In addition to heteromers between opioid receptor types, heteromers involving opioid receptors and other GPCRs including adrenoceptors, metabotropic glutamate receptors, sensory neuron‐specific receptors, have been described (Gomes et al ., 2013a ). In this review, we describe in vitro and in vivo evidence for heteromers involving opioid receptors, the development of heteromer‐selective ligands and the therapeutic potential of GPCR heteromers as target molecules for novel drug development.

Over the last decade, an increasing number of studies have explored the ability of GPCRs including opioid receptors to heteromerize (either with members of the same family or related families). In the case of opioid receptors, early ‘indirect’ evidence for the presence of heteromers was provided by radioligand binding and electrophysiological studies that suggested interactions between μ and δ receptors (Zieglgänsberger et al ., 1982 ; Rothman et al ., 1985; 1988 ; Metcalf et al ., 2012 ; Akgun et al ., 2013 ). Moreover, studies examining the effect of pretreatment with leucine‐enkephalin on morphine‐mediated analgesia (Vaught and Takemori, 1979a,b ), and of δ receptor antagonists on the development of morphine tolerance and dependence (Abdelhamid and Takemori, 1991 ; Zhu et al ., 1999 ) further supported a functional interaction between μ and δ receptors. Recent studies using heteromer‐selective reagents, such as antibodies (Gupta et al ., 2010 ), ligands (Daniels et al ., 2005 ; Waldhoer et al ., 2005 ; Gomes et al ., 2013b ) or agents that disrupt the heteromer in vivo (He et al ., 2011 ), have begun to provide ‘direct’ evidence for opioid receptor heteromerization.

The physical interaction between opioid receptors was first reported in 1997 using δ receptors, and these studies showed that agonist treatment modulated the level of receptor homodimers (Cvejic and Devi, 1997 ). Similar studies also showed that the κ receptors existed as detergent‐insensitive dimers (Jordan and Devi, 1999 ) and μ receptors existed in interacting complexes with distinct trafficking properties (He et al ., 2002 ). The most recent X‐ray crystallization analysis of μ receptors (Manglik et al ., 2012 ) has revealed a twofold symmetrical dimer through a four‐helix bundle motif formed by transmembrane segments 5 and 6. Although this dimeric arrangement of μ receptors could be due to the conditions used to crystallize the receptor, these structural findings are exciting as they enable the development of structure‐based approaches to complement the more conventional drug discovery programmes, in addition to providing novel insights into the roles of oligomerization in GPCR function.

It is becoming generally accepted that GPCRs including opioid receptors interact with each other to form homomers and heteromers, opening new therapeutic possibilities for identifying drugs targeting GPCRs.

A naltrexone derivative, IBN tx A, has been identified as a putative μ‐NOP heteromer‐selective ligand based on the high‐affinity binding of radiolabelled IBN tx A in cells coexpressing μ1G receptors (a μ receptor with mutations in the sixth transmembrane region) and NOP receptors (Majumdar et al ., 2011 ) (Table 4 ). This compound was 10 times more potent as an antinociceptive agent than morphine and did not display side effects, such as respiratory depression, physical dependence and appreciable constipation. Moreover, IBN tx A did not appear to be either rewarding or aversive in conditioned place preference studies (Majumdar et al ., 2011 ) (Table 1 ). Taken together, these results suggest that targeting the μ‐NOP heteromer could provide a major advance in the design and development of new highly potent opiate analgesics, without many side effects.

Examination of the pharmacological properties of μ‐NOP complexes showed an increase in the binding affinity of μ receptor agonists including DAMGO and fentanyl, compared with cells expressing only μ receptors (Pan et al ., 2002 ) (Table 1 ). In addition, signalling assays showed that while the EC 50 for inhibition of adenylyl cyclase (AC) activity and for activation of ERK1/2 phosphorylation by DAMGO was significantly increased in cells coexpressing μ and NOP receptors, compared with cells expressing only μ receptors, the EC 50 for the NOP receptor agonist was not affected (Wang et al ., 2005b ). These findings taken together with the data from co‐immunoprecipitation studies suggest that μ and NOP receptors form heteromers that selectively impair the potency of μ‐induced signal transduction pathways. In addition, while pretreatment with the NOP receptor agonist caused desensitization of not only NOP‐ but also DAMGO‐induced inhibition of AC activity, pretreatment with DAMGO did not affect desensitization of NOP receptor‐mediated pathways (Wang et al ., 2005b ) (Table 1 ). These results suggest that μ‐NOP heteromerization selectively causes cross‐desensitization of μ receptor‐mediated signal transduction.

Studies showing that ligands for the nociceptin/orphanin F/Q (NOP) receptor modulated the antinociceptive effects of morphine (King et al ., 1998 ; Rizzi et al ., 2000 ) led to investigations on whether μ receptors and NOP receptors formed heteromers. Co‐immunoprecipitation studies showed that μ receptors formed interacting complexes with NOP receptors in heterologous cells coexpressing both receptors and in adult rat dorsal root ganglions (Pan et al ., 2002 ; Wang et al ., 2005b ; Evans et al ., 2010 ). In addition, studies using C‐terminal deletion mutants suggested an involvement of the intracellular C‐terminal region in the formation of these μ‐NOP receptor complexes (Wang et al ., 2005b ) (Table 1 ).

A few studies have focused on identifying μ‐κ heteromer‐selective ligands (Chakrabarti et al ., 2010 ; Yekkirala et al ., 2011 ). Although strong evidence for the role of dynorphin 1–17 at μ‐κ heteromers is not available, it has been reported that N ‐naphthoyl‐β‐naltrexamine (NNTA) selectively activates μ‐κ heteromers (Table 4 ), and it is 50 times more potent than morphine, as an antinociceptive agent (Yekkirala et al ., 2011 ) (Table 1 ). Moreover, intrathecal administration of NNTA leads to greater antinociceptive effect (∼two orders of magnitude) than intracerebroventricular administration, an effect not seen in μ receptor‐knockout mice, which would suggest a higher degree of functional coupling between μ and κ receptors in the spinal cord (Yekkirala et al ., 2011 ). In addition, the administration of NNTA does not lead to the development of physical dependence while antinociceptive tolerance to NNTA is low upon chronic intracerebroventricular administration and not observed upon chronic intrathecal administration (Yekkirala et al ., 2011 ) (Table 1 ). This suggests that μ‐κ heteromers play important roles in pain regulation and that they may be viable targets for the development of analgesics devoid of the unwanted side effects associated with chronic morphine administration.

Examination of the properties of the μ‐κ heteromer using radioligand binding and [ 35 S]GTPγS assays revealed that the binding affinity of μ receptor agonists, such as DAMGO and endomorphin‐1, is lower in cells coexpressing μ and κ receptors compared with cells expressing only μ receptors. In the case of the κ receptor agonists, U69593 and U50488H, no differences were observed between cells expressing μ‐κ heteromers or κ receptors (Wang et al ., 2005a ) (Table 1 ). These findings indicate that heteromerization with κ receptors alters the binding properties of μ receptors.

A few studies have investigated possible heteromerization between μ and κ receptors (μ‐κ heteromers). Early co‐immunoprecipitation studies using antibodies to the epitope tags on the receptors were unable to detect the presence of interacting complexes between μ and κ receptors in heterologous cells (Jordan and Devi, 1999 ). However, a study used antibodies to endogenous μ and κ receptors to detect the presence of μ and κ interacting complexes in spinal cord membranes from female but not male rats (Chakrabarti et al ., 2010 ) (Table 1 ). This study reported that the levels of μ‐κ interacting complexes were dependent upon the stage of the oestrous cycle (Chakrabarti et al ., 2010 ). These results suggest that the detection of μ‐κ interacting complexes may be susceptible to the detergent conditions used in co‐immunoprecipitation studies or may depend on the tissue used. In addition, it is possible that in cells/tissues expressing μ and κ receptors the μ‐κ interacting complexes may be inducible only under certain physiological conditions or may be under the regulation of sex hormones. Further support for the probable formation of μ‐κ heteromers came from BRET assays showing that the two receptors are close enough to directly interact in live cells (Wang et al ., 2005a ). Thus it appears that μ and κ receptors can form physiologically relevant heteromers and studies to explore the functional role of this heteromer in biological systems are needed.

In order to understand the role of δ‐κ heteromers in vivo , heteromer‐selective reagents such as antibodies have been generated. A recent study detected the presence of δ‐κ heteromers in peripheral sensory neurons following thermal allodynia (Berg et al ., 2012 ). Moreover, the study found that a δ‐κ heteromer‐selective antibody could enhance the anti‐allodynic effects of the δ receptor agonist, DPDPE; this suggests a role for δ‐κ heteromers in modulation of thermal allodynia (Berg et al ., 2012 ) (Table 1 ). Ligands targeting the δ‐κ heteromer include a bivalent ligand named KDN‐21 and 6′‐guanidinonaltrindole (6′‐GNTI) (Table 4 ). KDN‐21 comprises a κ receptor‐selective antagonist pharmacophore, 5′‐guanidinonaltrindole, which is tethered through a 21‐atom spacer arm to the δ receptor‐selective antagonist pharmacophore, naltrindole. KDN‐21 exhibits selective δ1 receptor and κ2 receptor antagonistic activity and does not induce antinociception (Bhushan et al ., 2004 ) (Table 1 ). In contrast, 6′‐GNTI functions as an agonist that induces ∼50‐times more potent antinociception than the selective κ receptor agonist U50488H when administered intrathecally but not intracerebroventricularly (Waldhoer et al ., 2005 ) (Table 1 ). However, a recent study reported that 6′‐GNTI also exhibits biased agonistic properties at κ receptors (Rives et al ., 2012 ); this would suggest that the behavioural outcomes from studies with 6′‐GNTI could be due to its activity at either κ receptors alone or δ‐κ heteromers. Taken together, these studies suggest that δ‐κ heteromers form a distinct functional signalling unit that could provide a target for the development of tissue‐selective opiate analgesics.

Examination of the binding, signalling and trafficking properties of the δ‐κ heteromer showed that they were distinct from δ or κ homomers. For example, the binding affinities for δ or κ receptor agonists were lower at δ‐κ heteromers compared with the respective receptor homomers (Jordan and Devi, 1999 ). An increase not only in the binding affinity but also in intracellular signalling was observed when the δ‐κ heteromer was treated with a combination of δ and κ receptor agonists (Jordan and Devi, 1999 ) (Table 1 ). An increase in the binding affinity was also observed when the δ‐κ heteromer was treated with a combination of δ or κ receptor antagonists (Jordan and Devi, 1999 ). These findings suggest the possibility of allosteric interactions between δ and κ receptors. With regard to the trafficking properties of the δ‐κ heteromer, studies with etorphine, a potent non‐selective opioid agonist that binds to both κ and δ receptors, show that it does not induce δ receptor internalization in cells expressing the δ‐κ heteromer, while it induces receptor internalization in cells expressing only δ receptors (Jordan and Devi, 1999 ). These findings suggest that δ‐κ heteromerization alters the trafficking of properties of δ receptors (Table 1 ).

The δ and κ receptor heteromer (δ‐κ heteromer) was the first opioid receptor heteromer to be reported. Co‐immunoprecipitation studies using κ receptors tagged with a myc epitope and δ receptors tagged with a Flag epitope detected the presence of interacting complexes only in cells coexpressing both receptors (Jordan and Devi, 1999 ). In addition, BRET assays showed that the two receptors existed in close proximity and could directly interact in live cells (Ramsay et al ., 2002 ) (Table 1 ).

In order to understand the role of δ‐μ heteromers in vivo , heteromer‐selective reagents (ligands, antibodies) as well as agents that selectively disrupt the heteromer have been developed. Studies with these reagents suggest a possible involvement of δ‐μ heteromers in the development of tolerance to morphine. For example, δ‐μ heteromer‐selective antibodies detect increased heteromer levels in discrete brain regions following chronic morphine administration (Gupta et al ., 2010 ). Moreover, administration of a membrane permeable peptide TAT peptide (YGRKKRRQRRR) fused to the peptide representing transmembrane domain 1 of μ receptors disrupts the δ‐μ heteromer and leads to an increase in morphine‐mediated antinociception and a decrease in the development of tolerance to morphine (He et al ., 2011 ) (Table 1 ). These results suggest that the δ‐μ heteromer could be a target for the development of antinociceptive therapeutics as potent as morphine but with lesser side effects such as antinociceptive tolerance and dependence. This is supported by studies with MDANs that differ in the length of the spacer arm between pharmacophores and selectively target the δ‐μ heteromer. Of these, MDAN‐21 was found to exhibit 100 times more potent antinociception and to be less rewarding than morphine (Daniels et al ., 2005 ; Lenard et al ., 2007 ). Moreover, chronic administration of MDAN‐21 did not lead to the development of tolerance or dependence (Daniels et al ., 2005 ). In addition to MDANs, bivalent ligands comprising of a high‐affinity μ receptor agonist (oxymorphone) joined by a spacer arm to a low affinity δ receptor antagonist (ENTI) or of a high‐affinity μ receptor antagonist (naltrexone) joined by a spacer arm to a low affinity δ receptor agonist (DM‐SNC80) have been generated (Harvey et al ., 2012 ) (Table 4 ). However, the antinociceptive effects of these ligands and their side effects have not been evaluated. More recently, a high throughput screening of a small molecule library for a δ‐μ heteromer‐selective ligand led to the identification of CYM51010 as a biased δ‐μ heteromer agonist (Table 4 ); this study also showed that the compound exhibited potent antinociception with reduced antinociceptive tolerance (Gomes et al ., 2013b ). The antinociceptive effect of CYM51010 was significantly blocked by δ‐μ heteromer‐selective antibodies (Gomes et al ., 2013b ), which would suggest that, in vivo , this heteromer plays an important role in pain regulation (Table 1 ). Taken together, these studies support the δ‐μ heteromer as a novel therapeutic target for pain attenuation with reduced side effects.

It is well known that following continued exposure to agonists, μ or δ receptors are phosphorylated; this leads to the recruitment of β‐arrestin, receptor endocytosis to acidic endosomal compartments and termination of Gα i/o protein‐mediated signalling. In the endosomes, the internalized receptors are either dephosphorylated and recycled back to the cell surface to undergo another round of signalling or are targeted to lysosomes for degradation. Very few studies have investigated how heteromerization modulates the endocytosis of the δ‐μ heteromer. While one study found that each protomer in the δ‐μ heteromer internalized independently from the other protomer, other studies found that the heteromer as a whole could be endocytosed by some selective agonists (DAMGO, deltorphin II, methadone) but not others, such as D‐penicillamine(2,5)‐enkephalin (DPDPE) or [(D‐Ser 2 , Leu 5 ]enkephalin‐Thr 6 (Law et al ., 2005 ; Hasbi et al ., 2007 ; Milan‐Lobo and Whistler, 2011 ). Moreover, methadone, a μ receptor agonist that induces homomer internalization and recycling, induced endocytosis of the δ‐μ heteromer leading to its degradation and this effect could be blocked by co‐treatment with naltriben, a δ receptor antagonist (Milan‐Lobo and Whistler, 2011 ). Another study found that a bivalent ligand selective for the δ‐μ heteromer, MDAN‐21 (comprising a δ receptor antagonist pharmacophore, DN‐20, separated by a 21‐atom spacer from the μ receptor agonist pharmacophore, MA‐19) (Table 4 ), did not induce heteromer internalization (Yekkirala et al ., 2013 ). As co‐administration of the individual monovalent pharmacophores (DN‐20 and MA‐19) induced internalization of δ‐μ heteromers, it has been suggested that the spacer arm in MDAN‐21 by bridging both protomers in the δ‐μ heteromer, immobilizes the latter thereby preventing its endocytosis (Table 1 ). In addition to modulation of receptor internalization and endocytosis, heteromerization appears to modulate the maturation and cell surface expression of the δ‐μ heteromer. In cells coexpressing μ and δ receptors, the heteromer is localized to the Golgi apparatus and its cell surface expression requires a chaperone named receptor transport protein 4 (RTP4) (Decaillot et al ., 2008 ). RTP4 protects the receptor heteromer during folding and maturation from ubiquitination and proteasomal degradation (Decaillot et al ., 2008 ). Further studies are needed to examine if RTP4 affects ligand binding and signalling by the δ‐μ heteromer.

Comparison of the intracellular signalling between the δ‐μ heteromers and δ or μ homomers showed interesting differences. For example, occupancy of one of the protomers in the δ‐μ heteromer enhanced signalling mediated via the activation of the partner protomer (Gomes et al ., 2000 ). In addition, while δ or μ homomers are coupled to and signal via Gα i/o proteins, studies have reported that the δ‐μ heteromers could be associated with a Pertussis toxin‐insensitive G‐protein such as Gz (George et al ., 2000 ; Fan et al ., 2005 ; Hasbi et al ., 2007 ) and/or with β‐arrestin 2 (Rozenfeld and Devi, 2007 ). The latter study showed that activation of a protomer in δ‐μ heteromers leads to β‐arrestin 2‐mediated signalling. This is characterized by the presence of a second phase of ERK activation that is PKC‐independent and can be blocked by small interfering RNA to β‐arrestin 2 (Rozenfeld and Devi, 2007 ) (Table 1 ). Moreover, δ‐μ heteromer‐mediated β‐arrestin 2 signalling leads to changes in the spatiotemporal dynamics of ERK1/2 phosphorylation. For example, in cells expressing the δ‐μ heteromer, ERK1/2 which was phosphorylated following treatment with the μ receptor agonist, [D‐Ala 2 , N‐MePhe 4 , Gly‐ol]‐enkephalin (DAMGO), was not translocated to the nucleus (as seen with cells expressing only μ receptors). This leads to the phosphorylation of the cytoplasmic and not the nuclear ERK1/2 substrates and ultimately differential activation of transcription factors (Rozenfeld and Devi, 2007 ). Finally, a study showed that in GH3 cells expressing δ‐μ heteromers, the μ receptor agonist, DAMGO, stimulated Ca +2 ‐mediated signalling instead of Gα i/o ‐mediated signalling (Charles et al ., 2003 ). Taken together, these studies show that heteromerization increases the repertoire of signalling of μ and δ receptors.

Examination of the ligand‐binding properties of the δ‐μ heteromers showed that the binding affinity of agonists to individual protomers was decreased in δ‐μ heteromers when compared with individual receptors (George et al ., 2000 ) (Table 1 ). Interestingly, this was increased in the presence of agonists or antagonists selective for the partner protomer (Gomes et al ., 2000; 2004; 2011 ). Further analysis revealed that this was due to allosteric modulation; the occupancy of one protomer allosterically modulated ligand binding to the partner protomer by affecting the rate of dissociation of the ligand (Gomes et al ., 2011 ). These changes in the pharmacological properties of δ‐μ heteromers compared with δ or μ homomers suggested possible differences in signalling between heteromers and homomers.

Antinociception (i.t, i.c.v. and s.c.) with MDAN21 is more potent than morphine without development of tolerance revealed and it is less rewarding than morphine.

The δ and μ receptor heteromer (δ‐μ heteromer) is the most extensively studied opioid receptor heteromer. The ‘direct’ interaction between δ and μ receptors was first demonstrated by co‐immunoprecipitation studies (George et al ., 2000 ; Gomes et al ., 2000 ) where Flag‐tagged μ receptors were found to form interacting complexes (∼150 kDa) with myc ‐tagged δ receptors in HEK‐293 cells coexpressing both receptors (Gomes et al ., 2000 ). Endogenous δ‐μ complexes could also be detected by co‐immunoprecipitation studies with the spinal cord membranes from wild‐type, but not δ receptor knockout mice (Gomes et al ., 2004 ). Furthermore, BRET assays demonstrated that both receptors exist within 100Å of each other in live cells, which is close enough to allow for direct receptor–receptor interactions (Gomes et al ., 2004 ) (Table 1 ).

In order to understand the physiological role of μ‐CB 1 heteromers, bivalent ligands comprising a selective μ receptor agonist connected to a CB 1 receptor‐selective antagonist/inverse agonist, via spacers of different lengths, have been generated (Le Naour et al ., 2013 ). Among these, the ligand having a 20‐atom spacer was found to bridge both receptors in the μ‐CB 1 heteromer (Table 4 ) and this compound exhibited potent antinociceptive effects without development of antinociceptive tolerance (Le Naour et al ., 2013 ) (Table 2 ). Because the development of antinociceptive tolerance is a key side effect of opiates and cannabinoids that limits their clinical use, the μ‐CB 1 heteromer is a potential target for the development of analgesics with reduced side effects.

Examination of the properties of the μ‐CB 1 heteromer in heterologous and endogenous systems shows that while agonists to individual protomers can activate G‐protein‐mediated signalling and ERK phosphorylation, a combination of agonists to both protomers causes a decrease in signalling (Rios et al ., 2006 ). Moreover, a study used receptors fused to chimeric G‐proteins to show that the agonist for either protomer in the μ‐CB 1 heteromer induces signalling by activating the same G‐protein (Hojo et al ., 2008 ). Heteromerization between μ and CB 1 receptors is of physiological relevance given that while agonists to either protomer cause an increase in neurite outgrowth in Neuro 2A cells, a combination of agonists to both receptors reduces neurite outgrowth by decreasing the phosphorylation of Src and STAT3 (Rios et al ., 2006 ) (Table 2 ). Taken together, these studies show that μ‐CB 1 heteromerization leads to modulation of individual protomer signalling and that this heteromer may have a physiological role.

Possible formation of heteromers between μ receptors and CB 1 receptors was suggested by studies showing functional interactions between these receptors. For example, antinociceptive synergy was reported when using a combination of morphine and a CB 1 receptor agonist (Cichewicz, 2004 ). Moreover, μ receptor‐knockout mice do not exhibit conditioned place preference for CB 1 receptor agonists (Ghozland et al ., 2002 ) while the reinforcing effects of morphine as well as the severity of withdrawal symptoms from this drug are absent in CB 1 receptor‐knockout mice (Ledent et al ., 1999 ). Direct evidence for the formation of μ and CB 1 receptor heteromers (μ‐CB 1 heteromer) came from proximity‐based assays such as BRET and FRET that showed these two receptors were close enough to directly interact in live cells (Rios et al ., 2006 ; Hojo et al ., 2008 ). Furthermore, co‐immunoprecipitation studies demonstrated the presence of interacting μ‐CB 1 receptor complexes in heterologous cells coexpressing both receptors (Hojo et al ., 2008 ).

The observation that δ‐CB 1 heteromer levels are altered under pathophysiological conditions is of great importance. In a rodent model of neuropathic pain where animals exhibit mechanical allodynia, δ‐CB 1 heteromer levels (detected using a δ‐CB 1 heteromer‐selective monoclonal antibody) are significantly elevated in cortex, hypothalamus and midbrain (Bushlin et al ., 2012 ). Moreover, in this model of neuropathic pain, the activation of G‐protein mediated signalling by a CB 1 receptor‐selective agonist was increased while that of a δ receptor‐selective agonist was decreased; however, the latter was restored in the presence of a CB 1 receptor‐selective agonist (Bushlin et al ., 2012 ). These findings suggest that the pharmacological effects of δ and CB 1 receptors could be altered under conditions of neuropathic pain and that heteromer formation might be involved in these changes (Bushlin et al ., 2012 ) (Table 2 ). Taken together, these studies suggest that the δ‐CB 1 heteromer could be a target for the development of novel therapeutics to treat neuropathic pain.

Heteromerization with δ receptors modulates the subcellular localization of CB 1 receptors as the latter receptor exhibits an intracellular localization when expressed alone and when coexpressed with δ receptors, it was found on the cell surface (Rozenfeld et al ., 2012 ). This differential localization requires the association of CB 1 receptors with the adaptor protein‐2 whereas, in the absence of δ receptors, CB 1 receptors associate with adaptor protein‐3 (Rozenfeld et al ., 2012 ). Signalling assays showed that the δ receptor and CB 1 receptor heteromer (δ‐CB 1 heteromer) exhibited signalling distinct from that of receptor homomers. For example, the signalling potency of a CB 1 receptor agonist was decreased in cells expressing the heteromer compared with the CB 1 receptor homomer and this decrease in potency was not seen following knockdown of δ receptors (Rozenfeld et al ., 2012 ). Also, while δ or CB 1 receptor homomers signal via activation of Gα i/o proteins, δ‐CB 1 heteromer‐mediated signalling involves PLC‐mediated recruitment of β‐arrestin 3 and the activation of signalling pathways that promote cell survival (Rozenfeld et al ., 2012 ) (Table 2 ). Taken together, these findings show that δ‐CB 1 heteromerization expands the signalling repertoire of individual receptors.

Controversial. One study showed no internalization of the heteromer but another study showed internalization of the heteromer and cross‐desensitization by α 2 adrenoceptor or μ agonist via p38 MAPK.

Several lines of evidence have suggested interactions between δ‐opioid receptors and CB 1 receptors. These included studies showing that (i) δ receptor agonists decrease CB 1 receptor signalling, (ii) δ receptor antagonists attenuate CB 1 receptor‐mediated anxiolytic effects, (iii) CB 1 receptor levels and signalling increase in the substantia nigra of δ receptor‐knockout mice and (iv) δ receptor activity increases in the caudate putamen of CB 1 receptor‐knockout mice (Shapira et al ., 1998 ; Berrendero and Maldonado, 2002 ; Berrendero et al ., 2003 ; Uriguen et al ., 2005 ). Direct interactions between δ and CB 1 receptors were suggested by BRET assays carried out in heterologous cells coexpressing luciferase‐tagged CB 1 receptors and yellow fluorescent protein (YFP)‐tagged δ receptors showing both receptors in close proximity in live cells (Rios et al ., 2006 ). This was further supported by co‐immunoprecipitation studies using epitope‐tagged receptors and by immunofluorescence studies showing colocalization of δ receptors with CB 1 receptors in cortical neurons (Rozenfeld et al ., 2012 ) (Table 2 ).

Although studies have shown that the analgesic potency of spinally administered morphine is decreased in mice lacking α 2A adrenoceptors (Stone et al ., 1997 ), very little is known about the physiological relevance of μ‐α 2A heteromers. Thus further studies, using either heteromer‐selective ligands/antibodies or agents that disrupt the heteromer, are needed to elucidate the role of the μ‐α 2A heteromer during normal physiology and pathology in particular during pain attenuation.

Examination of G‐protein and MAPK activation in cells expressing the μ‐α 2A heteromer shows that the morphine‐mediated signalling is enhanced compared with cells expressing only μ receptors; however, this enhancement of signalling is not seen when a combination of morphine with clonidine (α 2 adrenoceptor agonist) is used (Jordan et al ., 2003 ). Furthermore, the G‐protein as well as MAPK activation by noradrenaline (α 2A adrenoceptor agonist) is decreased in the presence of morphine (Vilardaga et al ., 2008 ). Similar findings were made with spinal cord neurons (Jordan et al ., 2003 ), suggesting that the interactions between μ receptors and α 2A adrenoceptors also take place in endogenous systems and that the signalling of μ receptors can be modulated by α 2A adrenoceptors (Table 2 ). This is also supported by studies carried out in mouse dorsal root ganglion neurons which show that prolonged treatment with the μ receptor agonist, DAMGO, or the α 2 adrenoceptor agonist, clonidine, induced cross‐desensitization between μ and α 2A receptor‐mediated inhibition of voltage‐gated Ca +2 current and this was associated with the co‐internalization of μ and α 2A adrenoceptors (Tan et al ., 2009 ).

Studies showing functional interactions between μ receptors and α 2A adrenoceptors suggested that these two receptors could form heteromers. These included studies showing that a combination of μ receptor and α 2A adrenoceptor agonists resulted in antinociceptive synergy and that the potency of morphine‐mediated antinociception is decreased in mice lacking α 2A adrenoceptors (Drasner and Fields, 1988 ; Ossipov et al ., 1997 ; Stone et al ., 1997 ). Several lines of evidence support heteromerization between μ receptors and α 2A adrenoceptors. For example, immunohistochemical analysis using Flag‐tagged μ and hemagglutinin‐tagged α 2A adrenoceptors demonstrate colocalization of both receptors not only at the plasma membrane but also within intracellular vesicles (Jordan et al ., 2003 ). In addition, co‐immunoprecipitation studies detect the presence of interacting μ‐α 2A complexes in heterologous cells and in primary hippocampal neurons, and BRET and FRET assays show that the two receptors are close enough to form interacting complexes in live cells (Jordan et al ., 2003 ; Zhang and Limbird, 2004 ; Vilardaga et al ., 2008 ). Moreover, FRET analysis revealed that binding of an agonist to μ receptors suppressed the α 2A adrenoceptor agonist (norepinephrine)‐induced FRET signal probably through a conformational change transmitted from the μ receptors to the α 2A adrenoceptors (Vilardaga et al ., 2008 ) (Table 2 ). Interestingly, while treatment with agonists to individual receptors, such as morphine (μ receptor) or clonidine (α 2A adrenoceptor), leads to an apparent increase in the levels of μ‐α 2A complexes, a combination of agonists to the two receptors leads to a decrease to below the basal levels (Jordan et al ., 2003 ). This suggests that either co‐occupancy of both protomers disrupts heteromer formation or makes the latter more susceptible to the effects of detergents used for cell lysis.

Very little is known about the physiological relevance of κ‐β 2 heteromers. Thus further studies are needed to evaluate the role of this heteromer pair in normal physiology and pathology and particularly in cardiac pathology, given the presence of both receptors in the heart.

Examination of trafficking properties show that treatment with a β 2 adrenoceptor agonist, that induces β 2 adrenoceptor internalization in cells expressing only this receptor, does not induce β 2 adrenoceptor internalization in cells expressing the κ‐β 2 heteromer (Jordan et al ., 2001 ) (Table 2 ). This suggests that heteromerization between κ receptors and β 2 adrenoceptors modulates the trafficking properties of the β 2 adrenoceptors.

The signalling properties of the κ‐β 2 heteromer were investigated using the AC and the MAPK phosphorylation assays. In the case of the AC assay, in cells expressing the κ‐β 2 heteromer, there were no changes in the ability of κ receptor agonists to inhibit and of β 2 adrenoceptor agonists to stimulate enzyme activity compared with cells expressing individual receptors (Jordan et al ., 2001 ). In the case of agonist‐mediated MAPK activation, it was observed that in cells expressing the κ‐β 2 heteromer, the κ receptor agonist but not the β 2 adrenoceptor agonist could induce ERK1/2 phosphorylation. These findings suggest that while heteromerization with κ receptors does not significantly affect the functional G‐protein coupling properties of β 2 adrenoceptors, it may promote biased signalling at β 2 adrenoceptors by preserving G‐protein‐mediated signalling (i.e. AC activity) but impairing MAPK signalling (Jordan et al ., 2001 ) (Table 2 ).

Examination of the ligand‐binding properties of κ‐β 2 heteromers shows that there are no changes in binding affinity of individual receptor ligands, such as U‐69593, nor‐binaltorphimine and isoprenaline, when comparing with cells expressing either κ receptors or β 2 adrenoceptors (Jordan et al ., 2001 ); this suggests that heteromerization between these two receptors does not lead to alterations in the pharmacological properties of individual receptor protomers (Table 2 ).

Studies showing that κ receptors and β 2 adrenoceptors are present in the heart, and that a β 2 adrenoceptor agonist modulates radiolabelled ligand binding to κ receptors suggested possible heteromerization between these two receptors (Ventura et al ., 1989 ; Tai et al ., 1991 ). Co‐immunoprecipitation studies carried out in HEK‐293 cells coexpressing myc ‐tagged κ receptors and Flag‐tagged β 2 adrenoceptors detected the presence of interacting complexes at the cell surface (Jordan et al ., 2001 ). Furthermore, BRET assays show that the two receptors are close enough to directly interact in live cells (Ramsay et al ., 2002 ) (Table 2 ). Taken together, these studies indicate that κ receptors and β 2 adrenoceptors can form heteromers in cells coexpressing both receptors.

Very little ‘direct evidence’ is available about the physiological role of δ‐β 2 heteromers. Studies suggest that interactions between δ receptors and β 2 adrenoceptors may play a role in pathological conditions such as myocardial ischaemia (Huang et al ., 2007 ). Thus further studies, using either heteromer‐selective ligands/antibodies or agents that disrupt the heteromer, are needed to elucidate the role of the δ‐β 2 heteromer during normal physiology and pathology.

Examination of the trafficking properties of the δ‐β 2 heteromer shows that treatment with either δ receptor or β 2 adrenoceptor agonists induces heteromer internalization (Jordan et al ., 2001 ), while in cells expressing individual receptors, δ receptor agonists do not induce β 2 adrenoceptor endocytosis and β 2 adrenoceptor agonists do not induce δ‐receptor endocytosis (Jordan et al ., 2001 ). These findings suggest that heteromerization between δ receptors and β 2 adrenoceptors leads to alterations in the trafficking of individual receptors (Table 2 ).

Examination of the ligand‐binding properties of the δ‐β 2 heteromer shows that heteromerization between these two receptors does not lead to alterations in the pharmacological properties of individual protomers (Jordan et al ., 2001 ) (Table 2 ). In addition, the signalling properties of δ receptoragonists (inhibition of AC activity) are similar in cells expressing either δ receptors or the δ‐β 2 heteromer (Jordan et al ., 2001 ). Similarly, the signalling properties of β 2 adrenoceptor agonists (stimulation of AC activity) are similar in cells expressing either β 2 adrenoceptors or the δ‐β 2 heteromer (Jordan et al ., 2001 ). Moreover, the activation of MAPK induced by δ receptor or β 2 adrenoceptor agonists in δ‐β 2 heteromer‐expressing cells was similar to that observed in cells expressing individual receptors (Jordan et al ., 2001 ) (Table 2 ). These results indicate that heteromerization of δ receptors with β 2 adrenoceptors does not significantly affect signalling by agonists to individual protomers (Table 2 ).

Studies showing that [Leu]enkephalin (a δ‐receptor agonist) modulates β 2 adrenoceptor signalling and contraction in the heart suggested possible heteromerization between these two receptors (Pepe et al ., 1997 ; Xiao et al ., 1997 ). Co‐immunoprecipitation studies showed that these receptors form interacting complexes at the cell surface of HEK‐293 cells (Jordan et al ., 2001 ). In addition, BRET studies showed that δ receptors and β 2 adrenoceptors were in close proximity and could directly interact in live cells. Moreover, the BRET signal was not changed in the absence or presence of agonists to both receptors leading to the suggestion that δ‐β 2 heteromers are constitutively formed in cells coexpressing both receptors (Ramsay et al ., 2002 ) (Table 2 ).

The physiological relevance of probable δ‐α 2A heteromerization was examined by investigating agonist‐mediated neurite outgrowth in Neuro 2A cells. These studies showed that coexpression of α 2A adrenoceptors could increase deltorphin II‐(a δ receptor agonist) mediated neurite outgrowth in Neuro 2A cells. This suggests that α 2A adrenoceptors could allosterically modulate δ‐receptor function (Rios et al ., 2004 ). Another study examined the effects of δ receptor and α 2A adrenoceptor agonists on antinociception and found that a combination of agonists to the two receptors resulted in ∼30‐fold increase in antinociceptive potency compared with administration of individual receptor agonists (Overland et al ., 2009 ). Moreover, this increase in antinociceptive potency by a combination of δ receptor and α 2A adrenoceptor agonists was blocked by a PKC and not by a PKA inhibitor, whereas when each agonist was individually administered its potency was blocked by a PLC and not by a PKC inhibitor (Overland et al ., 2009 ). The PKC isoform involved in these interactions between δ and α 2A adrenoceptor agonists has been recently identified as PKCε (Schuster et al ., 2013 ). The antinociceptive synergy observed with a combination of δ and α 2A adrenoceptor agonists is thought to be due to the synergistic inhibition of the release of calcitonin gene‐related peptide (CGRP) from the terminals of primary afferent neurons in the spinal cord (Overland et al ., 2009 ) (Table 2 ). Together, these studies suggest a role for δ‐α2A interacting receptor complexes in pain modulation but additional studies showing that disruption of the heteromer pair leads to changes in associating complexes and pain modulation would further support this point.

Functional interactions between δ and α 2A adrenoceptors suggested possible heteromerization between these two receptors. This included studies showing that attenuation of substance P‐mediated antinociception was potentiated by a combination of α 2A adrenoceptor and δ receptor agonists (Fairbanks et al ., 2000 ), and that synergistic antinociceptive interactions between these two receptors were observed in μ receptor‐knockout but not in α 2A adrenoceptor‐knockout mice (Stone et al ., 1997 ; Fairbanks et al ., 2002 ; Guo et al ., 2003 ). Colocalization studies suggested that δ and α 2A adrenoceptors could form interacting complexes as these receptors were extensively colocalized to the same cells in the terminals of capsaicin‐sensitive substance P‐expressing primary afferent neurons (Riedl et al ., 2009 ). In addition, BRET assays show that these two receptors were close enough to directly interact in live cells (Rios et al ., 2004 ). Furthermore, co‐immunoprecipitation studies using epitope‐tagged receptors show that the two receptors form interacting complexes (Rios et al ., 2004 ) (Table 2 ). Although a combination of colocalization, co‐immunoprecipitation and BRET studies suggest the formation of δ‐α 2A heteromers, not much is known about how interactions between these two receptors modulate the binding, signalling and trafficking properties of individual protomers. Such information is necessary in order to consider this receptor pair as a bona fide heteromer.

Other heteromers involving opioid receptors

δ opioid and chemokine receptor heteromers The formation of heteromers between δ opioid receptors and the chemokine receptor CXCR4 (δ‐CXCR4 heteromer) is suggested by FRET and co‐immunoprecipitation studies carried out using heterologous cells and primary monocytes from healthy donors (Pello et al., 2008) (Table 3). The FRET signal and the level of interacting δ‐CXCR4 complexes did not change in the presence or absence of receptor‐selective agonists such as DPDPE (δ receptor agonist) or CXCL12 (CXCR4 agonist), suggesting that these heteromers are constitutively formed (Pello et al., 2008). Table 3. Heteromers involving opioid receptors and GPCRs other than cannabinoid or catecholamine receptors Heteromer pair In vitro heteromer properties (binding, signalling, trafficking) In vivo effects of reagents targeting heteromers References δ‐CXCR4 Detection Co‐IP, FRET Binding No change. Signalling Inactivated by δ + CXCR4 agonists. No association with G‐proteins in the presence of δ + CXCR4 agonists. Trafficking No change. Pello et al., 2008 δ‐SNSR‐4 Detection BRET Binding Remains to be determined Signalling Preferential Gα q signalling and attenuation of Gα i signalling. Trafficking ↓ in δ receptor endocytosis by BAM22 in the presence of SNSR‐4. Breit et al., 2006 κ‐APJ Detection Colocalization, Co‐IP, BRET Binding Remains to be determined. Signalling ↑ in heteromer‐mediated PKC signalling. Trafficking Remains to be determined. Li et al., 2012 μ‐CCR5b Detection Co‐IP Binding Binding affinity for a ligand to one protomer not changed in the presence of ligand to partner protomer. Signalling ↓ in μ receptor ‐mediated G‐protein activation by CCR5 receptor agonist and vice versa. Trafficking μ receptor internalization by μ and not by CCR5 receptor agonists and vice versa. Bivalent ligand (Bivalent ligand 1) More potent inhibition of viral entry compared with naltrexone + maraviroc in antiviral activity assay. Suzuki et al., 2002 et al., 2004 et al., 2012; 2013 μ1D‐GRPRc Detection Co‐IP Binding Not reported. Signalling μ receptor‐mediated Ca+2 signalling only in cells expressing the heteromer. Trafficking ↑ in μ receptor ‐mediated GRPR internalization. TAT‐fusion protein (TAT‐μ1D CT ) TAT‐μ1D CT disrupts μ1D‐GRP receptor heteromers and blocks morphine‐induced scratching without affecting analgesia. Liu et al., 2011 μ‐mGlu 5 d Detection Co‐IP Binding No change in binding affinity for μ receptor agonist. ↑ in binding affinity for mGlu 5 receptor antagonist. Signalling No change in μ receptor agonist‐mediated inhibition of adenylate cyclase activity. mGlu 5 receptor antagonist ↓ μ receptor agonist‐mediated phosphorylation and desensitization of μ receptors. Trafficking mGlu 5 receptor antagonist ↓ μ receptor agonist‐mediated internalization of μ receptors. Bivalent ligand (MMG22) MMG22 antinociception (i.t. and i.c.v.) is equipotent to morphine in naïve mice, and more potent in LPS‐treated mice with less tolerance and respiratory depression. MMG22 shows antinociception (i.t.) in CFA‐induced inflammatory pain or bone cancer pain model. Schroder et al., 2009 et al., 2013 μ‐5‐HT 1A Detection Colocalization, Co‐IP, BRET Binding Remains to be determined. Signalling Transactivation of G‐protein fused to 5‐HT 1A receptor by μ receptor agonist. Activation of ERK1/2 by μ receptor agonist is blocked by 5‐HT 1A receptor agonist pretreatment. Trafficking No co‐internalization by protomer‐selective agonists. Daval et al., 1987 et al., 1992 et al., 1998 et al., 2000 et al., 2001 et al., 2012 μ‐NK1d Detection Colocalization, Co‐IP, BRET Binding ↑ in affinity for μ receptor agonist. No change in affinity for NK 1 receptor agonist. Signalling Pre‐incubation with μ agonist ↓ NK 1 receptor‐mediated ERK phosphorylation and vice versa. Trafficking Co‐internalization by protomer‐selective agonists. Bivalent peptide Assay shows that the peptide exhibits μ agonist and NK 1 receptor antagonist activity. Small molecule ligands Assay shows that the ligands exhibit μ agonist and NK 1 receptor antagonist activity. Multifunctional μ/δ agonist/NK 1 receptor antagonist compound (TY027) TY027 exhibits antinociception (i.c.v., i.t.) in naïve mice. TY027 exhibits antinociception (i.t., i.v.) against spinal nerve ligation‐induced hyperalgesia. TY027 produced antinociception with low tolerance, dependence or rewarding effects and was not accompanied by opioid‐related emesis or constipation. Aicher et al., 2000a,b et al., 2003 et al., 2007 et al., 2011 et al., 2013 μ‐sst 2A Detection Colocalization, Co‐IP Binding ↓ in binding affinity for sst 2 receptor agonists. No change in binding affinity for μ receptor agonist. Signalling ↑ inhibition of adenylate cyclase activity. No change in ERK1/2 activation. Pretreatment with the protomer agonist causes cross‐desensitization. Trafficking sst 2 receptor agonist induces heteromer internalization. μ agonist internalizes μ receptors but not sst 2A receptors. Pfeiffer et al., 2002 Table 4. List of ligands targeting opioid receptor heteromers Target Heteromer Pair Ligands Pharmacophores Spacer length References δ‐μ Bivalent ligand MDAN21 δ antagonist: DN‐21 μ agonist: MA‐19 21‐atom (Daniels et al., 2005 et al., 2013b et al., 2012 Bivalent ligand L2 δ antagonist:ENTI μ agonist: oxymorphone optimized for the heteromer(19–22‐atom) (not exactly mentioned) Bivalent ligand L4 δ agonist: DM‐SNC80 μ antagonist: naltrexone Biased agonist CYM51010 δ‐μ agonist: CYM51010 Not applicable (N/A) δ‐κ Bivalent ligand KDN‐21 δ antagonist: naltrindole (NTI) κ antagonist: 5′‐guanidinonaltrindole (5′‐GNTI) 21‐atom (Bhushan et al., 2004 et al., 2005 Heteromer targeting agonist 6′‐GNTI* δ‐κ agonist: 6′‐guanidinonaltrindole (6′‐GNTI) N/A μ‐κ Heteromer targeting agonist NNTA μ‐κ agonist: N‐naphthoyl‐β‐naltrexamine (NNTA) N/A (Yekkirala et al., 2011 μ1G‐NOP Heteromer targeting ligand IBN tx A μ1G‐NOP agonist: iodobenzoylnaltrexamide (IBN tx A) N/A (Majumdar et al., 2011 μ‐CB 1 R Bivalent ligand μ agonist: α‐oxymorphamine CB1 antagonist: SR141716 20‐atom (Le Naour et al., 2013 μ‐CCR5 Bivalent ligand Bivalent ligand 1 μ antagonist: naltrexone CCR5 antagonist: maraviroc 21‐atom (Yuan et al., 2012 μ‐mGluR5 Bivalent ligand MMG22 μ agonist: oxymorphone mGluR5 antagonist: m‐methoxy‐2‐methyl‐6‐(phenylethynyl) pyridine (M‐MPEP) 22‐atom (Akgun et al., 2013 μ‐NK1 Bivalent peptides Opioid agonist: H‐Tyr‐D‐Ala‐Gly‐Phe NK1 antagonist: Pro‐Leu‐Trp‐O‐3,5‐Bzl(CF 3 ) 2 N/A (Largent‐Milnes et al., 2013 et al., 2011 et al., 2007 Bivalent ligands small molecule μ agonist: fentanyl NK1 antagonist: L732138 N/A Multifunctional μ/δ agonist/NK1 antagonist compound TY027 μ‐NK1 agonist: H‐Tyr‐D‐Ala‐Gly‐Phe‐Met‐Pro‐Leu‐Trp‐NH‐3,5Bn(CF 3 ) 2 (TY027) N/A Examination of intracellular signalling shows that although selective agonists (DPDPE or CXCL12) lead to Gα i/o ‐protein activation in cells that coexpress both receptors, a combination of these two agonists inhibits receptor association with Gα i/o protein (Pello et al., 2008). These observations suggest that while δ‐CXCR4 heteromers are fully functional when activated by agonists to either protomer, a combination of agonists to both protomers inactivates the heteromer. Furthermore, CXCR12‐induced phosphorylation of CXCR4 (or desensitization of CXCR4) was not altered by cotreatment with DPDPE (Pello et al., 2008) (Table 3), suggesting that the simultaneous activation of both protomers in δ‐CXCR4 heteromers does not promote heterologous desensitization. Very little is known about the physiological role of δ‐CXCR4 heteromers. However, both δ receptors and CXCR4 are widely distributed in brain tissues and immune cells, and play key roles in inflammation processes and in pain sensation. As activation of both protomers in the δ‐CXCR4 heteromer appears to result in a ‘silent’ receptor complex, further studies to evaluate the role of this heteromer in vivo, particularly under inflammatory conditions are needed.

δ opioid and sensory neuron‐specific receptor (SNSR4) heteromers The sensory neuron‐specific receptor 4 is a GPCR with many names including SNSR3, SNSR4 and the official name of MRGPRX1 receptor; for brevity here it will be referred to as SNSR4. Heteromerization between δ receptors and SNSR4 was investigated based on studies showing that both receptors are present in dorsal root ganglia, and are activated by the bovine medulla adrenal peptide 22 (BAM22; a cleavage product of proenkephalin) although δ receptors mediate antinociceptive responses while SNSR4 mediates nociceptive responses (Lembo et al., 2002; Grazzini et al., 2004). BRET assays carried out in heterologous cells coexpressing δ and SNSR4 show that both receptors are in close proximity to one another and could directly interact in live cells (Breit et al., 2006) (Table 3). While δ receptor‐selective agonists activate Gα i/o ‐mediated signalling, SNSR4‐selective agonists activate Gα q ‐mediated signalling in cells expressing either δ receptors or SNSR4 or in cells coexpressing both receptors; this suggests that each receptor in the heteromeric complex acts as an independent signalling unit (Breit et al., 2006). Interestingly, naltrexone, an opioid receptor antagonist, can block BAM22‐mediated Gα q activation (Breit et al., 2006) suggesting transinhibition of SNSR4 signalling by δ receptors within the δ‐SNSR4 heteromer. Furthermore, costimulation of both protomers in the δ‐SNSR4 heteromer leads to preferential activation of Gα q ‐mediated signalling (PLC activation) and inhibition of Gα i/o ‐mediated signalling (Breit et al., 2006) (Table 3). This regulatory influence of SNSR4 on δ receptor signalling is not due to the PKC‐mediated δ receptor desensitization (Breit et al., 2006). Similar observations were made with cultured dorsal root ganglia from rat embryos (Breit et al., 2006), indicating that these changes in δ receptor coupling and signalling because of heteromerization with SNSR4 also occur in vivo. As both δ and SNSR4 contribute to the regulation of pain sensation (antinociception and nociception, respectively), further investigation on the role of the δ‐SNSR4 heteromer in normal physiology and pathology is needed.

κ opioid and apelin receptor (APJ) heteromers Studies have suggested the formation of heteromers between κ receptors and APJ receptors. Immunocytochemical studies in heterologous cells coexpressing both receptors show that both are colocalized predominantly at the plasma membrane (Li et al., 2012). Co‐immunoprecipitation studies show that both receptors form interacting complexes and BRET assays show that they are in close enough proximity to directly interact in live cells (Li et al., 2012). Interestingly, treatment with receptor‐specific agonists such as dynorphin A1–13 (for κ receptors) and apelin‐13 (for APJ receptors) increased the BRET ratio, indicating that either the heteromerization between these two receptors was facilitated by receptor occupancy or that the latter induced conformational changes that decreased the distance between the epitope tags on individual protomers (i.e. between luciferase and YFP tags) (Li et al., 2012) (Table 3). Signalling by the κ‐APJ heteromer was examined in heterologous cells and in cells that endogenously express both receptors. These studies show that treatment with an agonist to either receptor induces a PKC‐dependent ERK1/2 activation that is two‐ to threefold higher in cells coexpressing both receptors compared with cells expressing individual receptors (Li et al., 2012). In addition, heteromerization between κ and APJ receptors leads to an increase in PKC‐mediated signalling and a decrease in PKA‐mediated signalling compared with cells expressing individual receptors (Li et al., 2012) (Table 3). Taken together, these studies show that heteromerization between κ and APJ receptors leads to modulation of signalling by individual protomers. The functional consequence of κ‐APJ heteromerization was observed at the level of cell proliferation where treatment with either dynorphin A1–13 or apelin‐13 significantly increased the proliferation of cells expressing the heteromer compared with cells expressing individual receptors (Li et al., 2012). Although not much is known about the role of the κ‐APJ heteromer in vivo either in normal physiology or during pathology, the distribution of the dynorphin/κ receptor system and of the apelin/APJ system in the nuclei of the hypothalamus involved in regulation of arginine vasopressin release as well in the cardiovascular system (Sherman et al., 1986; Tsushima et al., 1993; Reaux et al., 2001), suggests a potential role for κ‐APJ heteromers in cardiovascular regulation.

μ opioid and chemokine receptor CCR5 heteromers Studies showing that μ‐opioid receptors are present in immune cells and that morphine treatment increased the expression of the chemokine receptor CCR5 in lymphocytes led to investigations on heteromerization between these two receptors (Chuang et al., 1995; Miyagi et al., 2000). Co‐immunoprecipitation studies show that CCR5 forms an interacting complex with μ receptors in cell lines that coexpress both receptors and that this is not modulated by treatment with receptor‐selective ligands (Suzuki et al., 2002; Chen et al., 2004) (Table 3). Examination of the pharmacological properties of the μ‐CCR5 heteromer shows that pretreatment with the μ receptor agonist, DAMGO, did not change the binding of radiolabelled CCL4 (MIP‐1β) to CCR5. Similarly, CCL5, another CCR5 ligand, did not change the binding properties of radiolabelled ligands to μ receptors (Chen et al., 2004) (Table 3). Interestingly, pretreatment with either DAMGO or CCL5 reduced CCL5‐ or DAMGO‐mediated [35S]GTPγS binding respectively (Chen et al., 2004). These results indicate that pretreatment with agonist to one protomer in the heteromeric complex reduces the ability of the partner receptor to activate G‐proteins. Examination of the trafficking properties of μ‐CCR5 heteromers shows that the μ agonist, DAMGO, induced internalization of μ receptors and not of CCR5 while the CCR5 agonist, CCL5, induced internalization of CCR5 and not of μ receptors (Chen et al., 2004) (Table 3) suggesting that agonists selective for one receptor do not affect internalization of the other receptor in μ‐CCR5 heteromers. Interestingly, pretreatment with either DAMGO or CCL5 can enhance the phosphorylation of both receptors in the heteromer, suggesting heterologous desensitization or cross‐desensitization (Chen et al., 2004). In this context, activation of PKCζ has been reported to be involved in the cross‐desensitization between μ and CCL5 (Song et al., 2011) (Table 3). The cross‐desensitization between μ receptors and CCR5 within the heteromeric complex may modulate the physiological effects of opioids and chemokines in pathological conditions such as HIV infection or opiate addiction (Table 3). A bivalent ligand targeting the μ‐CCR5 heteromer that comprises a μ‐selective antagonist pharmacophore, naltrexone, tethered through a 21‐atom spacer to the CCR5‐selective antagonist pharmacophore, maraviroc, has been developed (Yuan et al., 2012) (Table 4). This bivalent ligand is reported to be twice as potent as an inhibitor of viral entry, as a mixture of both antagonists in vitro (Yuan et al., 2013) (Table 3), suggesting a possible clinical usefulness of bivalent ligands targeting the μ‐CCR5 heteromer against infection by HIV.

μ opioid 1D (μ1D) receptor and gastrin‐releasing peptide (GRP) receptor heteromers μ1D is a μ receptor isoform comprising exons 1–3 and 8–9 of the Oprm gene and μ1D receptors colocalize with GRP receptors (also known as bombesin BB 2 receptors) in the dorsal horn of the spinal cord (Liu et al., 2011). Co‐immunoprecipitation studies with heterologous cells coexpressing both receptors or with spinal cord membranes show that μ1D and GRP receptors form interacting complexes (Liu et al., 2011) (Table 3). Examination of the signalling properties of the μ1D‐GRPR heteromer shows activation of the PLC‐mediated Ca2+ signalling pathway by either morphine or GRP in cells coexpressing μ1D and GRP receptors (Liu et al., 2011). Moreover, morphine or GRP receptor‐induced calcium spikes are blocked by the GRP receptor antagonist or by naloxone, indicating that morphine cross‐activates GRP receptors through μ1D receptors (Liu et al., 2011) (Table 3). The μ1D‐GRPR heteromer exhibits unique trafficking properties in that morphine treatment induces GRP receptor internalization in cells coexpressing both receptors but not in cells expressing only GRP receptors while a GRP receptor agonist did not induce μ1D receptor internalization in cells coexpressing both receptors (Liu et al., 2011). Taken together, these results suggest that heteromerization leads to modulation of protomer signalling and trafficking properties. In order to elucidate the physiological roles of μ1D‐GRPR heteromers, a membrane‐permeable peptide consisting of TAT fused to μ1D CT (TAT‐μ1D CT ), that disrupts heteromer formation, has been developed. Intrathecal administration of TAT‐μ1D CT specifically blocks morphine‐induced scratching without affecting morphine‐induced analgesia (Liu et al., 2011) (Table 3). This suggests that the μ1D‐GRPR heteromer may play a role in morphine‐induced scratching. Moreover, the uncoupling of morphine‐induced analgesia and morphine‐induced scratching by the TAT‐μ1D CT peptide underscores the necessity for elucidating the function of individual μ receptor isoforms, which could be useful in the development of novel analgesics without side effects.

μ opioid and metabotropic glutamate mGlu 5 receptor heteromers Co‐immunoprecipitation studies show that μ and mGlu 5 receptors can form interacting complexes in HEK‐293 cells coexpressing both receptors (Schroder et al., 2009). Interestingly, treatment with 2‐methyl‐6‐(phenylethynyl) pyridine (MPEP), a mGlu 5 receptor antagonist, increases the levels of interacting complexes (Schroder et al., 2009); this suggests that occupancy of mGlu 5 receptors by MPEP affects the conformation of μ receptors which either facilitates the formation of μ‐mGlu 5 heteromers or stabilizes the heteromer under the conditions used for receptor solubilization. Examination of the pharmacological properties of μ‐mGlu 5 heteromers shows that the binding affinity of the μ receptor agonist, DAMGO, is not changed when compared with cells expressing only μ receptors, while the binding affinity of the mGlu 5 receptor specific antagonist is increased when compared with cells expressing only mGlu 5 receptors (Schroder et al., 2009). In addition, while the presence of mGlu 5 receptors does not affect binding and signalling by μ receptors, occupancy of mGlu 5 receptors with the inhibitor MPEP causes a decrease in DAMGO‐mediated phosphorylation, internalization and desensitization of μ receptors (Schroder et al., 2009). These findings suggest that a change in the conformation of mGlu 5 receptors by MPEP might allosterically regulate μ receptor function. Given the wide expression of μ receptors and mGlu 5 receptors in the CNS and their role in regulation of pain transmission, opioid analgesia, dependence and withdrawal, ligands targeting this heteromer pair could play a role in pain regulation. In this context, a bivalent ligand targeting the μ‐mGlu 5 heteromer has been developed. This ligand, MMG22, comprises a μ receptor agonist pharmacophore, oxymorphone, and a mGlu 5 receptor antagonist pharmacophore, m‐methoxy‐MPEP linked via a 22‐atom spacer arm (Table 4). MMG22 exhibits antinociception similar to morphine in naïve mice (Akgun et al., 2013). However, it exhibits 4000 times more potent antinociception and less tolerance and respiratory depression compared with morphine in LPS‐treated mice, an inflammatory pain model (Akgun et al., 2013). Taken together, these results suggest that MMG22 may be useful as a pharmacological tool to investigate μ‐mGlu 5 heteromers in vivo, and in the development of novel drugs to treat inflammatory pain (Table 3).

μ opioid and 5‐HT 1A receptor heteromers Heteromerization between μ receptors and 5‐HT 1A receptors was examined based on studies showing that (i) acute treatment with morphine increased 5‐HT synthesis in different brain regions (Sastre‐Coll et al., 2002), (ii) the inhibition of 5‐HT synthesis by 5‐HT 1A receptor agonists was enhanced in morphine‐dependent animals (Sastre‐Coll et al., 2002), (iii) chronic administration of a 5‐HT 1A receptor agonist to the dorsal raphe nucleus delayed the development of tolerance to morphine (Nayebi and Charkhpour, 2006), (iv) chronic morphine administration increased 5‐HT 1A receptor activity in the medial prefrontal cortex, and decreased 5‐HT1A receptor activity in the dorsal raphe nucleus (Lutz et al., 2011) and (v) μ and 5‐HT 1A receptors colocalize in discrete brain regions (Daval et al., 1987; Pompeiano et al., 1992; Wang et al., 1998; Zhang et al., 2000; Kishimoto et al., 2001). Co‐immunoprecipitation and proximity‐based assays carried out in cells coexpressing both receptors suggest that μ and 5‐HT 1A receptors could form heteromers (μ‐5‐HT 1A ) (Cussac et al., 2012) (Table 3). Not much is known about the pharmacological properties of the μ‐5‐HT 1A heteromer. Examination of the signalling properties showed that the μ receptor agonist could activate a Gα o protein that was covalently fused to 5‐HT 1A receptors only in cells coexpressing both receptors (Cussac et al., 2012). In addition, phosphorylation of ERK1/2 induced following activation of μ receptors was blocked in the presence of the 5‐HT 1A receptor agonist (Cussac et al., 2012). Examination of the trafficking properties of this heteromer shows that treatment with the agonist to one protomer did not induce internalization of the partner protomer (Cussac et al., 2012). Although these studies indicate that μ‐5‐HT 1A heteromerization may modulate the signalling properties of the μ receptor, further studies are needed to not only characterize the pharmacological properties of the μ‐5‐HT 1A heteromer but also its role during normal physiology and pathology.

μ opioid and substance P receptor heteromers Electron microscopy studies demonstrating colocalization of μ receptors with the substance P NK 1 receptor, in the dendrites of the dorsal horn, together with co‐immunoprecipitation and proximity‐based assays showing that μ and NK 1 receptors form interacting complexes provide evidence for μ‐NK 1 heteromerization (Aicher et al., 2000a,b; Pfeiffer et al., 2003) (Table 3). Examination of the pharmacological properties of the μ‐NK 1 heteromer shows that the μ receptor agonist, DAMGO, exhibits ∼threefold higher affinity compared with cells expressing only μ receptors, while substance P showed similar affinity as cells expressing only NK 1 receptors (Pfeiffer et al., 2003). Competition‐binding assays showed that DAMGO or substance P did not compete with [3H]substance P or [3H]DAMGO, respectively, in μ‐NK 1 heteromer expressing cells (Pfeiffer et al., 2003) (Table 3). However, both receptors in the μ‐NK 1 heteromer can be cross‐phosphorylated and co‐internalized into the same endosomal compartment by protomer‐selective agonists. This involves the recruitment of β‐arrestin and formation of stable β‐arrestin receptor complexes that are co‐internalized (Pfeiffer et al., 2003) (Table 3). Taken together, these results indicate that although μ‐NK 1 heteromerization does not affect the binding properties of the individual receptor protomers, it modulates their trafficking properties. Activation of the μ‐NK 1 heteromer by either DAMGO or substance P leads to rapid and transient MAPK activation (ERK1/2 phosphorylation) that is comparable with cells expressing individual receptors (Pfeiffer et al., 2003). However, pre‐incubation with either DAMGO or substance P significantly attenuates either NK 1 or μ receptor‐dependent ERK1/2 phosphorylation, respectively, suggesting that the μ‐NK 1 heteromer undergoes homologous cross‐desensitization (Pfeiffer et al., 2003). Moreover, the resensitization of μ receptor‐mediated MAPK signalling is severely delayed in cells expressing μ‐NK 1 heteromers as compared with cells expressing only μ receptors (Pfeiffer et al., 2003) (Table 3). These results indicate that the formation of heteromers between μ and NK 1 receptors influences the kinetics of signalling by individual protomers. Both μ and NK 1 receptors play important roles in modulation of nociceptive responses. Therefore, targeting the μ‐NK 1 heteromer could lead to the development of novel therapeutics to treat pain. In this context, attempts have been made to develop ligands that selectively target μ‐NK 1 heteromers. These include bivalent peptides designed to possess the peptide sequences for both μ/δ receptor agonist and NK 1 receptor antagonist (Table 4). These bivalent peptides exhibited potent binding affinity and G‐protein activation similar to that of either DPDPE or DAMGO alone (Yamamoto et al., 2007). Furthermore, the bivalent peptides exhibited agonistic activity for opioid receptors and antagonistic activity for NK 1 receptors in the guinea pig isolated ileum assay (Yamamoto et al., 2007). Further studies are needed to elucidate the effects of these peptides on pain regulation in vivo. Other ligands developed to selectively target the μ‐NK 1 heteromer include small molecules that represent variations of combinations of structures of the opioid agonist fentanyl and the NK 1 receptor antagonist pharmacophore L732138 (Vardanyan et al., 2011); however, very little is known about the role of these small molecules in pain modulation. More recently, TY027, a multifunctional μ/δ receptor agonist/ NK 1 receptor antagonist compound, has been shown to have a preclinical profile of excellent antinociceptive efficacy, low abuse liability and no opioid‐related emesis or constipation (Largent‐Milnes et al., 2013). TY027 exhibited antinociceptive efficacy in both non‐injured and spinal nerve‐ligated animals (Largent‐Milnes et al., 2013). In non‐injured animals, the antinociceptive effect of TY027 was similar to that of morphine in the tail‐flick test (Largent‐Milnes et al., 2013). Moreover, repeated administration of TY027 did not lead to development of antinociceptive tolerance, dependence or reward (Largent‐Milnes et al., 2013). Taken together, compounds targeting μ‐NK 1 heteromers could be a promising therapeutic approach in treating patients who suffer from acute and chronic pain (Table 3).