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  • REVIEW ARTICLE

    Molecular Mechanisms of Opioid Receptor-dependentSignaling and Behavior

    Ream Al-Hasani, Ph.D.,* Michael R. Bruchas, Ph.D.†

    ABSTRACT

    Opioid receptors have been targeted for the treatment of painand related disorders for thousands of years and remain themost widely used analgesics in the clinic. Mu (�), kappa (�),and delta (�) opioid receptors represent the originally classi-fied receptor subtypes, with opioid receptor like-1 (ORL1)being the least characterized. All four receptors are G-proteincoupled and activate inhibitory G proteins. These receptorsform homo- and heterodimeric complexes and signal to ki-nase cascades and scaffold a variety of proteins.

    The authors discuss classic mechanisms and developments inunderstanding opioid tolerance and opioid receptor signalingand highlight advances in opioid molecular pharmacology, be-havioral pharmacology, and human genetics. The authors putinto context how opioid receptor signaling leads to the modu-lation of behavior with the potential for therapeutic interven-tion. Finally, the authors conclude there is a continued need formore translational work on opioid receptors in vivo.

    O PIOIDS are the most widely used and effective anal-gesics for the treatment of pain and related disorders.Opiates have been used for thousands of years for the treat-

    ment of pain, and in the last century we have made hugestrides in the development of opioids derived from naturallyoccurring opiates within the fields of receptor pharmacologyand medicinal chemistry. In addition, opioids are used fre-quently in the treatment of numerous other disorders, includingdiarrhea, cough, postoperative pain, and cancer (table 1).

    Opioid systems are critical in the modulation of pain behav-ior and antinociception. Opioid peptides and their receptors areexpressed throughout the nociceptive neural circuitry and criti-cal regions of the central nervous system included in reward andemotion-related brain structures. To date, four different opioidreceptor systems mu (�), delta (�), kappa (�), opioid receptorlike-1 (ORL1) and their genes have been characterized at thecellular, molecular, and pharmacologic levels.1

    The most commonly used opioids for pain managementact on � opioid receptor (MOR) systems (fig. 1). Although �opioids continue to be some of the most effective analgesics,they are also mood enhancers and cause activation of centraldopamine reward pathways that modulate euphoria. Theseunwanted side effects have driven researchers at basic andclinical levels to actively pursue other opioid receptors asputative drug targets for pain relief (table 1).

    The opioid receptor subtypes were identified pharmaco-logically and genetically more than 2 decades ago.1 From thatpoint on, numerous studies have implicated all four opioidreceptors in an array of behavioral effects, including analge-sia, reward, depression, anxiety, and addiction. In addition,all four receptor subtypes have been characterized at cellularlevels with respect to the downstream signal transductionpathways they activate. However, there are fewer studies thathave directly linked opioid signal transduction to behavioralevents. One of the “holy grails” in opioid pharmacology re-search has been to identify pathway-specific opiate receptoragonists that could activate antinociceptive signaling withoutcausing � agonist-mediated euphorigenic responses or � ag-onist-mediated dysphoria.2,3 Understanding the diversity ofsignaling at opioid receptors and how second messenger ac-tivation leads to modulation of pain and reward could revealnovel opioid receptor drug candidates.

    In this review, we highlight the current status of in vitromolecular pharmacology at opioid receptors and discussmany of the recent advances that connect these molecular stud-

    * Postdoctoral Researcher, † Assistant Professor, Departments of Anes-thesiology and Anatomy/Neurobiology, Washington University School ofMedicine, Washington University Pain Center, St. Louis, Missouri.

    Received from the Departments of Anesthesiology and Anato-my/Neurobiology, Washington University Pain Center, WashingtonUniversity School of Medicine, St. Louis, Missouri. Submitted forpublication May 1, 2011. Accepted for publication September 13,2011. Supported by the National Institute on Drug Abuse, NationalInstitutes of Health, Bethesda, Maryland, grant MRB DA025182 (toDr. Bruchas). Figures 1-3 were prepared by Annemarie B. Johnson,C.M.I., Medical Illustrator, Wake Forest University School of Medi-cine Creative Communications, Wake Forest University MedicalCenter, Winston-Salem, North Carolina.

    Address correspondence to Dr. Bruchas: Department of An-esthesiology, Washington University School of Medicine, 660South Euclid Avenue, Box 8054, St. Louis, Missouri [email protected] Information on purchasing reprints maybe found at www.anesthesiology.org or on the masthead page atthe beginning of this issue. ANESTHESIOLOGY’s articles are madefreely accessible to all readers, for personal use only, 6 monthsfrom the cover date of the issue.

    Copyright © 2011, the American Society of Anesthesiologists, Inc. LippincottWilliams & Wilkins. Anesthesiology 2011; 115:1363–81

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  • ies with opioid behavioral pharmacology. We discuss the ad-vances in opioid receptor pharmacology and highlight the con-nections between signaling at opioid receptors, tolerance toopioids, and behavioral responses. The review’s primary aim isto discuss recent efforts in understanding how opioid receptorsmediate a diverse array of molecular or cellular responses whilealso modulating behaviors such as analgesia, reward, depression,and anxiety. We summarize the modern advances in opioidreceptor signaling to mitogen-activated protein kinases(MAPK) and receptor protein–protein interaction networksand propose that there is a strong potential for selective ligandintervention at opioid receptors to treat a variety of central andperipheral nervous system disorders by using biased ligandsand pathway-selective pharmacology. Moreover, we highlighthow a greater connection between these advances at the molec-ular levels and behavioral pharmacology is imperative to fullyunderstand the field of opioid pharmacology.

    Opioid Tolerance in the ClinicBefore a detailed understanding of the molecular and cel-lular actions of opioid receptors is developed, it is impor-

    tant to consider their general effects and those observed indaily clinical settings. Different potencies of opiate drugformulations have been effective in the treatment of avariety of acute, chronic, and cancer-related pain disor-ders. The clinical utility of opioids continues to be limitedby a compromise between efficacy and side effects. Themost common side effects of opiates can be divided intoperipheral effects (constipation, urinary retention, hives,bronchospasm) and central effects (nausea, sedation, re-spiratory depression, hypotension, miosis, cough suppres-sion), all of which seriously affect the agents’ clinical util-ity and patients’ quality of life4,5 (table 1). There havebeen many attempts to develop better opioid drugs, butthese have been largely unsuccessful because of our incom-plete understanding about the development of toleranceto the analgesic effects.6

    Opioid tolerance is defined typically in the clinic as the needto increase a dose to maintain the analgesic effects. However,this increase in dose can exacerbate the perpetual problem of theside effects mentioned. This continual cycle of insufficient an-algesia and side effects is among the greatest challenges of using

    Table 1. Organ System Effects of Morphine and Its Surrogates

    Organ Systems Effects Additional Information

    Central nervoussystem

    1Analgesia —1Euphoria Leading to risk of addiction and abuse1Sedation —2Rate of respiration —2Cough reflex Codeine used for treatment of pathologic cough1Miosis—constriction of the pupils1Truncal rigidity1Nausea and vomiting

    Most apparent when using fentanyl,sufentanil, alfentanil

    Peripheral Gastrointestinal system —1Constipation2Gastric motility2Digestion in the small intestine2Peristaltic waves in the colon1Constriction of biliary smooth muscle1Esophageal reflux

    Other smooth muscle1Depression of renal function2Uterine tone1Urinary retention

    Skin1Itching and sweating1Flushing of the face, neck, and thorax

    Cardiovascular system2Blood pressure and heart rate if cardiovascular

    system is stressed

    Immune system2Formation of rosettes by human lymphocytes2Cytotoxic activity of natural killer cells

    OtherBehavioral restlessness

    The actions summarized in this table are observed for all clinically available opioid agonists.

    Opioid Signaling

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  • opioids in the clinic. Because of these limitations, opioid toler-ance can ultimately lead to low patient compliance and treat-ment discontinuation. These clinical problems highlight thecontinued need for a better understanding of the molecular andpharmacologic mechanisms of opioid receptor tolerance, regu-lation, and signal transduction.

    Classic Opioid Receptors SignalingOpioid receptors are expressed in pain-modulating descend-ing pathways, which include the medulla, locus coeruleus,and periaqueductal gray area. They are also expressed in lim-bic, midbrain, and cortical structures (fig. 1). The activationof opioid receptors at these locations directly inhibits neu-rons, which in turn inhibit spinal cord pain transmission.4,5

    The process by which these receptors engage in disinhibitionis understood mostly with respect to analgesia; however, re-search is still active in this area because investigators continueto unravel novel modulatory mechanisms in these opioidcircuits.

    All four opioid receptors are seven-transmembrane span-ning proteins that couple to inhibitory G proteins. Afterbeing activated by an agonist, such as the endogenous �-opi-oid peptide endorphin, or exogenous agonists, such as mor-phine and fentanyl, the G� and G�� subunits dissociatefrom one another and subsequently act on various intracel-lular effector pathways.7,8 Early work in opioid receptorpharmacology demonstrated that guanine nucleotides such

    as guanosine triphosphate (GTP) modulate agonist bindingto opioid receptors in membrane preparations from braintissue. It was later determined that GTPase activity is stimu-lated by opioid agonists and endogenous opioid peptides.9

    Agonist stimulation of opioid receptors was also shown toinhibit cyclic adenosine monophosphate (cAMP) produc-tion in a manner similar to that of other types of G protein-coupled receptors (GPCR).10 When pertussis toxin was usedto selectively adenosine diphosphate (ADP)-ribosylate the Gprotein, the inhibitory function of opioid receptors oncAMP signaling was found to be G�i dependent.

    11,12 Todayit is widely accepted that all four opioid receptor types coupleto pertussis-toxin–sensitive G proteins, including G�i, tocause inhibition of cAMP formation.

    The classic and perhaps most important aspect of opioidreceptor signal transduction relates to opioids’ ability tomodulate calcium and potassium ion channels (fig. 2). AfterG�i dissociation from G��, the G� protein subunit moveson to directly interact with the G-protein gated inwardlyrectifying potassium channel, Kir3. Channel deactivationhappens after GTP to guanosine diphosphate hydrolysis andG�� removal from interaction with the channel.13–15 Thisprocess causes cellular hyperpolarization and inhibits tonicneural activity. In several reports, the inhibitory effects ofopioids on neural excitability were shown to be mediated byinteractions of opioid receptors with G protein-regulated in-wardly rectifying potassium channel (Kir3).16,17

    When activated, all four opioid receptors cause a reduc-tion in Ca�2 currents that are sensitive to P/Q-type, N-type,and L-type channel blockers.18 Opioid receptor-induced in-hibition of calcium conductance is mediated by binding ofthe dissociated G�� subunit directly to the channel. Thisbinding event is thought to reduce voltage activation of chan-nel pore opening.19,20 Numerous reports have shown thatopioid receptors interact with and modulate Ca�2 channels;this has led to the examination of specific Ca�2 channelsubunits that may be involved in opioid receptor modula-tion. For instance, it was reported that MOR stimulationresults in G protein-dependent inhibition of �1A and �1Bsubunits.21

    It is also clear that the acute administration of opioidagonists reduces Ca�2 content in synaptic vesicles and syn-aptosomes, with compensatory up-regulation of vesicularCa�2 content during the development of opiate toler-ance.22,23 In addition, because the activation of �, �, and �opioid receptors inhibits adenylyl cyclase activity, the cAMP-dependent Ca�2 influx is also reduced.

    The evidence for opioid receptors positively coupling topotassium channels while negatively modulating calciumchannels has been reported in numerous model systems andcell types. For many years this was thought to be the primaryaction of opioid receptors in the nervous system. This cou-pling of opioid receptors to potassium and calcium channelshas been demonstrated in a wide range of systems, fromneurons in the hippocampus, locus coeruleus, and ventral

    Fig. 1. Sites of action of opioid analgesics. The gray pathwayshows the sites of action on the pain transmission pathwayfrom periphery to central nervous system. The red pathwayshows the actions on pain-modulating neurons in the mid-brain and medulla. GABA � �-aminobutyric acid; MOR � �opioid receptor.

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  • tegmental area to the dorsal root ganglia, supporting thenotion that these channels are highly conserved opioid recep-tor substrates and represent one of the most important tar-gets for opioid receptor modulation. Newer findings, whichwe highlight later in this review (see Opioid Receptor Regu-lation), suggest that although opioid receptors have potenteffects on ion channel modulation, they also have slower yetrobust effects on other signal transduction pathways.

    Molecular Mechanisms of OpioidTolerance

    To date, the molecular and cellular mechanisms mediatingthe development of tolerance to morphine remain a matter ofcontroversy. Traditionally, it was thought that the down-regulation of opioid receptors after chronic agonist exposureinduces tolerance, as reported in in vitro studies.24,25 How-ever, recent in vivo studies show that down-regulation doesnot occur consistently with each and every agonist and maynot completely explain tolerance. In light of these findings, ithas been suggested that MOR proteins are in fact not down-regulated but instead may be desensitized and uncoupledfrom downstream signaling pathways.26 It has been observedthat after chronic morphine exposure, levels of the secondmessenger cAMP are increased. However, this elevation incAMP may not be attributable to opioid receptor uncouplingfrom inhibitory G proteins but instead could reflect cellularadaptive changes, including the up-regulation of adenylylcyclase, protein kinase A, and cAMP response element-bind-ing protein.27 It is this ineffective regulation of cAMP bymorphine that some believe induces tolerance.

    It has also been proposed that the regulation of opioid recep-tors by endocytosis reduces the development of tolerance andtherefore serves a protective role.28,29 After endocytosis, the cel-lular response is desensitized to the � agonist, but the receptorscan be recycled to the cell surface in an active state, resensitizingthe receptor to the agonist. Morphine-activated opioid receptorssignal for long periods of time, thereby enhancing the produc-tion of cAMP, which is thought to result in tolerance. In vivostudies have shown that facilitation of MOR endocytosis inresponse to morphine prevents the development of morphinetolerance.28 In addition, it has been shown in vivo that the lackof �-arrestin 2 prevents the desensitization of MOR afterchronic morphine treatment, and these mice also failed to de-velop antinociceptive tolerance.30

    Recent studies have identified how ligand-directed re-sponses, more commonly known as biased agonism, are cru-cial in understanding the complexity of opioid-induced tol-erance. The work of Bohn and colleagues showed how�-arrestin 1 and �-arrestin 2 differentially mediate the regu-lation of MOR. �-arrestins are required for internalization,but only �-arrestin 2 can rescue morphine-induced MORinternalization, whereas both �-arrestin 1 and �-arrestin 2can rescue [d-Ala2, N-Me-Phe4, Gly-ol5]enkephalin(DAMGO)-induced MOR internalization.31 These findingssuggest that MOR regulation is dependent on the agonistand may be critical in understanding the mechanism in-volved in the development of tolerance. Melief et al. furthershowed how acute analgesic tolerance to morphine is blockedby c-jun N-terminal kinase (JNK) inhibition but not G pro-tein-receptor kinase 3 (GRK-3) knockout. In contrast, usinga second class of � agonists (fentanyl, methadone, and oxy-

    Fig. 2. Summary of opioid receptor signaling. Figure depicts opioid receptor signal transduction and trafficking. In general, allfour opioid receptor subtypes (mu [�], delta [�], kappa [�], and opioid receptor like-1 [ORL1]) share these common pathways.New research indicates that selective ligands at each opioid receptor can direct opioid receptors to favor one or more of thesesignaling events (biased agonism or ligand-directed signaling). Arrows refer to activation steps; T lines refer to blockade orinhibition of function. �� � G protein �-� subunit; cAMP � cyclic adenosine monophosphate; ERK � extracellular signal-regulated kinase; JNK � c-jun N-terminal kinase; MAPK � mitogen-activated protein kinases; P � phosphorylation.

    Opioid Signaling

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  • codone), acute analgesic tolerance was blocked in GRK-3knockout but not JNK inhibition.32 Ligand-biased re-sponses are well documented in vitro but less so in vivo;however, a recent study addressed biased agonism at � opioidreceptors (DOR) in vivo showing that DOR agonists withsimilar binding and analgesic properties but different inter-nalization potencies lead to the development of differentialtolerance at DOR.33 These findings highlight the importantimplications of ligand-selective responses in GPCR biol-ogy33 and indicate the need for additional work to examinethe role and consequences of biased signaling in behavioralmodels.

    Opioid Receptor RegulationAgonist-induced receptor phosphorylation is believed to beone of the many critical molecular components of opioidtolerance. This process is well established in the GPCR liter-ature and typically occurs after chronic agonist exposure orsustained release of endogenous opioid peptides. Sustainedopioid treatment produces tolerance to the acute effects ofthe drug and can potentially lead to physical and psycholog-ical dependence. As a result of this problem, opioid-receptortrafficking, desensitization, and phosphorylation have beenextensively examined (for a detailed review see Bailey andConnor34). Here we highlight the key findings in this area asthey connect potential signaling to tolerance mechanisms.

    � Opioid Receptors (MOR)One common thread between the opioid receptor subtypes isthe interesting observation that receptor trafficking and regula-tion vary depending upon the agonist. For example, morphine isunable to promote receptor internalization, in contrast toDAMGO, which causes robust internalization.32,35,36 It isthought that morphine tolerance, a major problem in theclinic, is perhaps mediated by these differences in receptorregulatory activity. Several groups are actively working todiscern the various mechanisms for the differences in ligand-dependent MOR regulation, but controversy remains, withsome groups hypothesizing that MOR internalization doesnot actually uncouple the receptor from signal transductionpathways but instead induces recycling of uncoupled recep-tors to the plasma membrane. Alternatively, the morphine-bound receptor, although not internalized, may still signal atthe cell membrane, and because signaling is never attenuated,the cellular machinery adapts to produce tolerance. A recentstudy has shown that morphine acts as a “collateral agonist”to promote receptor G-protein uncoupling (“jamming”) andJNK activation (see MAPK Signaling at Opioid Receptors),whereas fentanyl and DAMGO internalize and desensitizenormally.35 It is plausible that many processes work togetherto produce receptor regulation and opioid tolerance, andadditional study is warranted to continue to decipher thesediscrepancies.

    � Opioid receptors contain more than 15 serine, threo-nine, and tyrosine residues that are accessible to protein ki-

    nases, which phosphorylate the receptor. All three intracel-lular loops and the carboxyl terminal tail contain thesesites.37 32P incorporation experiments have been critical toour understanding of MOR receptor phosphorylation, andcombined with our knowledge of site-directed mutagenesis,we now have a clear understanding of the key residues in-volved in MOR phosphorylation. Rat MORs are phosphor-ylated at Ser375 in the carboxy terminus,38,39 and treatmentwith both morphine and DAMGO causes robust phosphor-ylation of this residue. However, some reports have suggestedthat morphine and DAMGO induce different degrees ofphosphorylation of Ser375,39 suggesting that Ser375 maynot be the only amino acid residue phosphorylated and re-sponsible for MOR regulation. The highly conserved GPCR“DRY motif” in the second cytoplasmic loop of the � opioidreceptor has been implicated in regulation of agonist efficacy.Phosphorylation of Tyr166 reduced the efficacy ofDAMGO-mediated G-protein activation.40 It has beenshown recently that agonist-selective differences in MORregulation are in fact determined not only by net incorpora-tion of phosphates into the receptor population as a wholebut also by individual receptors achieving a critical numberof phosphorylated residues (multiphosphorylation) in a spe-cific region of the C-tail.41 In addition, the same group iden-tified that multiphosphorylation specifically involves the375STANT379 motif required for the efficient endocytosis ofMOR. These ligand-mediated differences highlight the li-gand-dependent nature of opioid receptor function and re-quire additional further study in vivo.

    � Opioid Receptors (KOR)� Opioid receptor trafficking shares some common featureswith MOR regulation because KOR is readily phosphory-lated, desensitized, and internalized. KOR is phosphory-lated, desensitized, and internalized by the agonists U50,488and dynorphin 1–17 but not by other agonists, such as etor-phine or levorphanol.42,43 Both dynorphin A and B havebeen shown to initiate significant receptor internalization inhuman KORs and three structurally distinct KOR ligands:Salvinorin A (salvA), TRK820, and 3FLB were shown toinduce KOR internalization with varying rank orders ofpotency.44 There have been conflicting data regarding ag-onist-induced KOR internalization, which seem to be de-pendent on the cell line, receptor species, or model systemused. In Chinese hamster ovary cells expressing KOR, theselective KOR agonists U50,488 and U69,593 did notcause robust receptor internalization45; however, inmouse pituitary tumor (AtT20) cells and human embry-onic kidney (HEK293) cells, U50,488 initiated stronginternalization of KOR-green fluorescent protein (GFP)proteins.46 – 48 Despite this, several groups have foundconsistency in the ability of KOR to become phosphory-lated, internalized, and desensitized by its endogenousopioid peptide dynorphin.

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  • � Opioid Receptors (DOR)In contrast to MOR and KOR, DOR were thought to existprimarily (more than 90%) at intracellular sites49–51 untilrecently, when mice expressing fluorescently tagged DORrevealed that there was strong membrane localization ofDORs in vivo.52 The reasons for this discrepancy betweenthe numerous studies showing intracellular DOR labelingand membrane labeling remain unclear and continue to bematters of controversy. It is plausible that previous studiesusing DOR antibodies were flawed because of antibody spec-ificity issues, despite the numerous controls conducted. Al-though DORs tagged with GFP are a powerful in vivo tool,they require careful interpretation given that GFP is a largeprotein that may interfere with the typical DOR traffickingmachinery. Additional investigation is required in both cases,and it is plausible that both concepts are indeed true; theconcentrations of DOR expressed on the cell surface maywell be higher than originally hypothesized, yet a large intra-cellular pool of DOR protein remains. Nevertheless, DORseems to be a dynamic opioid receptor that can readily trafficin response to agonists. Some reports have shown thatchronic morphine treatment promotes movement of DORsto the cell surface in the dorsal horn of the rat spinal cord.49

    This effect was dependent on MOR receptor activity becauseblocking or deleting MOR genetically (MOR knockout)prevents the effect.

    Like MOR and KOR, desensitization of DOR is con-trolled via phosphorylation, after recruitment of arrestinsand sequestration of arrestin-bound receptors.53,54 Phos-phorylation of DOR has been shown with both small-mole-cule organic ligands and peptide treatments. Once again,c-terminal phosphorylation was shown to be critical for opi-oid receptor regulation. In DOR, the Ser363 residue is thekey phosphorylation site.55,56 This phosphorylation eventwas shown to be mediated by GPCR kinase 2 (GRK-2).56,57

    Other studies have demonstrated that other amino acid res-idues are involved in DOR regulation. For example, Thr353was found to be important for [D-Ala2, D-Leu5]-Enkepha-lin (DADLE)-mediated down-regulation of DOR, andLeu245 and 246 act as lysosomal targeting motifs that par-take in determining agonist-bound DOR localization.58,59

    Furthermore, ligand-specific variability in agonist-depen-dent DOR phosphorylation has been observed with poten-tial differences between SCN80- and [D-Pen2,5]Enkephalin(DPDPE)-bound conformations recruiting kinases with var-ious efficacies and potencies.60

    Opioid Receptor Like-1 (ORL1)Opioid receptor like-1 (also called nociceptin or orphaninFQ) receptors are the newest members of the opioid receptorfamily, and few groups have examined their regulatory prop-erties. Agonist-induced internalization of ORL1 is rapid andconcentration- dependent.61 Both the endogenous agonist no-ciceptin and small-molecule selective ORL1 agonist Ro646198promote rapid internalization of ORL1. Agonist challenge also

    reduces the ability of ORL1 to couple to inhibition of forskolin-stimulated cAMP production, suggesting that ORL1 undergoesdesensitization mechanisms similar to those of the other threeopioid receptor subtypes. ORL1 internalization appears to bemore rapid than that of the other opioid receptors, with somegroups reporting internalization after only 2 min of agonist ex-posure in Chinese hamster ovary cells.61 However, this appearsto be dependent on ligand type and cell line expression becauseORL1 internalization in human neuroblastoma cells was slowerand occurred closer to a 30-min time point.62 ORL1 receptorsrecently were demonstrated to cointernalize with N-typeCav2.2 channels after a 30-min agonist treatment.63 The inter-nalization of the entire signaling complex is not unusual inGPCRs; however, the effect in the case of ORL1 is particularlypronounced and is believed to play a major role in how ORL1selectively removes N-type calcium channels from the plasmamembrane to inhibit calcium influx.

    Opioid receptor like-1 receptor regulation is increasinglystudied, but our understanding remains in the infant stagescompared with that of the other three opioid receptor sub-types. To date, few site-directed mutagenesis studies havebeen conducted, and receptor regulation in primary neurons,dorsal root ganglion, or dorsal horn neurons remains un-known. As we move forward in understanding opioid recep-tor signaling and identify novel opioid receptor targets,ORL1 receptors become likely candidates for the future ofopioid pharmacology.

    Opioid Receptors and Arrestin RecruitmentPhosphorylation by GRK-2 or -3 of �, �, and � opioidreceptors leads to arrestin 2 or 3 recruitment. Arrestin mol-ecules are key proteins that bind phosphorylated GPCRs toregulate their desensitization, sequestration, and sorting andultimately assist in determining receptor fate. Opioid recep-tors are regulated by arrestin 2 and arrestin 3 binding (alsocalled �-arrestin 1 and �-arrestin 2, respectively), and thisinteraction depends on the model system and agonist treat-ment procedure. Mice lacking arrestin 3 have been shown tohave a reduced tolerance to � opioids such as morphine,suggesting that MOR regulation requires arrestin 3.30,64

    With the use of surface plasmon resonance methods, glu-tathione s-transferase pull-down assays, and classic immuno-precipitation methods, the C-terminal tails of DOR, MOR,KOR have been shown to be crucial for arrestin 2 or 3 bind-ing. C-terminal carboxyl mutant opioid receptors have beenstudied widely, and these serine mutant receptors show de-creased agonist-induced receptor internalization and arrestinrecruitment. Dominant positive arrestins (such as Arrestin-2-R169E or Arrestin-3-R170E) that bind the nonphospho-rylated receptors can rescue internalization of serine mutatedMOR/DOR/KOR,48,65 further implicating arrestin depen-dence in opioid-receptor trafficking. Most studies implicat-ing arrestin have been conducted in heterologous expressionsystems using overexpressed arrestins and opioid receptorsubtypes. These conditions are atypical and do not represent

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  • the likely physiologic state of opioid receptors and arrestinsin vivo, so these data should be interpreted with caution, andadditional studies using in vivo approaches are needed toincrease our understanding of arrestin–opioid interactions.

    MAPK Signaling at Opioid ReceptorsIn the discussion above, we highlighted that sustained ago-nist treatment causes GRK phosphorylation at the carboxyl-terminal domain of opioid receptors activating arrestin-dependent receptor desensitization and internalization (fig.2). During the last several years, GPCR research has discov-ered that the phosphorylated arrestin-bound GPCR complexis not simply inactive, but that it recruits alternate signaltransduction cascades, including MAPKs.66 The merging ofour previous knowledge regarding opioid receptor phosphor-ylation, arrestin, and cellular mechanisms of tolerance withan understanding of opioid receptor signaling to MAPKs isbecoming more appreciated (table 2).

    Mitogen-activated protein kinase pathways are diversesignaling cassettes that govern cellular responses, includingcell proliferation, differentiation, apoptosis, transcriptionfactor regulation, channel phosphorylation, and protein scaf-folding.93 The MAPK family is composed of 12–15 geneproducts with the most well-described forms including ex-tracellular signal-regulated kinases 1 and 2 (ERK 1 and 2),JNK1–3, and p38 (�, �, �, �) stress kinase. The MAPKs aredistinct in that they have the capacity to respond to a varietyof stimuli and transmit a diverse array of intra- and extracel-lular signals.94 MAPK signaling is regulated by the kinetics ofactivation, nearby phosphatase activity, and the cellular do-main the MAPKs occupy.93 Initially, ERK MAPKs wereshown to require receptor tyrosine kinase transactivation,through epidermal growth factor or brain derived neu-rotrophic factor (also called TrkB receptors).95 Later reportsdirectly linked GPCRs to activation of MAPK signalingpathways, and now most, if not all, GPCRs have been foundto couple to this pathway.ERK 1 and 2 Signaling at Opioid Receptors. The mostfrequently examined opioid-induced MAPK cascade is ERK1 and 2. Coscia and colleagues have been crucial in develop-ing our understanding of the relationship between opioidreceptors and ERK 1 and 2 signaling. In one of the initialstudies, MOR and KOR stimulation was demonstrated toinitiate ERK 1 and 2 phosphorylation in astrocyte culturesand transfected cell lines.96 The kinetics of ERK 1 and 2phosphorylation by MOR and KOR systems vary, yet bothreceptors can activate ERK 1 and 2 within 5–10 min. MOR-mediated ERK 1 and 2 phosphorylation requires proteinkinase C (PKC�) activity, and MOR-dependent ERK 1 and2 signaling requires GRK-3 and arrestin in primary neurons,glial cells, and heterologous expression systems.67,68,97 Thedownstream substrates of MOR-mediated ERK 1 and 2 havebeen defined in some cases and remain unknown in others.In embryonic stem cells, MOR-dependent ERK 1 and 2signaling positively modulates and directs neural progenitor

    cell fate decisions.98,99 However, in astrocytes chronicmorphine can negatively regulate ERK 1 and 2 signaling bytyrosine kinase pathways to ultimately inhibit neuriteoutgrowth and synapse formation.69 Most studies useMAPK–ERK (MEK) inhibitors (the proximal upstream ki-nase) to determine substrates of ERK 1 and 2 signaling inGPCRs; however, few reports have shown direct interactionbetween �-opioid–induced ERK and a final substrate. (Thein vivo implications of MOR-dependent ERK signaling areexplored in Opioid Signaling and Behavior.) Several groupsare investigating the potential for ligand-specific ERK ago-nists at opioid receptors.

    � Opioid receptors have also been shown to activateERK 1 and 2 through G�� and Ras signaling cascades96

    and do not necessarily require receptor internalization orreceptor phosphorylation for signaling.100,101 DOR-me-diated ERK signaling recently was found to require integ-rin signal transduction through transactivation of epider-mal growth factor receptor pathways. DOR-mediatedepidermal growth factor receptor activation also initiatedphospholipase C signaling to stimulate ERK 1 and 2 phos-phorylation.83 DOR-dependent ERK 1 and 2 signalingrequires additional investigation because, coupled withDOR’s critical role in pain and mood regulation, ERKsignaling through DOR may reveal a novel mechanism forDOR regulation of neural activity.

    KOR-dependent ERK 1 and 2 phosphorylation occurs ina multiphase manner, with an early period of activity be-tween 5–15 min after agonist exposure and a late phase after2 h of agonist treatment. Similar to other GPCRs,102 thebiphasic ERK 1 and 2 activation for KOR contains an arres-tin-dependent late phase73 and an arrestin-independent earlyphase. This group identified G�� as a crucial mediator in theearly-phase ERK 1 and 2 activation by KOR, and showedthat arrestin 3 is required for late-phase ERK 1 and 2. KORsactivate ERK 1 and 2 through PI3-kinase, PKC�, and intra-cellular calcium.72 However, like MOR and DOR, the sub-strate for KOR-mediated ERK 1 and 2 has not been identi-fied, although a recent study suggests that KOR-inducedERK 1 and 2 also directs stem cell fate toward neural pro-genitor development. ORL1 receptor-dependent ERK 1 and2 activation has not been extensively examined, although onegroup has shown that ORL1 receptor activation does initiateERK 1 and 2 phosphorylation.89 The signaling pathways forORL1-mediated ERK 1 and 2 phosphorylation in neuronalcell types and in vivo need additional investigation.JNK. The JNK pathway is activated by environmental trig-gers, including stress, inflammation, cytokine activation, andneuropathic pain.103,104 Classically, JNK activity can resultin transactivation of c-jun, a component of the activatorprotein 1 transcription factor complex, and JNK phosphor-ylation is caused by cytokines, including tumor necrosis fac-tor and interleukin-1�.105 JNK activation has also been im-plicated in numerous other signaling cascades. JNK typicallyis activated by Ras-related GTP binding proteins in the

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  • family.106 JNK activation by GPCRs and opioid receptorshas not been examined thoroughly but has been demon-strated for all the opioid receptor subtypes. Like ERK 1 and2, arrestin 2 and arrestin 3 have been reported to scaffoldJNK signaling complexes, and it is believed that arrestin 3 hasJNK 3 specificity, although this remains a matter of contro-versy.107 The cellular mechanisms of arrestin-dependentJNK at GPCRs remain unresolved.

    Opioid-dependent JNK has been demonstrated by only afew groups. DOR causes protein kinase B (Akt)-dependentJNK phosphorylation through a PI3-kinase mechanism,88

    and JNK activity is PI3-kinase independent in others.108

    PI3-kinase is required for �-opioid–dependent JNK activa-tion. In contrast, U50,488-induced (KOR) JNK activationhas been shown to be independent of PI3-kinase.108 Thesubstrates and in vivo effects of opioid-induced JNK activa-

    Table 2. A Summary of Current Opioid Receptor-dependent Signaling

    Receptor Cascade/Signaling Pathway Model Reference

    � Opioid 1ERK 1 and 2 (GRK-3 and arrestin dependent) In vivo (murine) Macey et al. 200667

    1ERK 1 and 2 (arrestin dependent) Astrocyctes Miyatake et al. 200968

    2ERK 1 and 2 (chronic activation) Astrocyctes Ikeda et al. 201069

    1JNK 2 (PKC dependent) In vivo and HEK293 Melief et al. 201035

    Tan et al. 200970

    1Stat3 phosphorylation In vivo (murine) and CMT-93 Goldsmith et al.201171

    � Opioid 1ERK1 and 2 Astrocytes Belcheva et al. 200572

    In vivo Bruchas et al. 200648

    McLennan et al.200873

    Bruchas et al. 200874

    Potter et al. 201175

    1p38 MAPK (dependent on GRK-3 andarrestin)

    Striatal neurons Bruchas et al. 200648

    Astrocytes Bruchas et al. 200776

    In vivo Xu et al. 200777

    Bruchas et al. 201178

    1JNK 1 In vivo Melief et al. 201035

    Melief et al. 201132

    1JAK2/STAT3 and IRF2 signaling cascade PBMC Finley et al. 201179

    � Opioid 1ERK 1 and 2 HEK293 Eisinger et al. 200980

    Eisinger et al. 200481

    Audet et al. 200582

    1ERK 1 and 2 (integrin stimulated, EGFRmediated)

    HEK293 Eisinger et al. 200883

    1ERK 1 and 2 (integrin stimulated, Trk1mediated)

    NG108-15 Eisinger et al. 200883

    1 PI3K/AKT/2GSK-3� DOR-transfected CHO cells Olianas et al. 201184

    Rat NAcNG108-15NG108-15 Heis et al. 200985

    1PI3K/2GSK-3� (SRC and AMPK dependent) DOR-transfected CHO cells Olianas et al. 201186

    1PI3K (SRC and IGF-1 dependent) DOR-transfected CHO cells Olianas et al. 201187

    1JNK (AKT dependent Pi3K mediated) T cells Shahabi et al. 200688

    ORL1 1ERK 1 and 2 Neuro-2a cells Harrison et al. 201024

    Rats NAc Chen et al. 200889

    In vivo (porcine) Ross et al. 200590

    1p38 MAPK NG108-15 Zhang et al. 199991

    1JNK COS7 and NG108-15 Chan et al. 200092

    In vivo Ross et al. 200590

    1 � activation; 2 � deactivation; AKT � serine threonine protein kinase; AMPK � 5� adenosine monophosphate-activated proteinkinase; CHO � Chinese hamster ovary cells; CMT-93 � mouse rectum carcinoma cells; COS7 � monkey kidney fibroblast cell line;DOR � � opioid receptors; EGFR � epidermal growth factor receptor; ERK1 and 2 � extracellular signal-regulated kinases 1 and 2;GRK-3 � G protein-receptor kinase 3; GSK � glycogen synthase kinase 3; HEK293 � human embryonic kidney cells; IGF-1 �insulin-like growth factor 1; IRF2 � interferon regulatory factor 2; JAK2 � Janus kinase 2; JNK 1 and 2 � c-jun N-terminal kinase;MAPK � p38 mitogen-activated protein kinase; NG108-15 � neuroblastoma glioma hybrid cell line; ORL1 � opioid receptor like-1; p38STAT3 � signal transducer and activator transcription 3; PBMC � peripheral blood mononuclear cell; PI3K � phosphoinositide3-kinase; SRC � proto-oncogene tyrosine-protein kinase.

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  • tion are being studied by several groups. KOR (U50,488,dynorphin) agonists activate JNK in a pertussis toxin-sensi-tive (G�i) manner.35,108,109 U50,488-mediated JNK re-quires focal adhesion kinases and the GTPase Rac in immunecell types. MOR-induced JNK activation recently was shownto require PKC activity.35

    In two recent studies, KOR- and MOR-induced JNKphosphorylation by norbinaltorphimine and morphine wereshown to act as “collateral agonists” to cause JNK phosphor-ylation and initiate uncoupling of the G protein to blockG�i-mediated transduction.35,109 The persistent actions ofnorbinaltorphimine on KOR-agonist–mediated analgesia(21 days) were shown to require JNK because JNK 1 isoformknockout mice show an absence of norbinaltorphimine-de-pendent 21-day KOR blockade, and selective JNK inhibitorsprevented the long-lasting norbinaltorphimine effect. It wasalso recently identified that the long duration of action ofsmall molecule KOR antagonists in vivo is determined bytheir efficacy in activating JNK 1. The persistent KOR inac-tivation by these small-molecule collateral agonists did notrequire sustained JNK phosphorylation,32 implicating inter-mediate protein(s) or alternate JNK substrates in this pro-cess. In contrast, acute morphine tolerance was shown torequire JNK 2 because JNK 2 knockout mice showed anabsence of MOR inactivation. How ligand-dependent JNKactivation causes receptor uncoupling from G�i signalingremains unresolved, and proteomic biochemical approacheswill need to be used to identify ligand-dependent proteininteractions. Together, this work highlights the remarkablenature of opioid receptor sensitivity to a variety of ligand-stabilizing conformations.p38 MAPK. The p38 MAPK pathway also plays a key role inenvironmental stress and inflammation and is activated bycytokine production.110 In glial cells particularly, p38MAPK activity is required for an array of cellular responses,including interleukin-6 and interleukin-1� production, in-hibitory nitric oxide synthase activity, and tumor necrosisfactor � secretion. Activation of p38 MAPK is involved inproliferative and chemotactic responses in some systems andhas been shown to play a major role in neuropathic painresponses.77,111

    Opioid receptor-mediated p38 phosphorylation has beenmost widely demonstrated for the MOR and KOR systems.KOR-induced p38 MAPK has been observed in heterolo-gous expression systems, striatal neurons, astrocytes, and invivo.2,3,8,77,76,99 KOR-mediated p38 MAPK activation re-quires serine 369 phosphorylation by GRK-3 and arrestin 3recruitment.48,76 � Opioid receptor internalization recentlyhas been shown to require Rab5 signaling and p38 MAPK.This process seems to be ligand dependent because morphinewill not cause p38-dependent receptor internalization, butDAMGO will readily cause internalization.70 In addition, �opioid receptor cross regulation of �2A-adrenergic receptorshas been shown to require p38 MAPK, and p38 MAPKinhibition blocks DAMGO-induced MOR internaliza-

    tion.70 This cross activity between MOR and �2A-adrenergicreceptors requires arrestin 3, suggesting that arrestin 3 scaf-folding of p38 is likely to be conserved across opioid recep-tors. To date, few studies have identified a role for DOR orORL1 in mediating p38 phosphorylation. In one report,both ORL1 and DOR were shown to cause p38 phosphor-ylation through activation of protein kinase A and proteinkinase C.91 Opioid-induced p38 has several potential targets,including modulation of ion channels and transcription fac-tors. Recently, the potassium channel Kir3.1 was demon-strated to become tyrosine phosphorylated via KOR-depen-dent p38 MAPK Src activation.112 How p38 specificallyinteracts with various substrates will be an interesting nextstep and will reveal how such a ubiquitously expressed kinasecan selectively modulate the large variety of cellular events.

    Protein–Protein Networks and Opioid ReceptorsIn addition to intracellular signaling and receptor modifica-tion by phosphorylation, newer biochemical studies stronglysuggest that opioid receptors interact with one another, al-ternate GPCRs, and a whole host of anchoring and mem-brane protein sets. These interactions are becoming increas-ingly appreciated as critical to the ultimate functional role ofthe opioid receptor families. In many ways, the field of pro-tein–protein interactions is at the forefront of opioid recep-tor molecular pharmacology, as research moves from previ-ous work in heterologous expression systems to in vivoapproaches.Opioid Dimerization. Numerous reports have demonstratedthat GPCRs exist as dynamic protein complexes with largeinteractions between proteins and other receptor types. Sev-eral studies have shown that GPCRs can form dimers andoligomers. This oligomerization includes two varieties: ho-modimers (same receptor) and heterodimers (different re-ceptor type) (fig. 3). The existence of these GPCR homomersand heteromers has been shown in transfected cell line sys-tems, cell lines, and primary cultures and in some cases invivo (for review see Rios et al.113 and Prinster et al.114). De-spite that GPCR oligomerization remains a matter of con-troversy, it continues to generate interest because opioid re-ceptor dimers may reveal novel targets for the developmentof new opioid drugs.

    Devi and colleagues pioneered research into opioiddimerization and originally identified opioid receptor het-erodimers.115 They found that � receptors can exist as ho-modimers, and agonist stimulation causes their dissocia-tion.115,116 In this seminal work, the authors also found thatKOR and DOR form heterodimeric complexes, which ap-pear to alter the trafficking properties of these receptors.They showed how agonist-induced internalization of DORreceptors is reduced substantially in cells expressing DOR/KOR receptors.115 Moreover, it was shown that 6�-guanidi-nonaltrindole, which selectively targets the KOR or DORheterodimer, generates a unique signaling entity, giving ad-ditional evidence for the existence of opioid heterodimers.117

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  • It has been shown that MOR can heterodimerize withORL1,118 but the existence of MOR or KOR heterodimersremains a matter of controversy.115,119 The observation thatthe antagonism or absence of DOR diminishes the develop-ment of morphine tolerance and dependence suggests theremay be an interaction between the two receptors, althoughfuture biochemical work in vivo is needed to validate theseconcepts. Studies not only identified the existence of MORand DOR heterodimers but also revealed that MOR andDOR heterodimers have distinct ligand binding and signaltransduction properties,120 suggesting that heterodimeriza-tion may represent an alternative mechanism for the cell totune and control second messenger activity.

    It was hypothesized that the mechanisms and/or proteinsthat modulate the level of MOR or DOR complexes arecritical in the development of tolerance121; this theory in-spired research into the events that lead to dimerization. Deviand colleagues recently identified additional signaling pro-teins, such as RTP4, that partake in opioid receptor oligomertrafficking from the Golgi to distribute opioid receptor com-plexes at the cell membrane.121 In addition, it was found thatMOR activation promotes the formation of complexes be-tween RGS9–2 and G� subunits. It was shown that phar-macological manipulations were able to disrupt RGS9–2complexes formed after repeated morphine administra-tion.122 These data provide a better understanding of phar-

    macologic approaches that can be used to improve chronicanalgesic responses and tolerance.

    Some studies have shown that opioid receptors can het-erodimerize with other classes of GPCR. For example, MORcan interact and potentially heterodimerize with cannabi-noid receptor 1 (CB1) (fig. 3).123,124 Interactions betweenMOR and cannabinoid receptor 1 receptors appear to mod-ulate their effects, as evidenced by the administration of � 9-tetrahydrocannabinol, a cannabinoid receptor 1 agonist,which can enhance the potency of opioids such as mor-phine.125 In addition, the concomitant activation of MORor cannabinoid receptor 1 heterodimers leads to significantattenuation of ERK activity compared with the response af-ter the activation of each individual receptor.123

    A number of previous studies have noted interesting func-tional interactions between the MOR and �2A-adrenergicreceptor systems.126–128 These studies reported that the pres-ence of �2A receptors is sufficient to potentiate the phosphor-ylation of MAP kinases in response to morphine, whereas thecombination of ligands abolishes this effect. The interactionsbetween MOR and �2A receptors provide an alternate mech-anism for the control of receptor function and could haveprofound effects in the development of opioid-adrenergicanalgesics. Neurokinin 1 and MOR have also been shown toheterodimerize. The interaction between these two receptortypes does not alter ligand binding or signal transduction butdoes change internalization and resensitization.129 In addi-tion, substance P (neurokinin 1 selective ligand) caused crossphosphorylation and cointernalization of MOR.130 As neu-rokinin 1 and MOR coexist in the trigeminal dorsal horn, ithas been suggested that they may interact functionally withina signaling complex in these neurons during nociceptive neu-rotransmission.130 The functional consequences of opioidreceptor oligomerization in vivo are largely unknown, unex-plored, and matters of controversy. New technological ad-vances in mouse genetics and imaging are crucial in resolvingthese issues. One major area of continued interest is the invivo demonstration of opioid receptor homo- and het-erodimerization, as well as the development of additionalbiochemical tools to demonstrate unequivocally that thesereceptor proteins directly interact with one another.

    In addition to receptor–receptor interactions, it is increas-ingly clear that opioid receptors are highly complex systemsand that they interact with a whole host of extracellular,intracellular, and membrane proteins. The notion of opioidreceptors existing as dynamic signaling complexes sits at theforefront of the future of opioid-based therapeutics. The rea-sons for this include the notion that different opioid receptorligands can induce the formation of a diverse array of recep-tor complexes. In addition, it is increasingly appreciated thatthe opioid receptor’s native environment (i.e., cell type, neu-ral circuit) greatly affects the receptor’s ability to signal, traf-fic, and function. The idea of a binary GPCR as a simpleswitch mechanism, from off to on, is becoming widely dis-

    Fig. 3. Opioid dimerization. Figure depicts opioid receptorhomodimers and opioid receptor heterodimers (A); het-erodimers between opioid receptors and other G-protein–coupled receptors (B); protein–protein interactions involvedin opioid receptor signal transduction (C). �� � G protein �-�subunit; � � � opioid receptor; � � � opioid receptor; CB1 �cannabinoid receptor type 1; GPCR � G protein coupledreceptors; � � � opioid receptor; ORL-1 � opioid receptorlike-1; arrow � movement; T lines � blockade.

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  • regarded as new protein–protein interaction networks andligand-dependent properties are uncovered.32,33,35,76,95,131

    Other Protein–Protein Interactions. There are multiplelines of evidence pointing to arrestin molecules as crucialproteins that network and engage opioid receptor signaltransduction and orchestrate the interaction of proteinswithin the cellular milieu. The isolation of other opioid-selective protein-interaction networks has been slow, al-though more studies are examining the many important rolesin receptor fate. For one, MOR has been shown to interactwith numerous cytoskeletal trafficking proteins, most ofwhich participate in membrane protein endocytosis, includ-ing GASP-1, spinophilin, glycoprotein M6A, and tama-lin.132–134 MOR also has been shown to interact with cal-modulin, which is a highly sensitive Ca2� binding proteinimplicated in cytoplasmic enzyme activity, including adeny-lyl cyclases and CAM kinases.135 DORs are similar becausethey also use GASP-1 and glycoprotein M6A for regulatingsurface trafficking and endocytosis. KORs have been shownto interact with GEC-1 and EBP50-NHERF proteins, po-tentially acting to enhance receptor recovery and recyclingrates.136,137 Given that ORL1 has not been extensively stud-ied, most of our knowledge about its signaling complex cen-ters around the work of the Zamponi group demonstratingORL1-Cav2.2 complex formation.63,138 The increasingspecificity and affordability of proteomic technologies, suchas tandem affinity purification (TAP tag) approaches,139 willhelp to advance our understanding of opioid receptor com-plexes. Validating protein–protein interactions in vivo con-tinues to be a challenge, but it is expected that with newermouse genetic tools, proteomic dissection of opioid receptorcomplexes in vivo will become an easier task.

    Opioid Signaling and Behavior� Opioid Receptors. The most common behavioral func-tion linked to opioid receptors has been their ability to me-diate analgesic effects. Numerous reports have examined howopioid signaling causes opioid-induced analgesia (see Bod-nar140 and Walwyn et al.3). It is generally accepted thatMOR signaling to pertussis toxin-sensitive G�i is requiredfor morphine antinociception. In addition, in vitro blockadeof arrestin 3 expression improves morphine-mediated anal-gesic responses and acts to prevent morphine tolerance overtime.141 Spinal cord expression of G�� is required for MORcoupling to analgesic responses and is thought to play a keyrole in how MORs mediate antinociception.142 This is likelyto be through the modulation of potassium and calciumchannels in the dorsal root ganglion and dorsal horn. Mor-phine-induced analgesia and tolerance have been linked tonumerous signaling pathways, including interaction with ad-enylyl cyclases AC1, AC8, and AC5.143,144

    � Opioid receptor-dependent behavioral studies linkedto MAPK signaling have begun to become more common inthe literature. ERK 1 and 2 phosphorylation has beenshown to be up-regulated by chronic morphine treatment

    and in opioid withdrawal145; consistent with this idea,MOR-induced ERK 1 and 2 activity in the periaqueductalgray region acts as a mechanism to counteract morphinetolerance.146 Recently, reports have implicated ERK 1 and 2signal transduction in morphine reward and plasticity, in-cluding place preference and psychomotor sensitiza-tion.147,148 ERK 1 and 2 activity in the amygdala was foundto mediate anxiety-like behaviors during morphine with-drawal.149 Together, these reports strongly support the con-cept that ERK 1 and 2 signaling is an essential mediator of �opioid-induced plasticity in the brain and spinal cord.

    � Opioid receptors signaling via other protein kinasesand protein–protein interactions to modulate reward andanalgesia, such as PKC or protein kinase A and JNK, hasbeen demonstrated. For example, PKC 1 (also calledRACK1) is required for morphine reward in mouse models,and activation of IRS2-Akt signaling in dopaminergic ven-tral tegmental neurons is required for the behavioral andcellular actions of � opioids, including morphine.150 Thissame group has demonstrated that morphine action on rewardrequires the activation of transcription factors, includingpCREB and DeltaFosB.151 However, we still lack direct infor-mation regarding the substrates for these MOR-dependenttranscription factors and kinase-signaling pathways shown to berequired in behaviors such as analgesia and reward.� Opioid Receptors. Like MOR, DOR signaling researchhas been focused primarily on mechanisms of opioid analge-sia. In addition, DOR research in vivo has been more com-monly centered around DOR localization and anatomicalcharacterization, with far fewer studies linking DOR signal-ing with behavioral effects. Ligand-dependent DORsignaling has been an active area of research, with reportssuggesting that ligand-mediated trafficking governs agonist-induced analgesic tolerance to � opioids.33,152 These studiesdemonstrated that the DOR agonists SNC80 and ARM390differ in their ability to cause receptor internalization anddown-regulation of DOR-mediated Ca�2 channel modula-tion. DOR antinociception to thermal stimuli requires phos-pholipase C, and PKC activation also determines DOR-�2Asynergistic effects in the spinal cord.153 DOR agonists havebeen studied increasingly for their potential antidepressantand anxiolytic effects in rodent behavioral models.154 How-ever, it is not yet known how or where DOR agonists medi-ate antidepressant-like behavioral responses.� Opioid Receptors. Contemporary studies linking � opioidreceptor signaling and behavior have been centered aroundthe role of � opioids in stress (Bruchas and Chavkin155 andKnoll and Carlezon156). Stress-induced opioid peptide re-lease has been reported for all of the major opioid systems,and this release causes stress-induced analgesia via action atopioid receptors. In a few crucial reports, it was demon-strated that KOR activation after stress cannot only increaseanalgesic responses but also can modulate numerous behav-iors, including reward and depression.73,157,158

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  • � Opioid receptor activation of analgesic responses isthought to require G�� signal transduction,159 whereasKOR-induced potentiation of reward and dysphoria arethought to be mediated by more complex events, includingbut not limited to MAPK activation.76 Chartoff et al. showedthat the KOR agonist salvA has a biphasic effect on reward.The acute administration of salvA decreased the rewardingimpact of intracranial self-stimulation; however, repeatedKOR activation caused a net decrease in the reward-poten-tiating effects of cocaine.75 Both acute and repeated salvAadministration increased phosphorylated ERK, but onlyacute salvA increased c-fos, and only repeated salvA increasedcAMP response element-binding protein.75 These findingsprovide more information about the effects of KOR activa-tion on the reward-related effects of cocaine and will assist inthe dissection of the relationship between activation ofKOR- and ERK-signaling pathways. KOR-mediated p38MAPK activity has been shown to be required for condi-tioned place aversion and swim-stress immobility responses,whereas cAMP response element signaling is critical for pro-dynorphin gene induction and depression-like behavioral re-sponses.70,160 It is thought that KOR modulation of dopa-mine, serotonin, and noradrenergic systems plays a key rolein producing the negative behavioral affective re-sponses.155,156 Reports include KOR-mediated reductionson dopamine release, along with p38-dependent modulationof serotonergic output.111,159 It was shown recently thatKOR-induced p38� MAPK signaling in serotonergic cir-cuitry is required for stress-induced social avoidance, depres-sion-like behaviors, and reinstatement of drug-seeking be-havior. This report went on to show that KOR-inducedp38� MAPK causes a hyposerotonergic state through in-creased surface serotonin transporter expression.78 Themechanisms and neural circuits in KOR-mediated dysphoriaand analgesia are being studied by several groups; it is hopedthose studies will greatly assist in the development of poten-tial antidepressant ligands at KOR and novel analgesics thatbypass KOR-mediated dysphoria.161

    Opioids and GeneticsThe pathogenesis of addiction involves a series of complexinteractions among biological factors, including genetic vul-nerability and drug-induced alterations in gene expressionand proteins. Despite great efforts, the progress in findingcausal variants underlying drug addiction has been somewhatslow. Numerous case–control studies have investigated sin-gle nucleotide polymorphisms in opioid receptor genes andtheir correlation with addiction to opioids. However, thesestudies often have produced conflicting results. The mostextensively researched example is a polymorphism inOPRM1 (A118G, rs1799971), which results in the replace-ment of asparagine with aspartic acid at codon 40. Threestudies found an association with the variant 118G and opi-oid dependency,162–164 two studies observed an associationwith the common allele 118A and opioid dependency,165,166

    and nine studies found no overall association with opioiddependency.167–175 This polymorphism highlights the con-flicting results obtained from genetic studies of opioid recep-tor genes and drug dependence.

    We have summarized the genetic variants that may con-tribute to vulnerability to develop opioid addiction (table 3).Genetic testing has important clinical applications in theprevention of many diseases, but in the field of addiction,much work remains to be done to understand the associa-tions between these genetic variants and addiction-relatedphenotypes.

    Within the last decade it was learned that the gene encod-ing the MOR undergoes extensive alternative splicing, result-ing in the generation of multiple versions of this receptorprotein. However, correlating these splice variants to phar-macologically defined receptors has proven difficult. The rel-ative contribution to the pharmacologic effect of each splicevariant could vary from drug to drug and is dependent oneach splice variant’s potency and efficacy at a particular site.It has been suggested that the difference in the activationefficacies of various � opioids for the receptor splice variantsmay help explain the subtle but clear differences among var-ious � opioids in the clinic. In addition, understanding thefunctional significance of some of the truncated receptorsplice variants will be beneficial because they have been re-ported to modulate the activity of opioid receptors in othersystems (see Pasternak177,191).

    ConclusionsIn this review, we discuss a wide array of molecular, cellular,and in vivo studies in opioid receptor pharmacology. Wehighlight the traditional G protein, �� signaling pathways,and regulatory mechanisms and discuss recent advances inthe subfields of biochemistry, MAPK signal transduction,genetics, and behavior. It is important to note that we havenot attempted to discuss all of the fine details regarding theproperties of each receptor system.

    The most common thread in the reports reviewed is thata large body of our understanding of opioid receptor molec-ular pharmacology continues to stem from in vitro studies. Itis also increasingly clear that most molecular and cellularfeatures of opioid receptors remain disjointed and uncon-nected to any physiological or behavioral effects, which needsto be the focus of future work in this field.

    Many of the most important observations and discoveriessurrounding opioid receptors have relied on in vitro ap-proaches, and they continue to be the starting point for mostlaboratories in molecular pharmacology. However, given thediverse functionality of the opioid receptor family and thevariety of signaling pathways and interacting proteins, ourknowledge of how opioid receptors function in animal mod-els and, more importantly, human populations or disease islimited.

    Opioid receptor signaling has been a primary focus ofresearchers in this field since its discovery. The major reason

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  • Table 3. The Association of Polymorphisms in Opioid Receptor Genes with Opioid Addiction and FunctionalDifferences between These Variants

    Receptor(Gene) Polymorphism

    Synonymous/Nonsynonymous Effects and Associations Reference

    � Opioid(OPRM1)

    A118G(rs1799971)

    Nonsynonymous (Asn/Asp,Variant lacks the Nglycosylation site inOPRM1 extracellulardomain)

    118G allele associated with reduced ACTHresponse to metyrapone

    176

    118G associated with increasedendorphin- binding affinity and activity

    177

    118G allele reduces agonist-inducedreceptor-signaling efficacy

    178

    118G associated with lower OPRM1 expession 179118G altered downstream signaling of ERK

    1 and 2 and PKA compared with A118180

    118G associated with opioid dependency 162–164118A associated with opioid dependency 165–166118A associated with opioid and alcohol

    dependencyNo association with heroin dependency 167–175

    C17T(rs1799972)

    Nonsynonymous (Ala/Val) 17T allele associated with cocainedependence

    181

    TT genotype associated with cocaine andheroin use in African American women

    182

    No association with opiate addiction 162; 165;172; 174

    A/G(rs510769)

    Intron 1 G allele and heroin dependence* 168

    C/T(rs3778151)

    Intron 1 T allele and heroin dependence*

    C/T(rs6473797)

    Intron 2 C allele and heroin dependence*

    A/G(rs569356)

    Promoter G allele significantly higher reporter expression;altered transcription factor binding

    183

    � Opioid(OPRD1)

    G/T(rs1042114)

    Nonsynonymous(Cys27Phe)

    Cys27 compromised ATP-inducedintracellular Ca2� signaling

    184

    Cys27 2 HERPCys27 reduced maturation efficiency and

    differential subcellular localization185

    C/Trs2236861

    Intron 1 T allele and heroin dependence* —

    A/Grs3766951

    Intron 1 G allele and heroin dependence* 168

    A/Grs2236857

    Intron 1 G allele and heroin dependence*

    � Opioid(OPRK1)

    OPRK1Haplotype

    — No association between OPRK1 haplotypeand opioid dependency

    186

    187G36T

    (rs1051660)Synonymous Association of the T allele with heroin

    dependency188

    ORL1(OPRL1)

    G501C Nonsynonymous(Lys167Asn)

    167Asn impairs ERK 1 and 2 activation 189

    LDL-induced biosynthesis of LOX-1receptors is genotype dependent

    A/G(rs6512305)

    Intron Marginal association with opioid dependence 190

    C206T(rs6090043)

    5�UTR Marginal association with opioid dependence 190

    * No association after correcting for multiple testing.ACTH � adrenocorticotrophic hormone; ATP � adenosine triphosphate; ERK 1 and 2 � extracellular signal-regulated kinases 1 and2; LDL � low density lipoprotein; LOX-1 � low density lipoprotein-1; ORL1 � opioid receptor like-1; PKA � protein kinase A.

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  • for this interest is that it has been widely accepted that a clearunderstanding of opioid receptor synthesis, cellular localiza-tion, trafficking, and pharmacology will lead to novel thera-peutics that either directly act on opioid receptors or modu-late opioid receptor signaling pathways. With the advent ofconditional genetic approaches, receptor tags, antibodies,fluorescent tools, and optogenetic manipulation of neuralcircuitry, opioid receptor pharmacology is poised for somemajor breakthroughs in the next decade. It is hopeful thatthese new molecular and cellular discoveries will lead to bet-ter opioid analgesics in the clinic, with decreased risks ofaddiction and tolerance. In addition, it is likely that studies atthe forefront of molecular and behavioral pharmacology willcontinue to reveal novel uses for opioids in the treatment ofa variety of psychiatric and neurologic diseases.

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