPCRs exist as dimers in biochemical, functional and pharmacological assays. It is with these new tools that we seek to understand the mechanisms and implications of dimerization. Initial results of these studies have demonstrated that the dimerization of opioid receptors may help consolidate several pharmacological findings that have remained unanswered. In this review we present biochemical, pharmacological and functional evidence for opioid receptor complexes and add evidence from our recent studies on opioid receptor dimerization. We believe a thorough understanding of receptor dimerization is crucial in clarifying the mechanism of action of opioids and other drugs and may serve a more practical purpose in aiding the development of novel therapeutic drugs. [Neruopsychopharmacology 23:S5–S18, 2000]