Skip Navigation LinksHome > December 2011 - Volume 115 - Issue 6 > Molecular Mechanisms of Opioid Receptor-dependent Signaling...
Anesthesiology:
doi: 10.1097/ALN.0b013e318238bba6
Education: Review Articles

Molecular Mechanisms of Opioid Receptor-dependent Signaling and Behavior

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

Free Access
Article Outline
Collapse Box

Author Information

Collapse Box

Abstract

Opioid receptors have been targeted for the treatment of pain and related disorders for thousands of years and remain the most widely used analgesics in the clinic. Mu (μ), kappa (κ), and delta (δ) opioid receptors represent the originally classified receptor subtypes, with opioid receptor like-1 (ORL1) being the least characterized. All four receptors are G-protein coupled and activate inhibitory G proteins. These receptors form homo- and heterodimeric complexes and signal to kinase cascades and scaffold a variety of proteins.
The authors discuss classic mechanisms and developments in understanding opioid tolerance and opioid receptor signaling and highlight advances in opioid molecular pharmacology, behavioral pharmacology, and human genetics. The authors put into context how opioid receptor signaling leads to the modulation of behavior with the potential for therapeutic intervention. Finally, the authors conclude there is a continued need for more translational work on opioid receptors in vivo.
Table 1
Table 1
Image Tools
OPIOIDS are the most widely used and effective analgesics for the treatment of pain and related disorders. Opiates have been used for thousands of years for the treatment of pain, and in the last century we have made huge strides in the development of opioids derived from naturally occurring opiates within the fields of receptor pharmacology and medicinal chemistry. In addition, opioids are used frequently in the treatment of numerous other disorders, including diarrhea, cough, postoperative pain, and cancer (table 1).
Opioid systems are critical in the modulation of pain behavior and antinociception. Opioid peptides and their receptors are expressed throughout the nociceptive neural circuitry and critical regions of the central nervous system included in reward and emotion-related brain structures. To date, four different opioid receptor systems mu (μ), delta (δ), kappa (κ), opioid receptor like-1 (ORL1) and their genes have been characterized at the cellular, molecular, and pharmacologic levels.1
The most commonly used opioids for pain management act on μ opioid receptor (MOR) systems (fig. 1). Although μ opioids continue to be some of the most effective analgesics, they are also mood enhancers and cause activation of central dopamine reward pathways that modulate euphoria. These unwanted side effects have driven researchers at basic and clinical levels to actively pursue other opioid receptors as putative drug targets for pain relief (table 1).
The opioid receptor subtypes were identified pharmacologically and genetically more than 2 decades ago.1 From that point on, numerous studies have implicated all four opioid receptors in an array of behavioral effects, including analgesia, reward, depression, anxiety, and addiction. In addition, all four receptor subtypes have been characterized at cellular levels with respect to the downstream signal transduction pathways they activate. However, there are fewer studies that have directly linked opioid signal transduction to behavioral events. One of the “holy grails” in opioid pharmacology research has been to identify pathway-specific opiate receptor agonists that could activate antinociceptive signaling without causing μ agonist-mediated euphorigenic responses or κ agonist-mediated dysphoria.2,3 Understanding the diversity of signaling at opioid receptors and how second messenger activation leads to modulation of pain and reward could reveal novel opioid receptor drug candidates.
In this review, we highlight the current status of in vitro molecular pharmacology at opioid receptors and discuss many of the recent advances that connect these molecular studies with opioid behavioral pharmacology. We discuss the advances in opioid receptor pharmacology and highlight the connections between signaling at opioid receptors, tolerance to opioids, and behavioral responses. The review's primary aim is to discuss recent efforts in understanding how opioid receptors mediate a diverse array of molecular or cellular responses while also modulating behaviors such as analgesia, reward, depression, and anxiety. We summarize the modern advances in opioid receptor signaling to mitogen-activated protein kinases (MAPK) and receptor protein–protein interaction networks and propose that there is a strong potential for selective ligand intervention at opioid receptors to treat a variety of central and peripheral nervous system disorders by using biased ligands and pathway-selective pharmacology. Moreover, we highlight how a greater connection between these advances at the molecular levels and behavioral pharmacology is imperative to fully understand the field of opioid pharmacology.
Back to Top | Article Outline

Opioid Tolerance in the Clinic

Before a detailed understanding of the molecular and cellular actions of opioid receptors is developed, it is important to consider their general effects and those observed in daily clinical settings. Different potencies of opiate drug formulations have been effective in the treatment of a variety of acute, chronic, and cancer-related pain disorders. The clinical utility of opioids continues to be limited by a compromise between efficacy and side effects. The most common side effects of opiates can be divided into peripheral effects (constipation, urinary retention, hives, bronchospasm) and central effects (nausea, sedation, respiratory depression, hypotension, miosis, cough suppression), all of which seriously affect the agents' clinical utility and patients' quality of life4,5 (table 1). There have been many attempts to develop better opioid drugs, but these have been largely unsuccessful because of our incomplete understanding about the development of tolerance to the analgesic effects.6
Opioid tolerance is defined typically in the clinic as the need to increase a dose to maintain the analgesic effects. However, this increase in dose can exacerbate the perpetual problem of the side effects mentioned. This continual cycle of insufficient analgesia and side effects is among the greatest challenges of using opioids in the clinic. Because of these limitations, opioid tolerance can ultimately lead to low patient compliance and treatment discontinuation. These clinical problems highlight the continued need for a better understanding of the molecular and pharmacologic mechanisms of opioid receptor tolerance, regulation, and signal transduction.
Back to Top | Article Outline

Classic Opioid Receptors Signaling

Opioid receptors are expressed in pain-modulating descending pathways, which include the medulla, locus coeruleus, and periaqueductal gray area. They are also expressed in limbic, midbrain, and cortical structures (fig. 1). The activation of opioid receptors at these locations directly inhibits neurons, which in turn inhibit spinal cord pain transmission.4,5 The process by which these receptors engage in disinhibition is understood mostly with respect to analgesia; however, research is still active in this area because investigators continue to unravel novel modulatory mechanisms in these opioid circuits.
All four opioid receptors are seven-transmembrane spanning proteins that couple to inhibitory G proteins. After being activated by an agonist, such as the endogenous μ-opioid peptide endorphin, or exogenous agonists, such as morphine and fentanyl, the Gα and Gβγ subunits dissociate from one another and subsequently act on various intracellular effector pathways.7,8 Early work in opioid receptor pharmacology demonstrated that guanine nucleotides such as guanosine triphosphate (GTP) modulate agonist binding to opioid receptors in membrane preparations from brain tissue. It was later determined that GTPase activity is stimulated by opioid agonists and endogenous opioid peptides.9 Agonist stimulation of opioid receptors was also shown to inhibit cyclic adenosine monophosphate (cAMP) production in a manner similar to that of other types of G protein-coupled receptors (GPCR).10 When pertussis toxin was used to selectively adenosine diphosphate (ADP)-ribosylate the G protein, the inhibitory function of opioid receptors on cAMP signaling was found to be Gαi dependent.11,12 Today it is widely accepted that all four opioid receptor types couple to pertussis-toxin–sensitive G proteins, including Gαi, to cause inhibition of cAMP formation.
Fig. 2
Fig. 2
Image Tools
The classic and perhaps most important aspect of opioid receptor signal transduction relates to opioids' ability to modulate calcium and potassium ion channels (fig. 2). After Gαi dissociation from Gβγ, the Gα protein subunit moves on to directly interact with the G-protein gated inwardly rectifying potassium channel, Kir3. Channel deactivation happens after GTP to guanosine diphosphate hydrolysis and Gβγ removal from interaction with the channel.1315 This process causes cellular hyperpolarization and inhibits tonic neural activity. In several reports, the inhibitory effects of opioids on neural excitability were shown to be mediated by interactions of opioid receptors with G protein-regulated inwardly rectifying potassium channel (Kir3).16,17
When activated, all four opioid receptors cause a reduction in Ca+2 currents that are sensitive to P/Q-type, N-type, and L-type channel blockers.18 Opioid receptor-induced inhibition of calcium conductance is mediated by binding of the dissociated Gβγ subunit directly to the channel. This binding event is thought to reduce voltage activation of channel pore opening.19,20 Numerous reports have shown that opioid receptors interact with and modulate Ca+2 channels; this has led to the examination of specific Ca+2 channel subunits that may be involved in opioid receptor modulation. For instance, it was reported that MOR stimulation results in G protein-dependent inhibition of α1A and α1B subunits.21
It is also clear that the acute administration of opioid agonists reduces Ca+2 content in synaptic vesicles and synaptosomes, with compensatory up-regulation of vesicular Ca+2 content during the development of opiate tolerance.22,23 In addition, because the activation of μ, δ, and κ opioid receptors inhibits adenylyl cyclase activity, the cAMP-dependent Ca+2 influx is also reduced.
The evidence for opioid receptors positively coupling to potassium channels while negatively modulating calcium channels has been reported in numerous model systems and cell types. For many years this was thought to be the primary action of opioid receptors in the nervous system. This coupling of opioid receptors to potassium and calcium channels has been demonstrated in a wide range of systems, from neurons in the hippocampus, locus coeruleus, and ventral tegmental area to the dorsal root ganglia, supporting the notion that these channels are highly conserved opioid receptor substrates and represent one of the most important targets for opioid receptor modulation. Newer findings, which we highlight later in this review (see Opioid Receptor Regulation), suggest that although opioid receptors have potent effects on ion channel modulation, they also have slower yet robust effects on other signal transduction pathways.
Back to Top | Article Outline

Molecular Mechanisms of Opioid Tolerance

To date, the molecular and cellular mechanisms mediating the development of tolerance to morphine remain a matter of controversy. Traditionally, it was thought that the down-regulation of opioid receptors after chronic agonist exposure induces tolerance, as reported in in vitro studies.24,25 However, recent in vivo studies show that down-regulation does not occur consistently with each and every agonist and may not completely explain tolerance. In light of these findings, it has been suggested that MOR proteins are in fact not down-regulated but instead may be desensitized and uncoupled from downstream signaling pathways.26 It has been observed that after chronic morphine exposure, levels of the second messenger cAMP are increased. However, this elevation in cAMP may not be attributable to opioid receptor uncoupling from inhibitory G proteins but instead could reflect cellular adaptive changes, including the up-regulation of adenylyl cyclase, protein kinase A, and cAMP response element-binding protein.27 It is this ineffective regulation of cAMP by morphine that some believe induces tolerance.
It has also been proposed that the regulation of opioid receptors by endocytosis reduces the development of tolerance and therefore serves a protective role.28,29 After endocytosis, the cellular response is desensitized to the μ agonist, but the receptors can be recycled to the cell surface in an active state, resensitizing the receptor to the agonist. Morphine-activated opioid receptors signal for long periods of time, thereby enhancing the production of cAMP, which is thought to result in tolerance. In vivo studies have shown that facilitation of MOR endocytosis in response to morphine prevents the development of morphine tolerance.28 In addition, it has been shown in vivo that the lack of β-arrestin 2 prevents the desensitization of MOR after chronic morphine treatment, and these mice also failed to develop antinociceptive tolerance.30
Recent studies have identified how ligand-directed responses, more commonly known as biased agonism, are crucial in understanding the complexity of opioid-induced tolerance. The work of Bohn and colleagues showed how β-arrestin 1 and β-arrestin 2 differentially mediate the regulation of MOR. β-arrestins are required for internalization, but only β-arrestin 2 can rescue morphine-induced MOR internalization, whereas both β-arrestin 1 and β-arrestin 2 can rescue [d-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO)-induced MOR internalization.31 These findings suggest that MOR regulation is dependent on the agonist and may be critical in understanding the mechanism involved in the development of tolerance. Melief et al. further showed how acute analgesic tolerance to morphine is blocked by c-jun N-terminal kinase (JNK) inhibition but not G protein-receptor kinase 3 (GRK-3) knockout. In contrast, using a second class of μ agonists (fentanyl, methadone, and oxycodone), acute analgesic tolerance was blocked in GRK-3 knockout but not JNK inhibition.32 Ligand-biased responses are well documented in vitro but less so in vivo; however, a recent study addressed biased agonism at δ opioid receptors (DOR) in vivo showing that DOR agonists with similar binding and analgesic properties but different internalization potencies lead to the development of differential tolerance at DOR.33 These findings highlight the important implications of ligand-selective responses in GPCR biology33 and indicate the need for additional work to examine the role and consequences of biased signaling in behavioral models.
Back to Top | Article Outline

Opioid Receptor Regulation

Agonist-induced receptor phosphorylation is believed to be one of the many critical molecular components of opioid tolerance. This process is well established in the GPCR literature and typically occurs after chronic agonist exposure or sustained release of endogenous opioid peptides. Sustained opioid treatment produces tolerance to the acute effects of the drug and can potentially lead to physical and psychological dependence. As a result of this problem, opioid-receptor trafficking, desensitization, and phosphorylation have been extensively examined (for a detailed review see Bailey and Connor34). Here we highlight the key findings in this area as they connect potential signaling to tolerance mechanisms.
Back to Top | Article Outline
μ Opioid Receptors (MOR)
One common thread between the opioid receptor subtypes is the interesting observation that receptor trafficking and regulation vary depending upon the agonist. For example, morphine is unable to promote receptor internalization, in contrast to DAMGO, which causes robust internalization.32,35,36 It is thought that morphine tolerance, a major problem in the clinic, is perhaps mediated by these differences in receptor regulatory activity. Several groups are actively working to discern the various mechanisms for the differences in ligand-dependent MOR regulation, but controversy remains, with some groups hypothesizing that MOR internalization does not actually uncouple the receptor from signal transduction pathways but instead induces recycling of uncoupled receptors to the plasma membrane. Alternatively, the morphine-bound receptor, although not internalized, may still signal at the cell membrane, and because signaling is never attenuated, the cellular machinery adapts to produce tolerance. A recent study has shown that morphine acts as a “collateral agonist” to promote receptor G-protein uncoupling (“jamming”) and JNK activation (see MAPK Signaling at Opioid Receptors), whereas fentanyl and DAMGO internalize and desensitize normally.35 It is plausible that many processes work together to produce receptor regulation and opioid tolerance, and additional study is warranted to continue to decipher these discrepancies.
μ Opioid receptors contain more than 15 serine, threonine, and tyrosine residues that are accessible to protein kinases, which phosphorylate the receptor. All three intracellular loops and the carboxyl terminal tail contain these sites.37 32P incorporation experiments have been critical to our understanding of MOR receptor phosphorylation, and combined with our knowledge of site-directed mutagenesis, we now have a clear understanding of the key residues involved in MOR phosphorylation. Rat MORs are phosphorylated at Ser375 in the carboxy terminus,38,39 and treatment with both morphine and DAMGO causes robust phosphorylation of this residue. However, some reports have suggested that morphine and DAMGO induce different degrees of phosphorylation of Ser375,39 suggesting that Ser375 may not be the only amino acid residue phosphorylated and responsible for MOR regulation. The highly conserved GPCR “DRY motif” in the second cytoplasmic loop of the μ opioid receptor has been implicated in regulation of agonist efficacy. Phosphorylation of Tyr166 reduced the efficacy of DAMGO-mediated G-protein activation.40 It has been shown recently that agonist-selective differences in MOR regulation are in fact determined not only by net incorporation of phosphates into the receptor population as a whole but also by individual receptors achieving a critical number of phosphorylated residues (multiphosphorylation) in a specific region of the C-tail.41 In addition, the same group identified that multiphosphorylation specifically involves the 375STANT379 motif required for the efficient endocytosis of MOR. These ligand-mediated differences highlight the ligand-dependent nature of opioid receptor function and require additional further study in vivo.
Back to Top | Article Outline
κ Opioid Receptors (KOR)
κ Opioid receptor trafficking shares some common features with MOR regulation because KOR is readily phosphorylated, desensitized, and internalized. KOR is phosphorylated, desensitized, and internalized by the agonists U50,488 and dynorphin 1–17 but not by other agonists, such as etorphine or levorphanol.42,43 Both dynorphin A and B have been shown to initiate significant receptor internalization in human KORs and three structurally distinct KOR ligands: Salvinorin A (salvA), TRK820, and 3FLB were shown to induce KOR internalization with varying rank orders of potency.44 There have been conflicting data regarding agonist-induced KOR internalization, which seem to be dependent on the cell line, receptor species, or model system used. In Chinese hamster ovary cells expressing KOR, the selective KOR agonists U50,488 and U69,593 did not cause robust receptor internalization45; however, in mouse pituitary tumor (AtT20) cells and human embryonic kidney (HEK293) cells, U50,488 initiated strong internalization of KOR-green fluorescent protein (GFP) proteins.4648 Despite this, several groups have found consistency in the ability of KOR to become phosphorylated, internalized, and desensitized by its endogenous opioid peptide dynorphin.
Back to Top | Article Outline
δ Opioid Receptors (DOR)
In contrast to MOR and KOR, DOR were thought to exist primarily (more than 90%) at intracellular sites4951 until recently, when mice expressing fluorescently tagged DOR revealed that there was strong membrane localization of DORs in vivo.52 The reasons for this discrepancy between the numerous studies showing intracellular DOR labeling and membrane labeling remain unclear and continue to be matters of controversy. It is plausible that previous studies using DOR antibodies were flawed because of antibody specificity issues, despite the numerous controls conducted. Although DORs tagged with GFP are a powerful in vivo tool, they require careful interpretation given that GFP is a large protein that may interfere with the typical DOR trafficking machinery. Additional investigation is required in both cases, and it is plausible that both concepts are indeed true; the concentrations of DOR expressed on the cell surface may well be higher than originally hypothesized, yet a large intracellular pool of DOR protein remains. Nevertheless, DOR seems to be a dynamic opioid receptor that can readily traffic in response to agonists. Some reports have shown that chronic morphine treatment promotes movement of DORs to the cell surface in the dorsal horn of the rat spinal cord.49 This effect was dependent on MOR receptor activity because blocking or deleting MOR genetically (MOR knockout) prevents the effect.
Like MOR and KOR, desensitization of DOR is controlled via phosphorylation, after recruitment of arrestins and sequestration of arrestin-bound receptors.53,54 Phosphorylation of DOR has been shown with both small-molecule organic ligands and peptide treatments. Once again, c-terminal phosphorylation was shown to be critical for opioid receptor regulation. In DOR, the Ser363 residue is the key phosphorylation site.55,56 This phosphorylation event was shown to be mediated by GPCR kinase 2 (GRK-2).56,57 Other studies have demonstrated that other amino acid residues are involved in DOR regulation. For example, Thr353 was found to be important for [D-Ala2, D-Leu5]-Enkephalin (DADLE)-mediated down-regulation of DOR, and Leu245 and 246 act as lysosomal targeting motifs that partake in determining agonist-bound DOR localization.58,59 Furthermore, ligand-specific variability in agonist-dependent DOR phosphorylation has been observed with potential differences between SCN80- and [D-Pen2,5]Enkephalin (DPDPE)-bound conformations recruiting kinases with various efficacies and potencies.60
Back to Top | Article Outline
Opioid Receptor Like-1 (ORL1)
Opioid receptor like-1 (also called nociceptin or orphanin FQ) receptors are the newest members of the opioid receptor family, and few groups have examined their regulatory properties. Agonist-induced internalization of ORL1 is rapid and concentration- dependent.61 Both the endogenous agonist nociceptin and small-molecule selective ORL1 agonist Ro646198 promote rapid internalization of ORL1. Agonist challenge also reduces the ability of ORL1 to couple to inhibition of forskolin-stimulated cAMP production, suggesting that ORL1 undergoes desensitization mechanisms similar to those of the other three opioid receptor subtypes. ORL1 internalization appears to be more rapid than that of the other opioid receptors, with some groups reporting internalization after only 2 min of agonist exposure in Chinese hamster ovary cells.61 However, this appears to be dependent on ligand type and cell line expression because ORL1 internalization in human neuroblastoma cells was slower and occurred closer to a 30-min time point.62 ORL1 receptors recently were demonstrated to cointernalize with N-type Cav2.2 channels after a 30-min agonist treatment.63 The internalization of the entire signaling complex is not unusual in GPCRs; however, the effect in the case of ORL1 is particularly pronounced and is believed to play a major role in how ORL1 selectively removes N-type calcium channels from the plasma membrane to inhibit calcium influx.
Opioid receptor like-1 receptor regulation is increasingly studied, but our understanding remains in the infant stages compared with that of the other three opioid receptor subtypes. To date, few site-directed mutagenesis studies have been conducted, and receptor regulation in primary neurons, dorsal root ganglion, or dorsal horn neurons remains unknown. As we move forward in understanding opioid receptor signaling and identify novel opioid receptor targets, ORL1 receptors become likely candidates for the future of opioid pharmacology.
Back to Top | Article Outline
Opioid Receptors and Arrestin Recruitment
Phosphorylation by GRK-2 or -3 of μ, δ, and κ opioid receptors leads to arrestin 2 or 3 recruitment. Arrestin molecules are key proteins that bind phosphorylated GPCRs to regulate their desensitization, sequestration, and sorting and ultimately assist in determining receptor fate. Opioid receptors are regulated by arrestin 2 and arrestin 3 binding (also called β-arrestin 1 and β-arrestin 2, respectively), and this interaction depends on the model system and agonist treatment procedure. Mice lacking arrestin 3 have been shown to have a reduced tolerance to μ opioids such as morphine, suggesting that MOR regulation requires arrestin 3.30,64
With the use of surface plasmon resonance methods, glutathione s-transferase pull-down assays, and classic immunoprecipitation methods, the C-terminal tails of DOR, MOR, KOR have been shown to be crucial for arrestin 2 or 3 binding. C-terminal carboxyl mutant opioid receptors have been studied widely, and these serine mutant receptors show decreased agonist-induced receptor internalization and arrestin recruitment. Dominant positive arrestins (such as Arrestin-2-R169E or Arrestin-3-R170E) that bind the nonphosphorylated receptors can rescue internalization of serine mutated MOR/DOR/KOR,48,65 further implicating arrestin dependence in opioid-receptor trafficking. Most studies implicating arrestin have been conducted in heterologous expression systems using overexpressed arrestins and opioid receptor subtypes. These conditions are atypical and do not represent the likely physiologic state of opioid receptors and arrestins in vivo, so these data should be interpreted with caution, and additional studies using in vivo approaches are needed to increase our understanding of arrestin–opioid interactions.
Back to Top | Article Outline
MAPK Signaling at Opioid Receptors
Table 2
Table 2
Image Tools
In the discussion above, we highlighted that sustained agonist 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 discovered that the phosphorylated arrestin-bound GPCR complex is not simply inactive, but that it recruits alternate signal transduction cascades, including MAPKs.66 The merging of our previous knowledge regarding opioid receptor phosphorylation, arrestin, and cellular mechanisms of tolerance with an understanding of opioid receptor signaling to MAPKs is becoming more appreciated (table 2).
Mitogen-activated protein kinase pathways are diverse signaling cassettes that govern cellular responses, including cell proliferation, differentiation, apoptosis, transcription factor regulation, channel phosphorylation, and protein scaffolding.93 The MAPK family is composed of 12–15 gene products with the most well-described forms including extracellular signal-regulated kinases 1 and 2 (ERK 1 and 2), JNK1–3, and p38 (α, β, γ, δ) stress kinase. The MAPKs are distinct in that they have the capacity to respond to a variety of stimuli and transmit a diverse array of intra- and extracellular signals.94 MAPK signaling is regulated by the kinetics of activation, nearby phosphatase activity, and the cellular domain the MAPKs occupy.93 Initially, ERK MAPKs were shown to require receptor tyrosine kinase transactivation, through epidermal growth factor or brain derived neurotrophic factor (also called TrkB receptors).95 Later reports directly linked GPCRs to activation of MAPK signaling pathways, and now most, if not all, GPCRs have been found to couple to this pathway.
Back to Top | Article Outline
ERK 1 and 2 Signaling at Opioid Receptors.
The most frequently examined opioid-induced MAPK cascade is ERK 1 and 2. Coscia and colleagues have been crucial in developing our understanding of the relationship between opioid receptors and ERK 1 and 2 signaling. In one of the initial studies, MOR and KOR stimulation was demonstrated to initiate ERK 1 and 2 phosphorylation in astrocyte cultures and transfected cell lines.96 The kinetics of ERK 1 and 2 phosphorylation by MOR and KOR systems vary, yet both receptors can activate ERK 1 and 2 within 5–10 min. MOR-mediated ERK 1 and 2 phosphorylation requires protein kinase C (PKCε) activity, and MOR-dependent ERK 1 and 2 signaling requires GRK-3 and arrestin in primary neurons, glial cells, and heterologous expression systems.67,68,97 The downstream substrates of MOR-mediated ERK 1 and 2 have been defined in some cases and remain unknown in others. In embryonic stem cells, MOR-dependent ERK 1 and 2 signaling positively modulates and directs neural progenitor cell fate decisions.98,99 However, in astrocytes chronic morphine can negatively regulate ERK 1 and 2 signaling by tyrosine kinase pathways to ultimately inhibit neurite outgrowth and synapse formation.69 Most studies use MAPK–ERK (MEK) inhibitors (the proximal upstream kinase) to determine substrates of ERK 1 and 2 signaling in GPCRs; however, few reports have shown direct interaction between μ-opioid–induced ERK and a final substrate. (The in vivo implications of MOR-dependent ERK signaling are explored in Opioid Signaling and Behavior.) Several groups are investigating the potential for ligand-specific ERK agonists at opioid receptors.
δ Opioid receptors have also been shown to activate ERK 1 and 2 through Gβγ and Ras signaling cascades96 and do not necessarily require receptor internalization or receptor phosphorylation for signaling.100,101 DOR-mediated ERK signaling recently was found to require integrin signal transduction through transactivation of epidermal growth factor receptor pathways. DOR-mediated epidermal growth factor receptor activation also initiated phospholipase C signaling to stimulate ERK 1 and 2 phosphorylation.83 DOR-dependent ERK 1 and 2 signaling requires additional investigation because, coupled with DOR's critical role in pain and mood regulation, ERK signaling through DOR may reveal a novel mechanism for DOR regulation of neural activity.
KOR-dependent ERK 1 and 2 phosphorylation occurs in a multiphase manner, with an early period of activity between 5–15 min after agonist exposure and a late phase after 2 h of agonist treatment. Similar to other GPCRs,102 the biphasic ERK 1 and 2 activation for KOR contains an arrestin-dependent late phase73 and an arrestin-independent early phase. This group identified Gβγ as a crucial mediator in the early-phase ERK 1 and 2 activation by KOR, and showed that arrestin 3 is required for late-phase ERK 1 and 2. KORs activate ERK 1 and 2 through PI3-kinase, PKCζ, and intracellular calcium.72 However, like MOR and DOR, the substrate for KOR-mediated ERK 1 and 2 has not been identified, although a recent study suggests that KOR-induced ERK 1 and 2 also directs stem cell fate toward neural progenitor development. ORL1 receptor-dependent ERK 1 and 2 activation has not been extensively examined, although one group has shown that ORL1 receptor activation does initiate ERK 1 and 2 phosphorylation.89 The signaling pathways for ORL1-mediated ERK 1 and 2 phosphorylation in neuronal cell types and in vivo need additional investigation.
Back to Top | Article Outline
JNK.
The JNK pathway is activated by environmental triggers, including stress, inflammation, cytokine activation, and neuropathic pain.103,104 Classically, JNK activity can result in transactivation of c-jun, a component of the activator protein 1 transcription factor complex, and JNK phosphorylation is caused by cytokines, including tumor necrosis factor and interleukin-1β.105 JNK activation has also been implicated in numerous other signaling cascades. JNK typically is activated by Ras-related GTP binding proteins in the ρ family.106 JNK activation by GPCRs and opioid receptors has not been examined thoroughly but has been demonstrated for all the opioid receptor subtypes. Like ERK 1 and 2, arrestin 2 and arrestin 3 have been reported to scaffold JNK signaling complexes, and it is believed that arrestin 3 has JNK 3 specificity, although this remains a matter of controversy.107 The cellular mechanisms of arrestin-dependent JNK at GPCRs remain unresolved.
Opioid-dependent JNK has been demonstrated by only a few groups. DOR causes protein kinase B (Akt)-dependent JNK phosphorylation through a PI3-kinase mechanism,88 and JNK activity is PI3-kinase independent in others.108 PI3-kinase is required for μ-opioid–dependent JNK activation. In contrast, U50,488-induced (KOR) JNK activation has been shown to be independent of PI3-kinase.108 The substrates and in vivo effects of opioid-induced JNK activation are being studied by several groups. KOR (U50,488, dynorphin) agonists activate JNK in a pertussis toxin-sensitive (Gαi) manner.35,108,109 U50,488-mediated JNK requires focal adhesion kinases and the GTPase Rac in immune cell types. MOR-induced JNK activation recently was shown to require PKC activity.35
In two recent studies, KOR- and MOR-induced JNK phosphorylation by norbinaltorphimine and morphine were shown to act as “collateral agonists” to cause JNK phosphorylation and initiate uncoupling of the G protein to block Gαi-mediated transduction.35,109 The persistent actions of norbinaltorphimine on KOR-agonist–mediated analgesia (21 days) were shown to require JNK because JNK 1 isoform knockout mice show an absence of norbinaltorphimine-dependent 21-day KOR blockade, and selective JNK inhibitors prevented the long-lasting norbinaltorphimine effect. It was also recently identified that the long duration of action of small molecule KOR antagonists in vivo is determined by their efficacy in activating JNK 1. The persistent KOR inactivation by these small-molecule collateral agonists did not require sustained JNK phosphorylation,32 implicating intermediate protein(s) or alternate JNK substrates in this process. In contrast, acute morphine tolerance was shown to require JNK 2 because JNK 2 knockout mice showed an absence of MOR inactivation. How ligand-dependent JNK activation causes receptor uncoupling from Gαi signaling remains unresolved, and proteomic biochemical approaches will need to be used to identify ligand-dependent protein interactions. Together, this work highlights the remarkable nature of opioid receptor sensitivity to a variety of ligand-stabilizing conformations.
Back to Top | Article Outline
p38 MAPK.
The p38 MAPK pathway also plays a key role in environmental stress and inflammation and is activated by cytokine production.110 In glial cells particularly, p38 MAPK activity is required for an array of cellular responses, including interleukin-6 and interleukin-1β production, inhibitory nitric oxide synthase activity, and tumor necrosis factor α secretion. Activation of p38 MAPK is involved in proliferative and chemotactic responses in some systems and has been shown to play a major role in neuropathic pain responses.77,111
Opioid receptor-mediated p38 phosphorylation has been most widely demonstrated for the MOR and KOR systems. KOR-induced p38 MAPK has been observed in heterologous expression systems, striatal neurons, astrocytes, and in vivo.2,3,8,77,76,99 KOR-mediated p38 MAPK activation requires serine 369 phosphorylation by GRK-3 and arrestin 3 recruitment.48,76 μ Opioid receptor internalization recently has been shown to require Rab5 signaling and p38 MAPK. This process seems to be ligand dependent because morphine will not cause p38-dependent receptor internalization, but DAMGO will readily cause internalization.70 In addition, μ opioid receptor cross regulation of α2A-adrenergic receptors has been shown to require p38 MAPK, and p38 MAPK inhibition blocks DAMGO-induced MOR internalization.70 This cross activity between MOR and α2A-adrenergic receptors requires arrestin 3, suggesting that arrestin 3 scaffolding of p38 is likely to be conserved across opioid receptors. To date, few studies have identified a role for DOR or ORL1 in mediating p38 phosphorylation. In one report, both ORL1 and DOR were shown to cause p38 phosphorylation through activation of protein kinase A and protein kinase C.91 Opioid-induced p38 has several potential targets, including modulation of ion channels and transcription factors. Recently, the potassium channel Kir3.1 was demonstrated to become tyrosine phosphorylated via KOR-dependent p38 MAPK Src activation.112 How p38 specifically interacts with various substrates will be an interesting next step and will reveal how such a ubiquitously expressed kinase can selectively modulate the large variety of cellular events.
Back to Top | Article Outline
Protein–Protein Networks and Opioid Receptors
In addition to intracellular signaling and receptor modification by phosphorylation, newer biochemical studies strongly suggest that opioid receptors interact with one another, alternate GPCRs, and a whole host of anchoring and membrane protein sets. These interactions are becoming increasingly appreciated as critical to the ultimate functional role of the opioid receptor families. In many ways, the field of protein–protein interactions is at the forefront of opioid receptor molecular pharmacology, as research moves from previous work in heterologous expression systems to in vivo approaches.
Back to Top | Article Outline
Opioid Dimerization.
Numerous reports have demonstrated that GPCRs exist as dynamic protein complexes with large interactions between proteins and other receptor types. Several studies have shown that GPCRs can form dimers and oligomers. This oligomerization includes two varieties: homodimers (same receptor) and heterodimers (different receptor type) (fig. 3). The existence of these GPCR homomers and heteromers has been shown in transfected cell line systems, cell lines, and primary cultures and in some cases in vivo (for review see Rios et al.113 and Prinster et al.114). Despite that GPCR oligomerization remains a matter of controversy, it continues to generate interest because opioid receptor dimers may reveal novel targets for the development of new opioid drugs.
Devi and colleagues pioneered research into opioid dimerization and originally identified opioid receptor heterodimers.115 They found that δ receptors can exist as homodimers, and agonist stimulation causes their dissociation.115,116 In this seminal work, the authors also found that KOR and DOR form heterodimeric complexes, which appear to alter the trafficking properties of these receptors. They showed how agonist-induced internalization of DOR receptors is reduced substantially in cells expressing DOR/KOR receptors.115 Moreover, it was shown that 6′-guanidinonaltrindole, which selectively targets the KOR or DOR heterodimer, generates a unique signaling entity, giving additional evidence for the existence of opioid heterodimers.117 It has been shown that MOR can heterodimerize with ORL1,118 but the existence of MOR or KOR heterodimers remains a matter of controversy.115,119 The observation that the antagonism or absence of DOR diminishes the development of morphine tolerance and dependence suggests there may be an interaction between the two receptors, although future biochemical work in vivo is needed to validate these concepts. Studies not only identified the existence of MOR and DOR heterodimers but also revealed that MOR and DOR heterodimers have distinct ligand binding and signal transduction properties,120 suggesting that heterodimerization may represent an alternative mechanism for the cell to tune and control second messenger activity.
It was hypothesized that the mechanisms and/or proteins that modulate the level of MOR or DOR complexes are critical in the development of tolerance121; this theory inspired research into the events that lead to dimerization. Devi and colleagues recently identified additional signaling proteins, such as RTP4, that partake in opioid receptor oligomer trafficking from the Golgi to distribute opioid receptor complexes at the cell membrane.121 In addition, it was found that MOR activation promotes the formation of complexes between RGS9–2 and Gα subunits. It was shown that pharmacological manipulations were able to disrupt RGS9–2 complexes formed after repeated morphine administration.122 These data provide a better understanding of pharmacologic approaches that can be used to improve chronic analgesic responses and tolerance.
Some studies have shown that opioid receptors can heterodimerize with other classes of GPCR. For example, MOR can interact and potentially heterodimerize with cannabinoid receptor 1 (CB1) (fig. 3).123,124 Interactions between MOR and cannabinoid receptor 1 receptors appear to modulate their effects, as evidenced by the administration of Δ 9- tetrahydrocannabinol, a cannabinoid receptor 1 agonist, which can enhance the potency of opioids such as morphine.125 In addition, the concomitant activation of MOR or cannabinoid receptor 1 heterodimers leads to significant attenuation of ERK activity compared with the response after the activation of each individual receptor.123
A number of previous studies have noted interesting functional interactions between the MOR and α2A-adrenergic receptor systems.126128 These studies reported that the presence of α2A receptors is sufficient to potentiate the phosphorylation of MAP kinases in response to morphine, whereas the combination of ligands abolishes this effect. The interactions between MOR and α2A receptors provide an alternate mechanism for the control of receptor function and could have profound effects in the development of opioid-adrenergic analgesics. Neurokinin 1 and MOR have also been shown to heterodimerize. The interaction between these two receptor types does not alter ligand binding or signal transduction but does change internalization and resensitization.129 In addition, substance P (neurokinin 1 selective ligand) caused cross phosphorylation and cointernalization of MOR.130 As neurokinin 1 and MOR coexist in the trigeminal dorsal horn, it has been suggested that they may interact functionally within a signaling complex in these neurons during nociceptive neurotransmission.130 The functional consequences of opioid receptor oligomerization in vivo are largely unknown, unexplored, and matters of controversy. New technological advances in mouse genetics and imaging are crucial in resolving these issues. One major area of continued interest is the in vivo demonstration of opioid receptor homo- and heterodimerization, as well as the development of additional biochemical tools to demonstrate unequivocally that these receptor proteins directly interact with one another.
In addition to receptor–receptor interactions, it is increasingly clear that opioid receptors are highly complex systems and that they interact with a whole host of extracellular, intracellular, and membrane proteins. The notion of opioid receptors existing as dynamic signaling complexes sits at the forefront of the future of opioid-based therapeutics. The reasons for this include the notion that different opioid receptor ligands can induce the formation of a diverse array of receptor complexes. In addition, it is increasingly appreciated that the opioid receptor's native environment (i.e., cell type, neural circuit) greatly affects the receptor's ability to signal, traffic, and function. The idea of a binary GPCR as a simple switch mechanism, from off to on, is becoming widely disregarded as new protein–protein interaction networks and ligand-dependent properties are uncovered.32,33,35,76,95,131
Back to Top | Article Outline
Other Protein–Protein Interactions.
There are multiple lines of evidence pointing to arrestin molecules as crucial proteins that network and engage opioid receptor signal transduction and orchestrate the interaction of proteins within the cellular milieu. The isolation of other opioid-selective protein-interaction networks has been slow, although more studies are examining the many important roles in receptor fate. For one, MOR has been shown to interact with numerous cytoskeletal trafficking proteins, most of which participate in membrane protein endocytosis, including GASP-1, spinophilin, glycoprotein M6A, and tamalin.132134 MOR also has been shown to interact with calmodulin, which is a highly sensitive Ca2+ binding protein implicated in cytoplasmic enzyme activity, including adenylyl cyclases and CAM kinases.135 DORs are similar because they also use GASP-1 and glycoprotein M6A for regulating surface trafficking and endocytosis. KORs have been shown to interact with GEC-1 and EBP50-NHERF proteins, potentially acting to enhance receptor recovery and recycling rates.136,137 Given that ORL1 has not been extensively studied, most of our knowledge about its signaling complex centers around the work of the Zamponi group demonstrating ORL1-Cav2.2 complex formation.63,138 The increasing specificity and affordability of proteomic technologies, such as tandem affinity purification (TAP tag) approaches,139 will help to advance our understanding of opioid receptor complexes. Validating protein–protein interactions in vivo continues to be a challenge, but it is expected that with newer mouse genetic tools, proteomic dissection of opioid receptor complexes in vivo will become an easier task.
Back to Top | Article Outline
Opioid Signaling and Behavior
μ Opioid Receptors.
The most common behavioral function linked to opioid receptors has been their ability to mediate analgesic effects. Numerous reports have examined how opioid signaling causes opioid-induced analgesia (see Bodnar140 and Walwyn et al.3). It is generally accepted that MOR signaling to pertussis toxin-sensitive Gαi is required for morphine antinociception. In addition, in vitro blockade of arrestin 3 expression improves morphine-mediated analgesic responses and acts to prevent morphine tolerance over time.141 Spinal cord expression of Gβγ is required for MOR coupling to analgesic responses and is thought to play a key role in how MORs mediate antinociception.142 This is likely to be through the modulation of potassium and calcium channels in the dorsal root ganglion and dorsal horn. Morphine-induced analgesia and tolerance have been linked to numerous signaling pathways, including interaction with adenylyl cyclases AC1, AC8, and AC5.143,144
μ Opioid receptor-dependent behavioral studies linked to MAPK signaling have begun to become more common in the literature. ERK 1 and 2 phosphorylation has been shown 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 periaqueductal gray region acts as a mechanism to counteract morphine tolerance.146 Recently, reports have implicated ERK 1 and 2 signal transduction in morphine reward and plasticity, including place preference and psychomotor sensitization.147,148 ERK 1 and 2 activity in the amygdala was found to mediate anxiety-like behaviors during morphine withdrawal.149 Together, these reports strongly support the concept that ERK 1 and 2 signaling is an essential mediator of μ opioid-induced plasticity in the brain and spinal cord.
μ Opioid receptors signaling via other protein kinases and protein–protein interactions to modulate reward and analgesia, such as PKC or protein kinase A and JNK, has been demonstrated. For example, PKC 1 (also called RACK1) is required for morphine reward in mouse models, and activation of IRS2-Akt signaling in dopaminergic ventral tegmental neurons is required for the behavioral and cellular actions of μ opioids, including morphine.150 This same group has demonstrated that morphine action on reward requires the activation of transcription factors, including pCREB and DeltaFosB.151 However, we still lack direct information regarding the substrates for these MOR-dependent transcription factors and kinase-signaling pathways shown to be required in behaviors such as analgesia and reward.
Back to Top | Article Outline
δ Opioid Receptors.
Like MOR, DOR signaling research has been focused primarily on mechanisms of opioid analgesia. In addition, DOR research in vivo has been more commonly centered around DOR localization and anatomical characterization, with far fewer studies linking DOR signaling with behavioral effects. Ligand-dependent DOR signaling has been an active area of research, with reports suggesting that ligand-mediated trafficking governs agonist-induced analgesic tolerance to δ opioids.33,152 These studies demonstrated that the DOR agonists SNC80 and ARM390 differ in their ability to cause receptor internalization and down-regulation of DOR-mediated Ca+2 channel modulation. DOR antinociception to thermal stimuli requires phospholipase C, and PKC activation also determines DOR-α2A synergistic effects in the spinal cord.153 DOR agonists have been studied increasingly for their potential antidepressant and anxiolytic effects in rodent behavioral models.154 However, it is not yet known how or where DOR agonists mediate antidepressant-like behavioral responses.
Back to Top | Article Outline
κ Opioid Receptors.
Contemporary studies linking κ opioid receptor signaling and behavior have been centered around the role of κ opioids in stress (Bruchas and Chavkin155 and Knoll and Carlezon156). Stress-induced opioid peptide release has been reported for all of the major opioid systems, and this release causes stress-induced analgesia via action at opioid receptors. In a few crucial reports, it was demonstrated that KOR activation after stress cannot only increase analgesic responses but also can modulate numerous behaviors, including reward and depression.73,157,158
κ Opioid receptor activation of analgesic responses is thought to require Gβγ signal transduction,159 whereas KOR-induced potentiation of reward and dysphoria are thought to be mediated by more complex events, including but not limited to MAPK activation.76 Chartoff et al. showed that the KOR agonist salvA has a biphasic effect on reward. The acute administration of salvA decreased the rewarding impact of intracranial self-stimulation; however, repeated KOR activation caused a net decrease in the reward-potentiating effects of cocaine.75 Both acute and repeated salvA administration increased phosphorylated ERK, but only acute salvA increased c-fos, and only repeated salvA increased cAMP response element-binding protein.75 These findings provide more information about the effects of KOR activation on the reward-related effects of cocaine and will assist in the dissection of the relationship between activation of KOR- and ERK-signaling pathways. KOR-mediated p38 MAPK activity has been shown to be required for conditioned place aversion and swim-stress immobility responses, whereas cAMP response element signaling is critical for prodynorphin gene induction and depression-like behavioral responses.70,160 It is thought that KOR modulation of dopamine, serotonin, and noradrenergic systems plays a key role in producing the negative behavioral affective responses.155,156 Reports include KOR-mediated reductions on dopamine release, along with p38-dependent modulation of serotonergic output.111,159 It was shown recently that KOR-induced p38α MAPK signaling in serotonergic circuitry is required for stress-induced social avoidance, depression-like behaviors, and reinstatement of drug-seeking behavior. This report went on to show that KOR-induced p38α MAPK causes a hyposerotonergic state through increased surface serotonin transporter expression.78 The mechanisms and neural circuits in KOR-mediated dysphoria and analgesia are being studied by several groups; it is hoped those studies will greatly assist in the development of potential antidepressant ligands at KOR and novel analgesics that bypass KOR-mediated dysphoria.161
Back to Top | Article Outline
Opioids and Genetics
The pathogenesis of addiction involves a series of complex interactions among biological factors, including genetic vulnerability and drug-induced alterations in gene expression and proteins. Despite great efforts, the progress in finding causal variants underlying drug addiction has been somewhat slow. Numerous case–control studies have investigated single nucleotide polymorphisms in opioid receptor genes and their correlation with addiction to opioids. However, these studies often have produced conflicting results. The most extensively researched example is a polymorphism in OPRM1 (A118G, rs1799971), which results in the replacement of asparagine with aspartic acid at codon 40. Three studies found an association with the variant 118G and opioid dependency,162164 two studies observed an association with the common allele 118A and opioid dependency,165,166 and nine studies found no overall association with opioid dependency.167175 This polymorphism highlights the conflicting results obtained from genetic studies of opioid receptor genes and drug dependence.
Table 3
Table 3
Image Tools
We have summarized the genetic variants that may contribute to vulnerability to develop opioid addiction (table 3). Genetic testing has important clinical applications in the prevention of many diseases, but in the field of addiction, much work remains to be done to understand the associations between these genetic variants and addiction-related phenotypes.
Within the last decade it was learned that the gene encoding the MOR undergoes extensive alternative splicing, resulting in the generation of multiple versions of this receptor protein. However, correlating these splice variants to pharmacologically defined receptors has proven difficult. The relative contribution to the pharmacologic effect of each splice variant could vary from drug to drug and is dependent on each splice variant's potency and efficacy at a particular site. It has been suggested that the difference in the activation efficacies of various μ opioids for the receptor splice variants may help explain the subtle but clear differences among various μ opioids in the clinic. In addition, understanding the functional significance of some of the truncated receptor splice variants will be beneficial because they have been reported to modulate the activity of opioid receptors in other systems (see Pasternak177,191).
Back to Top | Article Outline

Conclusions

In this review, we discuss a wide array of molecular, cellular, and in vivo studies in opioid receptor pharmacology. We highlight the traditional G protein, βγ signaling pathways, and regulatory mechanisms and discuss recent advances in the subfields of biochemistry, MAPK signal transduction, genetics, and behavior. It is important to note that we have not attempted to discuss all of the fine details regarding the properties of each receptor system.
The most common thread in the reports reviewed is that a large body of our understanding of opioid receptor molecular pharmacology continues to stem from in vitro studies. It is also increasingly clear that most molecular and cellular features of opioid receptors remain disjointed and unconnected to any physiological or behavioral effects, which needs to be the focus of future work in this field.
Many of the most important observations and discoveries surrounding opioid receptors have relied on in vitro approaches, and they continue to be the starting point for most laboratories in molecular pharmacology. However, given the diverse functionality of the opioid receptor family and the variety of signaling pathways and interacting proteins, our knowledge of how opioid receptors function in animal models and, more importantly, human populations or disease is limited.
Opioid receptor signaling has been a primary focus of researchers in this field since its discovery. The major reason for this interest is that it has been widely accepted that a clear understanding of opioid receptor synthesis, cellular localization, trafficking, and pharmacology will lead to novel therapeutics that either directly act on opioid receptors or modulate opioid receptor signaling pathways. With the advent of conditional genetic approaches, receptor tags, antibodies, fluorescent tools, and optogenetic manipulation of neural circuitry, opioid receptor pharmacology is poised for some major breakthroughs in the next decade. It is hopeful that these new molecular and cellular discoveries will lead to better opioid analgesics in the clinic, with decreased risks of addiction and tolerance. In addition, it is likely that studies at the forefront of molecular and behavioral pharmacology will continue to reveal novel uses for opioids in the treatment of a variety of psychiatric and neurologic diseases.
Back to Top | Article Outline

References

1. Dhawan BN, Cesselin F, Raghubir R, Reisine T, Bradley PB, Portoghese PS, Hamon M: International Union of Pharmacology XII: Classification of opioid receptors. Pharmacol Rev 1996; 48:567–92

2. Bruchas MR, Chavkin C: Kinase cascades and ligand-directed signaling at the kappa opioid receptor. Psychopharmacology (Berl) 2010; 210:137–47

3. Walwyn WM, Miotto KA, Evans CJ: Opioid pharmaceuticals and addiction: The issues, and research directions seeking solutions. Drug Alcohol Depend 2010; 108:156–65

4. Ahlbeck K: Opioids: A two-faced Janus. Curr Med Res Opin 2011; 27:439–48

5. McNicol E, Horowicz-Mehler N, Fisk RA, Bennett K, Gialeli-Goudas M, Chew PW, Lau J, Carr D, American Pain Society: Management of opioid side effects in cancer-related and chronic noncancer pain: A systematic review. J Pain 2003; 4:231–56

6. Ballantyne JC, Mao J: Opioid therapy for chronic pain. N Engl J Med 2003; 349:1943–53

7. Childers SR, Snyder SH: Guanine nucleotides differentiate agonist and antagonist interactions with opiate receptors. Life Sci 1978; 23:759–61

8. Childers SR, Creese I, Snowman AM, Synder SH: Opiate receptor binding affected differentially by opiates and opioid peptides. Eur J Pharmacol 1979; 55:11–8

9. Barchfeld CC, Medzihradsky F: Receptor-mediated stimulation of brain GTPase by opiates in normal and dependent rats. Biochem Biophys Res Commun 1984; 121:641–8

10. Minneman KP, Iversen IL: Enkephalin and opiate narcotics increase cyclic GMP accumulation in slices of rat neostriatum. Nature 1976; 262:313–4

11. Hsia JA, Moss J, Hewlett EL, Vaughan M: ADP-ribosylation of adenylate cyclase by pertussis toxin. Effects on inhibitory agonist binding. J Biol Chem 1984; 259:1086–90

12. Taussig R, Iñiguez-Lluhi JA, Gilman AG: Inhibition of adenylyl cyclase by Gi alpha. Science 1993; 261:218–21

13. Wickman K, Clapham DE: Ion channel regulation by G proteins. Physiol Rev 1995; 75:865–85

14. Sadja R, Alagem N, Reuveny E: Gating of GIRK channels: Details of an intricate, membrane-delimited signaling complex. Neuron 2003; 39:9–12

15. Ippolito DL, Temkin PA, Rogalski SL, Chavkin C: N-terminal tyrosine residues within the potassium channel Kir3 modulate GTPase activity of Galphai. J Biol Chem 2002; 277:32692–6

16. Torrecilla M, Quillinan N, Williams JT, Wickman K: Pre- and postsynaptic regulation of locus coeruleus neurons after chronic morphine treatment: A study of GIRK-knockout mice. Eur J Neurosci 2008; 28:618–24

17. Torrecilla M, Marker CL, Cintora SC, Stoffel M, Williams JT, Wickman K: G-protein-gated potassium channels containing Kir3.2 and Kir3.3 subunits mediate the acute inhibitory effects of opioids on locus ceruleus neurons. J Neurosci 2002; 22:4328–34

18. Rusin KI, Giovannucci DR, Stuenkel EL, Moises HC: Kappa-opioid receptor activation modulates Ca2+ currents and secretion in isolated neuroendocrine nerve terminals. J Neurosci 1997; 17:6565–74

19. Zamponi GW, Snutch TP: Modulating modulation: Crosstalk between regulatory pathways of presynaptic calcium channels. Mol Interv 2002; 2:476–8

20. Zamponi GW, Snutch TP: Modulation of voltage-dependent calcium channels by G proteins. Curr Opin Neurobiol 1998; 8:351–6

21. Bourinet E, Soong TW, Stea A, Snutch TP: Determinants of the G protein-dependent opioid modulation of neuronal calcium channels. Proc Natl Acad Sci USA 1996; 93:1486–91

22. Díaz A, Ruíz F, Flórez J, Pazos A, Hurlé MA: Regulation of dihydropyridine-sensitive Ca++ channels during opioid tolerance and supersensitivity in rats. J Pharmacol Exp Ther 1995; 274:1538–44

23. Díaz A, Flórez J, Pazos A, Hurlé MA: Opioid tolerance and supersensitivity induce regional changes in the autoradiographic density of dihydropyridine-sensitive calcium channels in the rat central nervous system. Pain 2000; 86:227–35

24. Harrison RS, Ruiz-Go′mez G, Hill TA, Chow SY, Shepherd NE, Lohman RJ, Abbenante G, Hoang HN, Fairlie DP: Novel helix-constrained nociceptin derivatives are potent agonists and antagonists of ERK phosphorylation and thermal analgesia in mice. J Med Chem 2010; epub ahead of print

25. Dang VC, Christie MJ: Mechanisms of rapid opioid receptor desensitization, resensitization and tolerance in brain neurons. Br J Pharmacol 2011; epub ahead of print

26. Waldhoer M, Bartlett SE, Whistler JL: Opioid receptors. Annu Rev Biochem 2004; 73:953–90

27. Nestler EJ: Under siege: The brain on opiates. Neuron 1996; 16:897–900

28. He L, Fong J, von Zastrow M, Whistler JL: Regulation of opioid receptor trafficking and morphine tolerance by receptor oligomerization. Cell 2002; 108:271–82

29. Whistler JL, Chuang HH, Chu P, Jan LY, von Zastrow M: Functional dissociation of mu opioid receptor signaling and endocytosis: Implications for the biology of opiate tolerance and addiction. Neuron 1999; 23:737–46

30. Bohn LM, Gainetdinov RR, Lin FT, Lefkowitz RJ, Caron MG: Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence. Nature 2000; 408:720–3

31. Groer CE, Schmid CL, Jaeger AM, Bohn LM: Agonist-directed interactions with specific β-arrestins determine μ-opioid receptor trafficking, ubiquitination, and dephosphorylation. J Biol Chem 2011; 286:1731–41

32. Melief EJ, Miyatake M, Carroll FI, Beguin C, Carlezon WA, Cohen BM, Grimwood S, Mitch C, Rorick-Kehn LM, Chavkin C: Duration of action of a broad range of selective kappa opioid receptor antagonists is positively correlated with c-Jun N-Terminal kinase-1 activation. Mol Pharmacol 2011; epub ahead of print

33. Pradhan AA, Walwyn W, Nozaki C, Filliol D, Erbs E, Matifas A, Evans C, Kieffer BL: Ligand-directed trafficking of the δ-opioid receptor in vivo: Two paths toward analgesic tolerance. J Neurosci 2010; 30:16459–68

34. Bailey CP, Connor M: Opioids: Cellular mechanisms of tolerance and physical dependence. Curr Opin Pharmacol 2005; 5:60–8

35. Melief EJ, Miyatake M, Bruchas MR, Chavkin C: Ligand-directed c-Jun N-terminal kinase activation disrupts opioid receptor signaling. Proc Natl Acad Sci USA 2010; 107:11608–13

36. Finn AK, Whistler JL: Endocytosis of the mu opioid receptor reduces tolerance and a cellular hallmark of opiate withdrawal. Neuron 2001; 32:829–39

37. Chavkin C, McLaughlin JP, Celver JP: Regulation of opioid receptor function by chronic agonist exposure: Constitutive activity and desensitization. Mol Pharmacol 2001; 60:20–5

38. El Kouhen R, Kouhen OM, Law PY, Loh HH: The absence of a direct correlation between the loss of [D-Ala2, MePhe4, Gly5-ol]Enkephalin inhibition of adenylyl cyclase activity and agonist-induced mu-opioid receptor phosphorylation. J Biol Chem 1999; 274:9207–15

39. Schulz S, Mayer D, Pfeiffer M, Stumm R, Koch T, Höllt V: Morphine induces terminal micro-opioid receptor desensitization by sustained phosphorylation of serine-375. EMBO J 2004; 23:3282–9

40. Clayton CC, Bruchas MR, Lee ML, Chavkin C: Phosphorylation of the mu-opioid receptor at tyrosine 166 (Tyr3.51) in the DRY motif reduces agonist efficacy. Mol Pharmacol 2010; 77:339–47

41. Lau EK, Trester-Zedlitz M, Trinidad JC, Kotowski SJ, Krutchinsky AN, Burlingame AL, von Zastrow M: Quantitative encoding of the effect of a partial agonist on individual opioid receptors by multisite phosphorylation and threshold detection. Sci Signal 2011; 4:ra52

42. Blake AD, Bot G, Li S, Freeman JC, Reisine T: Differential agonist regulation of the human kappa-opioid receptor. J Neurochem 1997; 68:1846–52

43. Li JG, Zhang F, Jin XL, Liu-Chen LY: Differential regulation of the human kappa opioid receptor by agonists: Etorphine and levorphanol reduced dynorphin A- and U50,488H-induced internalization and phosphorylation. J Pharmacol Exp Ther 2003; 305:531–40

44. Wang Y, Tang K, Inan S, Siebert D, Holzgrabe U, Lee DY, Huang P, Li JG, Cowan A, Liu-Chen LY: Comparison of pharmacological activities of three distinct kappa ligands (Salvinorin A, TRK-820 and 3FLB) on kappa opioid receptors in vitro and their antipruritic and antinociceptive activities in vivo. J Pharmacol Exp Ther 2005; 312:220–30

45. Zhang F, Li J, Li JG, Liu-Chen LY: (-)U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzene acetamide] induces internalization and down-regulation of the human, but not the rat, kappa-opioid receptor: Structural basis for the differential regulation. J Pharmacol Exp Ther 2002;302:1184–92

46. McLaughlin JP, Xu M, Mackie K, Chavkin C: Phosphorylation of a carboxyl-terminal serine within the kappa-opioid receptor produces desensitization and internalization. J Biol Chem 2003; 278:34631–40

47. McLaughlin JP, Myers LC, Zarek PE, Caron MG, Lefkowitz RJ, Czyzyk TA, Pintar JE, Chavkin C: Prolonged kappa opioid receptor phosphorylation mediated by G-protein receptor kinase underlies sustained analgesic tolerance. J Biol Chem 2004; 279:1810–8

48. Bruchas MR, Macey TA, Lowe JD, Chavkin C: Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes. J Biol Chem 2006; 281:18081–9

49. Cahill CM, McClellan KA, Morinville A, Hoffert C, Hubatsch D, O'Donnell D, Beaudet A: Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: Evidence for somatodendritic labeling and antigen-specific cellular compartmentalization. J Comp Neurol 2001; 440:65–84

50. Wang H, Pickel VM: Preferential cytoplasmic localization of delta-opioid receptors in rat striatal patches: Comparison with plasmalemmal mu-opioid receptors. J Neurosci 2001; 21:3242–50

51. Gendron L, Lucido AL, Mennicken F, O'Donnell D, Vincent JP, Stroh T, Beaudet A: Morphine and pain-related stimuli enhance cell surface availability of somatic delta-opioid receptors in rat dorsal root ganglia. J Neurosci 2006; 26:953–62

52. Scherrer G, Imamachi N, Cao YQ, Contet C, Mennicken F, O'Donnell D, Kieffer BL, Basbaum AI: Dissociation of the opioid receptor mechanisms that control mechanical and heat pain. Cell 2009; 137:1148–59

53. Hasbi A, Polastron J, Allouche S, Stanasila L, Massotte D, Jauzac P: Desensitization of the delta-opioid receptor correlates with its phosphorylation in SK-N-BE cells: Involvement of a G protein-coupled receptor kinase. J Neurochem 1998; 70:2129–38

54. Law PY, Kouhen OM, Solberg J, Wang W, Erickson LJ, Loh HH: Deltorphin II-induced rapid desensitization of delta-opioid receptor requires both phosphorylation and internalization of the receptor. J Biol Chem 2000; 275:32057–65

55. Bradbury FA, Zelnik JC, Traynor JR: G protein independent phosphorylation and internalization of the delta-opioid receptor. J Neurochem 2009; 109:1526–35

56. Guo J, Wu Y, Zhang W, Zhao J, Devi LA, Pei G, Ma L: Identification of G protein-coupled receptor kinase 2 phosphorylation sites responsible for agonist-stimulated delta-opioid receptor phosphorylation. Mol Pharmacol 2000; 58:1050–6

57. Pei G, Kieffer BL, Lefkowitz RJ, Freedman NJ: Agonist-dependent phosphorylation of the mouse delta-opioid receptor: Involvement of G protein-coupled receptor kinases but not protein kinase C. Mol Pharmacol 1995; 48:173–7

58. Cvejic S, Trapaidze N, Cyr C, Devi LA: Thr353, located within the COOH-terminal tail of the delta opiate receptor, is involved in receptor down-regulation. J Biol Chem 1996; 271:4073–6

59. Trapaidze N, Keith DE, Cvejic S, Evans CJ, Devi LA: Sequestration of the delta opioid receptor: Role of the C terminus in agonist-mediated internalization. J Biol Chem 1996; 271:29279–85

60. Varga EV, Navratilova E, Stropova D, Jambrosic J, Roeske WR, Yamamura HI: Agonist-specific regulation of the delta-opioid receptor. Life Sci 2004; 76:599–612

61. Spampinato S, Baiula M, Calienni M: Agonist-regulated internalization and desensitization of the human nociceptin receptor expressed in CHO cells. Curr Drug Targets 2007; 8:137–46

62. Spampinato S, Di Toro R, Qasem AR: Nociceptin-induced internalization of the ORL1 receptor in human neuroblastoma cells. Neuroreport 2001; 12:3159–63

63. Altier C, Khosravani H, Evans RM, Hameed S, Peloquin JB, Vartian BA, Chen L, Beedle AM, Ferguson SS, Mezghrani A, Dubel SJ, Bourinet E, McRory JE, Zamponi GW: ORL1 receptor-mediated internalization of N-type calcium channels. Nat Neurosci 2006; 9:31–40

64. Bohn LM, Lefkowitz RJ, Gainetdinov RR, Peppel K, Caron MG, Lin FT: Enhanced morphine analgesia in mice lacking beta-arrestin 2. Science 1999; 286:2495–8

65. Lowe JD, Celver JP, Gurevich VV, Chavkin C: mu-Opioid receptors desensitize less rapidly than delta-opioid receptors due to less efficient activation of arrestin. J Biol Chem 2002; 277:15729–35

66. Lefkowitz RJ, Shenoy SK: Transduction of receptor signals by beta-arrestins. Science 2005; 308:512–7

67. Macey TA, Lowe JD, Chavkin C: Mu opioid receptor activation of ERK1/2 is GRK3 and arrestin dependent in striatal neurons. J Biol Chem 2006; 281:34515–24

68. Miyatake M, Rubinstein TJ, McLennan GP, Belcheva MM, Coscia CJ: Inhibition of EGF-induced ERK/MAP kinase-mediated astrocyte proliferation by mu opioids: Integration of G protein and beta-arrestin 2-dependent pathways. J Neurochem 2009; 110:662–74

69. Ikeda H, Miyatake M, Koshikawa N, Ochiai K, Yamada K, Kiss A, Donlin MJ, Panneton WM, Churchill JD, Green M, Siddiqui AM, Leinweber AL, Crews NR, Ezerskiy LA, Rendell VR, Belcheva MM, Coscia CJ: Morphine modulation of thrombospondin levels in astrocytes and its implications for neurite outgrowth and synapse formation. J Biol Chem 2010; 285:38415–27

70. Tan M, Walwyn WM, Evans CJ, Xie CW: p38 MAPK and beta-arrestin 2 mediate functional interactions between endogenous micro-opioid and alpha2A-adrenergic receptors in neurons. J Biol Chem 2009; 284:6270–81

71. Goldsmith JR, Uronis JM, Jobin C: Mu opioid signaling protects against acute murine intestinal injury in a manner involving Stat3 signaling. Am J Pathol 2011; 179:673–83

72. Belcheva MM, Clark AL, Haas PD, Serna JS, Hahn JW, Kiss A, Coscia CJ: Mu and kappa opioid receptors activate ERK/MAPK via different protein kinase C isoforms and secondary messengers in astrocytes. J Biol Chem 2005; 280:27662–9

73. McLennan GP, Kiss A, Miyatake M, Belcheva MM, Chambers KT, Pozek JJ, Mohabbat Y, Moyer RA, Bohn LM, Coscia CJ: Kappa opioids promote the proliferation of astrocytes via Gbetagamma and beta-arrestin 2-dependent MAPK-mediated pathways. J Neurochem 2008; 107:1753–65

74. Bruchas MR, Xu M, Chavkin C: Repeated swim stress induces kappa opioid-mediated activation of extracellular signal-regulated kinase 1/2. Neuroreport 2008; 19:1417–22
75. Potter DN, Damez-Werno D, Carlezon WA Jr, Cohen BM, Chartoff EH: Repeated exposure to the κ-opioid receptor agonist salvinorin A modulates extracellular signal-regulated kinase and reward sensitivity. Biol Psychiatry 2011; 70:744–53

76. Bruchas MR, Land BB, Aita M, Xu M, Barot SK, Li S, Chavkin C: Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria. J Neurosci 2007; 27:11614–23

77. Xu M, Bruchas MR, Ippolito DL, Gendron L, Chavkin C: Sciatic nerve ligation-induced proliferation of spinal cord astrocytes is mediated by kappa opioid activation of p38 mitogen-activated protein kinase. J Neurosci 2007; 27:2570–81

78. Bruchas MR, Schindler AG, Shankar H, Messinger DI, Miyatake M, Land BB, Lemos JC, Hagan CE, Neumaier JF, Quintana A, Palmiter RD, Chavkin C: Selective p38α MAPK deletion in serotonergic neurons produces stress resilience in models of depression and addiction. Neuron 2011; 71:498–511

79. Finley MJ, Steele A, Cornwell WD, Rogers TJ: Transcriptional regulation of the major HIV-1 coreceptor, CXCR4, by the kappa opioid receptor. J Leukoc Biol 2011; 90:111–21

80. Eisinger DA, Ammer H: Down-regulation of c-Cbl by morphine accounts for persistent ERK1/2 signaling in δ-opioid receptor-expressing HEK293 cells. J Biol Chem 2009; 284:34819–28

81. Eisinger DA, Schulz R: Extracellular signal-regulated kinase/mitogen-activated protein kinases block internalization of delta-opioid receptors. J Pharmacol Exp Ther 2004; 309:776–85

82. Audet N, Paquin-Gobeil M, Landry-Paquet O, Schiller PW, Piñeyro G: Internalization and Src activity regulate the time course of ERK activation by delta opioid receptor ligands. J Biol Chem 2005; 280:7808–16

83. Eisinger DA, Ammer H: Delta-opioid receptors activate ERK/MAP kinase via integrin-stimulated receptor tyrosine kinases. Cell Signal 2008; 20:2324–31

84. Olianas MC, Dedoni S, Onali P: Regulation of PI3K/Akt signaling by N-desmethylclozapine through activation of δ-opioid receptor. Eur J Pharmacol 2011; 660:341–50
85. Heiss A, Ammer H, Eisinger DA: delta-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108–15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation. Exp Cell Res 2009; 315:2115–25
86. Olianas MC, Dedoni S, Onali P: Signaling pathways mediating phosphorylation and inactivation of glycogen synthase kinase-3β by the recombinant human δ-opioid receptor stably expressed in Chinese hamster ovary cells. Neuropharmacology 2011; 60:1326–36

87. Olianas MC, Dedoni S, Onali P: δ-Opioid receptors stimulate GLUT1-mediated glucose uptake through Src- and IGF-1 receptor-dependent activation of PI3-kinase signalling in CHO cells. Br J Pharmacol 2011; 163:624–37
88. Shahabi NA, McAllen K, Sharp BM: Delta opioid receptors stimulate Akt-dependent phosphorylation of c-jun in T cells. J Pharmacol Exp Ther 2006; 316:933–9

89. Chen LY, Huang JX, Yu LC: Involvement of ORL1 receptor and ERK kinase in the orphanin FQ-induced nociception in the nucleus accumbens of rats. Regulatory Peptides 2008; 151:43–7

90. Ross J, Armstead WM: NOC/oFQ activates ERK and JNK but not p38 MAPK to impair prostaglandin cerebrovasodilation after brain injury. Brain Res 2005; 1054:95–102

91. Zhang Z, Xin SM, Wu GX, Zhang WB, Ma L, Pei G: Endogenous delta-opioid and ORL1 receptors couple to phosphorylation and activation of p38 MAPK in NG108-15 cells and this is regulated by protein kinase A and protein kinase C. J Neurochem 1999; 73:1502–9

92. Chan AS, Wong YH: Regulation of c-Jun N-terminal kinase by the ORL(1) receptor through multiple G proteins. J Pharmacol Exp Ther 2000; 295:1094–100

93. Raman M, Chen W, Cobb MH: Differential regulation and properties of MAPKs. Oncogene 2007; 26:3100–12

94. Karandikar M, Cobb MH: Scaffolding and protein interactions in MAP kinase modules. Cell Calcium 1999; 26:219–26

95. Pierce KL, Lefkowitz RJ: Classical and new roles of beta-arrestins in the regulation of G-protein-coupled receptors. Nat Rev Neurosci 2001; 2:727–33

96. Belcheva MM, Vogel Z, Ignatova E, Avidor-Reiss T, Zippel R, Levy R, Young EC, Barg J, Coscia CJ: Opioid modulation of extracellular signal-regulated protein kinase activity is ras-dependent and involves Gbetagamma subunits. J Neurochem 1998; 70:635–45

97. Groer CE, Tidgewell K, Moyer RA, Harding WW, Rothman RB, Prisinzano TE, Bohn LM: An opioid agonist that does not induce mu-opioid receptor–arrestin interactions or receptor internalization. Mol Pharmacol 2007; 71:549–57

98. Kim E, Clark AL, Kiss A, Hahn JW, Wesselschmidt R, Coscia CJ, Belcheva MM: Mu- and kappa-opioids induce the differentiation of embryonic stem cells to neural progenitors. J Biol Chem 2006; 281:33749–60

99. Hahn JW, Jagwani S, Kim E, Rendell VR, He J, Ezerskiy LA, Wesselschmidt R, Coscia CJ, Belcheva MM: Mu and kappa opioids modulate mouse embryonic stem cell-derived neural progenitor differentiation via MAP kinases. J Neurochem 2010; 112:1431–41

100. Kramer HK, Simon EJ: Mu and delta-opioid receptor agonists induce mitogen-activated protein kinase (MAPK) activation in the absence of receptor internalization. Neuropharmacology 2000; 39:1707–19

101. Kramer HK, Onoprishvili I, Andria ML, Hanna K, Sheinkman K, Haddad LB, Simon EJ: Delta opioid activation of the mitogen-activated protein kinase cascade does not require transphosphorylation of receptor tyrosine kinases. BMC Pharmacol 2002; 2:5

102. Gesty-Palmer D, Chen M, Reiter E, Ahn S, Nelson CD, Wang S, Eckhardt AE, Cowan CL, Spurney RF, Luttrell LM, Lefkowitz RJ: Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation. J Biol Chem 2006; 281:10856–64

103. Ji RR, Kawasaki Y, Zhuang ZY, Wen YR, Zhang YQ: Protein kinases as potential targets for the treatment of pathological pain. Handb Exp Pharmacol 2007; (177):359–89

104. Minden A, Karin M: Regulation and function of the JNK subgroup of MAP kinases. Biochim Biophys Acta 1997; 1333:F85–104

105. Baker SJ, Reddy EP: Modulation of life and death by the TNF receptor superfamily. Oncogene 1998; 17:3261–70

106. Nobes C, Hall A: Regulation and function of the Rho subfamily of small GTPases. Curr Opin Genet Dev 1994; 4:77–81

107. Song X, Coffa S, Fu H, Gurevich VV: How does arrestin assemble MAPKs into a signaling complex? J Biol Chem 2009; 284:685–95

108. Kam AY, Chan AS, Wong YH: Phosphatidylinositol-3 kinase is distinctively required for mu-, but not kappa-opioid receptor-induced activation of c-Jun N-terminal kinase. J Neurochem 2004; 89:391–402

109. Bruchas MR, Yang T, Schreiber S, Defino M, Kwan SC, Li S, Chavkin C: Long-acting kappa opioid antagonists disrupt receptor signaling and produce noncompetitive effects by activating c-Jun N-terminal kinase. J Biol Chem 2007; 282:29803–11

110. Tibbles LA, Woodgett JR: The stress-activated protein kinase pathways. Cell Mol Life Sci 1999; 55:1230–154

111. Watkins LR, Milligan ED, Maier SF: Glial activation: A driving force for pathological pain. Trends Neurosci 2001; 24:450–5

112. Clayton CC, Xu M, Chavkin C: Tyrosine phosphorylation of Kir3 following κ-opioid receptor activation of p38 MAPK causes heterologous desensitization. J Biol Chem 2009; 284:31872–81

113. Rios CD, Jordan BA, Gomes I, Devi LA: G-protein-coupled receptor dimerization: Modulation of receptor function. Pharmacol Ther 2001; 92:71–87

114. Prinster SC, Hague C, Hall RA: Heterodimerization of g protein-coupled receptors: Specificity and functional significance. Pharmacol Rev 2005; 57:289–98

115. Jordan BA, Devi LA: G-protein-coupled receptor heterodimerization modulates receptor function. Nature 1999; 399:697–700

116. Cvejic S, Devi LA: Dimerization of the delta opioid receptor: Implication for a role in receptor internalization. J Biol Chem 1997; 272:26959–64

117. Waldhoer M, Fong J, Jones RM, Lunzer MM, Sharma SK, Kostenis E, Portoghese PS, Whistler JL: A heterodimer-selective agonist shows in vivo relevance of G protein-coupled receptor dimers. Proc Natl Acad Sci USA 2005; 102:9050–5

118. Pan YX, Bolan E, Pasternak GW: Dimerization of morphine and orphanin FQ/nociceptin receptors: Generation of a novel opioid receptor subtype. Biochem Biophys Res Commun 2002; 297:659–63

119. Wang D, Sun X, Bohn LM, Sadée W: Opioid receptor homo- and heterodimerization in living cells by quantitative bioluminescence resonance energy transfer. Mol Pharmacol 2005; 67:2173–84

120. Gomes I, Jordan BA, Gupta A, Trapaidze N, Nagy V, Devi LA: Heterodimerization of mu and delta opioid receptors: A role in opiate synergy. J Neurosci 2000; 20:RC110

121. Décaillot FM, Rozenfeld R, Gupta A, Devi LA: Cell surface targeting of mu-delta opioid receptor heterodimers by RTP4. Proc Natl Acad Sci USA 2008; 105:16045–50

122. Psifogeorgou K, Psigfogeorgou K, Terzi D, Papachatzaki MM, Varidaki A, Ferguson D, Gold SJ, Zachariou V: A unique role of RGS9–2 in the striatum as a positive or negative regulator of opiate analgesia. J Neurosci 2011; 31:5617–24

123. Rios C, Gomes I, Devi LA: Mu opioid and CB1 cannabinoid receptor interactions: Reciprocal inhibition of receptor signaling and neuritogenesis. Br J Pharmacol 2006; 148:387–95

124. Hojo M, Sudo Y, Ando Y, Minami K, Takada M, Matsubara T, Kanaide M, Taniyama K, Sumikawa K, Uezono Y: mu-Opioid receptor forms a functional heterodimer with cannabinoid CB1 receptor: Electrophysiological and FRET assay analysis. J Pharmacol Sci 2008; 108:308–19

125. Cichewicz DL: Synergistic interactions between cannabinoid and opioid analgesics. Life Sci 2004; 74:1317–24

126. Ossipov MH, Lopez Y, Bian D, Nichols ML, Porreca F: Synergistic antinociceptive interactions of morphine and clonidine in rats with nerve-ligation injury. ANESTHESIOLOGY 1997; 86:196–204

127. Jordan BA, Gomes I, Rios C, Filipovska J, Devi LA: Functional interactions between mu opioid and alpha 2A-adrenergic receptors. Mol Pharmacol 2003; 64:1317–24

128. Drasner K, Fields HL: Synergy between the antinociceptive effects of intrathecal clonidine and systemic morphine in the rat. Pain 1988; 32:309–12

129. Pfeiffer M, Koch T, Schröder H, Laugsch M, Höllt V, Schulz S: Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and desensitization. J Biol Chem 2002; 277:19762–72

130. Pfeiffer M, Kirscht S, Stumm R, Koch T, Wu D, Laugsch M, Schröder H, Höllt V, Schulz S: Heterodimerization of substance P and mu-opioid receptors regulates receptor trafficking and resensitization. J Biol Chem 2003; 278:51630–7

131. Kenakin TP: Seven transmembrane receptors as nature's prototype allosteric protein: De-emphasizing the geography of binding. Mol Pharmacol 2008; 74:541–3

132. Milligan G: Opioid receptors and their interacting proteins. Neuromolecular Med 2005; 7:51–9

133. Whistler JL, Enquist J, Marley A, Fong J, Gladher F, Tsuruda P, Murray SR, Von Zastrow M: Modulation of postendocytic sorting of G protein-coupled receptors. Science 2002; 297:615–20

134. Charlton JJ, Allen PB, Psifogeorgou K, Chakravarty S, Gomes I, Neve RL, Devi LA, Greengard P, Nestler EJ, Zachariou V: Multiple actions of spinophilin regulate mu opioid receptor function. Neuron 2008; 58:238–47

135. Wang D, Quillan JM, Winans K, Lucas JL, Sadée W: Single nucleotide polymorphisms in the human mu opioid receptor gene alter basal G protein coupling and calmodulin binding. J Biol Chem 2001; 276:34624–30

136. Chen C, Wang Y, Huang P, Liu-Chen LY: Effects of C-terminal modifications of GEC1 protein and gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein (GABARAP), two microtubule-associated proteins, on kappa opioid receptor expression. J Biol Chem 2011; 286:15106–15

137. Huang P, Steplock D, Weinman EJ, Hall RA, Ding Z, Li J, Wang Y, Liu-Chen LY: kappa Opioid receptor interacts with Na(+)/H(+)-exchanger regulatory factor-1/Ezrin-radixin-moesin-binding phosphoprotein-50 (NHERF-1/EBP50) to stimulate Na(+)/H(+) exchange independent of G(i)/G(o) proteins. J Biol Chem 2004; 279:25002–9

138. Evans RM, You H, Hameed S, Altier C, Mezghrani A, Bourinet E, Zamponi GW: Heterodimerization of ORL1 and opioid receptors and its consequences for N-type calcium channel regulation. J Biol Chem 2010; 285:1032–40

139. Lyssand JS, Whiting JL, Lee KS, Kastl R, Wacker JL, Bruchas MR, Miyatake M, Langeberg LK, Chavkin C, Scott JD, Gardner RG, Adams ME, Hague C: Alpha-dystrobrevin-1 recruits alpha-catulin to the alpha1D-adrenergic receptor/dystrophin-associated protein complex signalosome. Proc Natl Acad Sci USA 2010; 107:21854–9

140. Bodnar RJ: Endogenous opiates and behavior: 2009. Peptides 2010; 31:2325–59

141. Raehal KM, Bohn LM: The role of beta-arrestin2 in the severity of antinociceptive tolerance and physical dependence induced by different opioid pain therapeutics. Neuropharmacology 2011; 60:58–65

142. Mathews JL, Smrcka AV, Bidlack JM: A novel Gbetagamma-subunit inhibitor selectively modulates mu-opioid-dependent antinociception and attenuates acute morphine-induced antinociceptive tolerance and dependence. J Neurosci 2008; 28:12183–9

143. Li S, Lee ML, Bruchas MR, Chan GC, Storm DR, Chavkin C: Calmodulin-stimulated adenylyl cyclase gene deletion affects morphine responses. Mol Pharmacol 2006; 70:1742–9

144. Kim KS, Lee KW, Lee KW, Im JY, Yoo JY, Kim SW, Lee JK, Nestler EJ, Han PL: Adenylyl cyclase type 5 (AC5) is an essential mediator of morphine action. Proc Natl Acad Sci USA 2006; 103:3908–13

145. Asensio VJ, Miralles A, García-Sevilla JA: Stimulation of mitogen-activated protein kinase kinases (MEK1/2) by mu-, delta- and kappa-opioid receptor agonists in the rat brain: Regulation by chronic morphine and opioid withdrawal. Eur J Pharmacol 2006; 539:49–56

146. Macey TA, Bobeck EN, Hegarty DM, Aicher SA, Ingram SL, Morgan MM: Extracellular signal-regulated kinase 1/2 activation counteracts morphine tolerance in the periaqueductal gray of the rat. J Pharmacol Exp Ther 2009; 331:412–8

147. Ramos-Miguel A, Esteban S, García-Sevilla JA: The time course of unconditioned morphine-induced psychomotor sensitization mirrors the phosphorylation of FADD and MEK/ERK in rat striatum: Role of PEA-15 as a FADD-ERK binding partner in striatal plasticity. Eur Neuropsychopharmacol 2010; 20:49–64

148. Lin X, Wang Q, Ji J, Yu LC: Role of MEK-ERK pathway in morphine-induced conditioned place preference in ventral tegmental area of rats. J Neurosci Res 2010; 88:1595–604

149. Hofford RS, Hodgson SR, Roberts KW, Bryant CD, Evans CJ, Eitan S: Extracellular signal-regulated kinase activation in the amygdala mediates elevated plus maze behavior during opioid withdrawal. Behav Pharmacol 2009; 20:576–83

150. Russo SJ, Bolanos CA, Theobald DE, DeCarolis NA, Renthal W, Kumar A, Winstanley CA, Renthal NE, Wiley MD, Self DW, Russell DS, Neve RL, Eisch AJ, Nestler EJ: IRS2-Akt pathway in midbrain dopamine neurons regulates behavioral and cellular responses to opiates. Nat Neurosci 2007; 10:93–9

151. Zachariou V, Bolanos CA, Selley DE, Theobald D, Cassidy MP, Kelz MB, Shaw-Lutchman T, Berton O, Sim-Selley LJ, Dileone RJ, Kumar A, Nestler EJ: An essential role for DeltaFosB in the nucleus accumbens in morphine action. Nat Neurosci 2006; 9:205–11

152. Pradhan AAA, Becker JAJ, Scherrer G, Tryoen-Toth P, Fillioi D, Matifas A, Massotte D, Gavériaux-Ruff C, Kieffer BL: In vivo delta opioid receptor internalization controls behavioral effects of agonists. PLoS One 2009; 4:e5425

153. Overland AC, Kitto KF, Chabot-Doré AJ, Rothwell PE, Fairbanks CA, Stone LS, Wilcox GL: Protein kinase C mediates the synergistic interaction between agonists acting at alpha2-adrenergic and delta-opioid receptors in spinal cord. J Neurosci. 2009; 29:13264–73

154. Jutkiewicz EM: RB101-mediated protection of endogenous opioids: Potential therapeutic utility? CNS Drug Rev 2007; 13:192–205

155. Bruchas MR, Land BB, Chavkin C: The dynorphin/kappa opioid system as a modulator of stress-induced and pro-addictive behaviors. Brain Res 2010; 1314:44–55

156. Knoll AT, Carlezon WA Jr: Dynorphin, stress, and depression. Brain Res 2010; 1314:56–73

157. Land BB, Bruchas MR, Lemos JC, Xu M, Melief EJ, Chavkin C: The dysphoric component of stress is encoded by activation of the dynorphin kappa-opioid system. J Neurosci 2008; 28:407–14

158. Carlezon WA Jr, Béguin C, DiNieri JA, Baumann MH, Richards MR, Todtenkopf MS, Rothman RB, Ma Z, Lee DY, Cohen BM: Depressive-like effects of the kappa-opioid receptor agonist salvinorin A on behavior and neurochemistry in rats. J Pharmacol Exp Ther 2006; 316:440–7

159. Land BB, Bruchas MR, Schattauer S, Giardino WJ, Aita M, Messinger D, Hnasko TS, Palmiter RD, Chavkin C: Activation of the kappa opioid receptor in the dorsal raphe nucleus mediates the aversive effects of stress and reinstates drug seeking. Proc Natl Acad Sci USA 2009; 106:19168–73

160. Pliakas AM, Carlson RR, Neve RL, Konradi C, Nestler EJ, Carlezon WA Jr: Altered responsiveness to cocaine and increased immobility in the forced swim test associated with elevated cAMP response element-binding protein expression in nucleus accumbens. J Neurosci 2001; 21:7397–403

161. Muschamp JW, Van't Veer A, Carlezon WA Jr: Tracking down the molecular substrates of stress: New roles for p38α MAPK and kappa-opioid receptors. Neuron 2011; 71:383–5

162. Kapur S, Sharad S, Singh RA, Gupta AK: A118g polymorphism in mu opioid receptor gene (oprm1): Association with opiate addiction in subjects of Indian origin. J Integr Neurosci 2007; 6:511–22

163. Bart G, Heilig M, LaForge KS, Pollak L, Leal SM, Ott J, Kreek MJ: Substantial attributable risk related to a functional mu-opioid receptor gene polymorphism in association with heroin addiction in central Sweden. Mol Psychiatry 2004; 9:547–9

164. Szeto CY, Tang NL, Lee DT, Stadlin A: Association between mu opioid receptor gene polymorphisms and Chinese heroin addicts. Neuroreport 2001; 12:1103–6

165. Tan EC, Tan CH, Karupathivan U, Yap EP: Mu opioid receptor gene polymorphisms and heroin dependence in Asian populations. Neuroreport 2003; 14:569–72

166. Schinka JA, Town T, Abdullah L, Crawford FC, Ordorica PI, Francis E, Hughes P, Graves AB, Mortimer JA, Mullan M: A functional polymorphism within the mu-opioid receptor gene and risk for abuse of alcohol and other substances. Mol Psychiatry 2002; 7:224–8

167. Nikolov MA, Beltcheva O, Galabova A, Ljubenova A, Jankova E, Gergov G, Russev AA, Lynskey MT, Nelson EC, Nesheva E, Krasteva D, Lazarov P, Mitev VI, Kremensky IM, Kaneva RP, Todorov AA: No evidence of association between 118A>G OPRM1 polymorphism and heroin dependence in a large Bulgarian case-control sample. Drug Alcohol Depend 2011; 117:62–5

168. Levran O, Londono D, O'Hara K, Nielsen DA, Peles E, Rotrosen J, Casadonte P, Linzy S, Randesi M, Ott J, Adelson M, Kreek MJ: Genetic susceptibility to heroin addiction: A candidate gene association study. Genes Brain Behav 2008; 7:720–9

169. Gelernter J, Kranzler H, Cubells J: Genetics of two mu opioid receptor gene (OPRM1) exon I polymorphisms: Population studies, and allele frequencies in alcohol- and drug-dependent subjects. Mol Psychiatry 1999; 4:476–83

170. Franke P, Wang T, Nöthen MM, Knapp M, Neidt H, Albrecht S, Jahnes E, Propping P, Maier W: Nonreplication of association between mu-opioid-receptor gene (OPRM1) A118G polymorphism and substance dependence. Am J Med Genet 2001; 105:114–9

171. Shi J, Hui L, Xu Y, Wang F, Huang W, Hu G: Sequence variations in the mu-opioid receptor gene (OPRM1) associated with human addiction to heroin. Hum Mutat 2002; 19:459–60

172. Crowley JJ, Oslin DW, Patkar AA, Gottheil E, DeMaria PA Jr, O'Brien CP, Berrettini WH, Grice DE: A genetic association study of the mu opioid receptor and severe opioid dependence. Psychiatr Genet 2003; 13:169–73

173. Franke P, Wendel B, Knapp M, Schwab SG, Neef D, Maier W, Wildenauer DB, Hoehe MR: Introducing a new recruitment approach to sample collection for genetic association studies in opioid dependence. Eur Psychiatry 2003; 18:18–22

174. Compton P, Geschwind DH, Alarcón M: Association between human mu-opioid receptor gene polymorphism, pain tolerance, and opioid addiction. Am J Med Genet B Neuropsychiatr Genet 2003; 121B:76–82

175. Bond C, LaForge KS, Tian M, Melia D, Zhang S, Borg L, Gong J, Schluger J, Strong JA, Leal SM, Tischfield JA, Kreek MJ, Yu L: Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: Possible implications for opiate addiction. Proc Natl Acad Sci USA 1998; 95:9608–13

176. Ducat E, Ray B, Bart G, Umemura Y, Varon J, Ho A, Kreek MJ. Mu-opioid receptor A118G polymorphism in healthy volunteers affects hypothalamic-pituitary-adrenal axis adrenocorticotropic hormone stress response to metyrapone. Addict Biol 2011: epub ahead of print
177. Pasternak GW: Insights into mu opioid pharmacology the role of mu opioid receptor subtypes. Life Sci 2001; 68:2213–9

178. Oertel BG, Kettner M, Scholich K, Renné C, Roskam B, Geisslinger G, Schmidt PH, Lötsch J: A common human micro-opioid receptor genetic variant diminishes the receptor signaling efficacy in brain regions processing the sensory information of pain. J Biol Chem 2009; 284:6530–5

179. Zhang Y, Wang D, Johnson AD, Papp AC, Sadée W: Allelic expression imbalance of human mu opioid receptor (OPRM1) caused by variant A118G. J Biol Chem 2005; 280:32618–24

180. Deb I, Chakraborty J, Gangopadhyay PK, Choudhury SR, Das S: Single-nucleotide polymorphism (A118G) in exon 1 of OPRM1 gene causes alteration in downstream signaling by mu-opioid receptor and may contribute to the genetic risk for addiction. J Neurochem 2010; 112:486–96

181. Berretini WH, Hoehe MR, Ferraro TN, Demaria PA, Gottheil E: Human mu opioid receptor gene polymorphisms and vulnerability to substance abuse. Addict Biol 1997; 2:303–8

182. Crystal HA, Hamon S, Randesi M, Cook J, Anastos K, Lazar J, Liu C, Pearce L, Golub E, Valcour V, Weber KM, Holman S, Ho A, Kreek MJ: A C17T polymorphism in the mu opiate receptor is associated with quantitative measures of drug use in African American women. Addict Biol 2010; epub ahead of print
183. Zhang H, Gelernter J, Gruen JR, Kranzler HR, Herman AI, Simen AA: Functional impact of a single-nucleotide polymorphism in the OPRD1 promoter region. J Hum Genet 2010; 55:278–84

184. Tuusa JT, Petäjä-Repo UE: Phe27Cys polymorphism of the human delta opioid receptor predisposes cells to compromised calcium signaling. Mol Cell Biochem 2011; 351:173–81

185. Leskelä TT, Markkanen PM, Alahuhta IA, Tuusa JT, Petäjä-Repo UE: Phe27Cys polymorphism alters the maturation and subcellular localization of the human delta opioid receptor. Traffic 2009; 10:116–29

186. Zhang H, Kranzler HR, Yang BZ, Luo X, Gelernter J: The OPRD1 and OPRK1 loci in alcohol or drug dependence: OPRD1 variation modulates substance dependence risk. Mol Psychiatry 2008; 13:531–43

187. Gerra G, Leonardi C, Cortese E, D'Amore A, Lucchini A, Strepparola G, Serio G, Farina G, Magnelli F, Zaimovic A, Mancini A, Turci M, Manfredini M, Donnini C: Human kappa opioid receptor gene (OPRK1) polymorphism is associated with opiate addiction. Am J Med Genet B Neuropsychiatr Genet 2007; 144B:771–5

188. Yuferov V, Fussell D, LaForge KS, Nielsen DA, Gordon D, Ho A, Leal SM, Ott J, Kreek MJ: Redefinition of the human kappa opioid receptor gene (OPRK1) structure and association of haplotypes with opiate addiction. Pharmacogenetics 2004; 14:793–804

189. Biocca S, Falconi M, Filesi I, Baldini F, Vecchione L, Mango R, Romeo F, Federici G, Desideri A, Novelli G: Functional analysis and molecular dynamics simulation of LOX-1 K167N polymorphism reveal alteration of receptor activity. PLoS One 2009; 4:e4648

190. Xuei X, Flury-Wetherill L, Almasy L, Bierut L, Tischfield J, Schuckit M, Nurnberger JI Jr, Foroud T, Edenberg HJ: Association analysis of genes encoding the nociceptin receptor (OPRL1) and its endogenous ligand (PNOC) with alcohol or illicit drug dependence. Addict Biol 2008; 13:80–7

191. Pasternak GW: Multiple opiate receptors: Déjà vu all over again. Neuropharmacology 2004; 47:312–23

Cited By:

This article has been cited 9 time(s).

Expert Opinion on Drug Metabolism & Toxicology
Metabolic and toxicological considerations of the opioid replacement therapy and analgesic drugs: methadone and buprenorphine
Moody, DE
Expert Opinion on Drug Metabolism & Toxicology, 9(6): 675-697.
10.1517/17425255.2013.783567
CrossRef
Neuropharmacology
Potentiation of morphine-induced mechanical antinociception by sigma(1) receptor inhibition: Role of peripheral sigma(1) receptors
Sanchez-Fernandez, C; Nieto, FR; Gonzalez-Cano, R; Artacho-Cordon, A; Romero, L; Montilla-Garcia, A; Zamanillo, D; Baeyens, JM; Entrena, JM; Cobos, EJ
Neuropharmacology, 70(): 348-358.
10.1016/j.neuropharm.2013.03.002
CrossRef
Journal of Biological Chemistry
Serine 363 Is Required for Nociceptin/Orphanin FQ Opioid Receptor (NOPR) Desensitization, Internalization, and Arrestin Signaling
Zhang, NR; Planer, W; Siuda, ER; Zhao, HC; Stickler, L; Chang, SD; Baird, MA; Cao, YQ; Bruchas, MR
Journal of Biological Chemistry, 287(): 42019-42030.
10.1074/jbc.M112.405696
CrossRef
Journal of Medicinal Chemistry
Development of kappa Opioid Receptor Antagonists
Carroll, FI; Carlezon, WA
Journal of Medicinal Chemistry, 56(6): 2178-2195.
10.1021/jm301783x
CrossRef
European Journal of Pharmacology
Nicotine-morphine interactions at alpha 4 beta 2, alpha 7 and alpha 3* nicotinic acetylcholine receptors
Talka, R; Salminen, O; Whiteaker, P; Lukas, RJ; Tuominen, RK
European Journal of Pharmacology, 701(): 57-64.
10.1016/j.ejphar.2013.01.005
CrossRef
Science Translational Medicine
Amygdala-Dependent Fear Is Regulated by Oprl1 in Mice and Humans with PTSD
Andero, R; Brothers, SP; Jovanovic, T; Chen, YT; Salah-Uddin, H; Cameron, M; Bannister, TD; Almli, L; Stevens, JS; Bradley, B; Binder, EB; Wahlestedt, C; Ressler, KJ
Science Translational Medicine, 5(): -.

Toxicology Letters
Comparison of tolerance to morphine-induced respiratory and analgesic effects in mice
Mohammed, W; Alhaddad, H; Marie, N; Tardy, F; Lamballais, F; Risede, P; Noble, F; Baud, FJ; Megarbane, B
Toxicology Letters, 217(3): 251-259.
10.1016/j.toxlet.2012.12.021
CrossRef
Molecular Pain
Chronic inflammatory injury results in increased coupling of delta opioid receptors to voltage-gated Ca2+ channels
Pradhan, A; Smith, M; McGuire, B; Evans, C; Walwyn, W
Molecular Pain, 9(): -.
ARTN 8
CrossRef
European Review for Medical and Pharmacological Sciences
Current practice and recent advances in pediatric pain management
Chiaretti, A; Pierri, F; Valentini, P; Russo, I; Gargiullo, L; Riccardi, R
European Review for Medical and Pharmacological Sciences, 17(): 112-126.

Back to Top | Article Outline

© 2011 American Society of Anesthesiologists, Inc.

Publication of an advertisement in Anesthesiology Online does not constitute endorsement by the American Society of Anesthesiologists, Inc. or Lippincott Williams & Wilkins, Inc. of the product or service being advertised.
Login

Article Tools

Images

Share