Wakako Fujita

G Protein–Coupled Receptor Heteromers

G protein-coupled receptors (GPCRs) compose one of the largest families of membrane proteins involved in intracellular signaling. They are involved in numerous physiological and pathological processes and are prime candidates for drug development. Over the past decade, an increasing number of studies have reported heteromerization between GPCRs. Many investigations in heterologous systems have provided important indications of potential novel pharmacology; however, the physiological relevance of these findings has yet to be established with endogenous receptors in native tissues. In this review, we focus on family A GPCRs and describe the techniques and criteria to assess their heteromerization. We conclude that advances in approaches to study receptor complex functionality in heterologous systems, coupled with techniques that enable specific examination of native receptor heteromers in vivo, are likely to establish GPCR heteromers as novel therapeutic targets.

Identification of a μ-δ opioid receptor heteromer-biased agonist with antinociceptive activity

G protein-coupled receptors play a pivotal role in many physiological signaling pathways. Mounting evidence suggests that G protein-coupled receptors, including opioid receptors, form dimers, and dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological role of dimerization in vivo has not been well-explored because of the lack of tools to study these dimers in endogenous systems. To address this problem, we previously generated antibodies to μ-δ opioid receptor (μOR-δOR) dimers and used them to study the pharmacology and signaling by this heteromer. We also showed that the heteromer exhibits restricted distribution in the brain and that its abundance is increased in response to chronic morphine administration. Thus, the μOR-δOR heteromer represents a potentially unique target for the development of therapeutics to treat pain. Here, we report the identification of compounds targeting μOR-δOR heteromers through high-throughput screening of a small-molecule library. These compounds exhibit activity in μOR-δOR cells but not μOR or δOR cells alone. Among them, CYM51010 was found to be a μOR-δOR–biased ligand, because its activity is blocked by the μOR-δOR heteromer antibody. Notably, systemic administration of CYM51010 induced antinociceptive activity similar to morphine, and chronic administration of CYM51010 resulted in lesser antinociceptive tolerance compared with morphine. Taken together, these results suggest that CYM51010, a μOR-δOR–biased ligand, could serve as a scaffold for the development of a unique type (heteromer-biased) of drug that is more potent and without the severe side effects associated with conventional clinical opioids.

Heteromers of μ-δ opioid receptors: new pharmacology and novel therapeutic possibilities

Several studies suggest that heteromerization between μ (MOP) and δ (DOP) opioid receptors modulates the signalling properties of the individual receptors. For example, whereas activation of MOP receptors by an agonist induces G protein‐mediated signalling, the same agonist induces β‐arrestin‐mediated signalling in the context of the MOP‐DOP receptor heteromer. Moreover, heteromer‐mediated signalling is allosterically modulated by a combination of MOP and DOP receptor ligands. This has implications in analgesia given that morphine‐induced antinociception can be potentiated by DOP receptor ligands. Recently reagents selectively targeting the MOP‐DOP receptor heteromer such as bivalent ligands, antibodies or membrane permeable peptides have been generated; these reagents are enabling studies to elucidate the contribution of endogenously expressed heteromers to analgesia as well as to the development of side‐effects associated with chronic opioid use. Recent advances in drug screening technology have led to the identification of a MOP‐DOP receptor heteromer‐biased agonist that activates both G protein‐mediated and β‐arrestin‐mediated signalling. Moreover, this heteromer‐biased agonist exhibits potent antinociceptive activity but with reduced side‐effects, suggesting that ligands targeting the MOP‐DOP receptor heteromer form a basis for the development of novel therapeutics for the treatment of pain. In this review, we summarize findings regarding the biological and functional characteristics of the MOP‐DOP receptor heteromer and the in vitro and in vivo properties of heteromer‐selective ligands.

Revolution in GPCR signalling: opioid receptor heteromers as novel therapeutic targets: IUPHAR Review 10

GPCRs can interact with each other to form homomers or heteromers. Homomers involve interactions with the same receptor type while heteromers involve interactions between two different GPCRs. These receptor–receptor interactions modulate not only the binding but also the signalling and trafficking properties of individual receptors. Opioid receptor heteromerization has been extensively investigated with the objective of identifying novel therapeutic targets that are as potent as morphine but without the side effects associated with chronic morphine use. In this context, studies have described heteromerization between the different types of opioid receptors and between opioid receptors and a wide range of GPCRs including adrenoceptors, cannabinoid, 5‐HT, metabotropic glutamate and sensory neuron‐specific receptors. Recent advances in the field involving the generation of heteromer‐specific reagents (antibodies or ligands) or of membrane‐permeable peptides that disrupt the heteromer interaction are helping to elucidate the physiological role of opioid receptor heteromers and the contribution of the partner receptor to the side effects associated with opioid use. For example, studies using membrane‐permeable peptides targeting the heteromer interface have implicated μ and δ receptor heteromers in the development of tolerance to morphine, and heteromers of μ and gastrin‐releasing peptide receptors in morphine‐induced itch. In addition, a number of ligands that selectively target opioid receptor heteromers exhibit potent antinociception with a decrease in the side effects commonly associated with morphine use. In this review, we summarize the latest findings regarding the biological and functional characteristics of opioid receptor heteromers both in vitro and in vivo.

Molecular characterization of eluxadoline as a potential ligand targeting mu-delta opioid receptor heteromers

Eluxadoline, an orally active mixed μ opioid receptor (μOR) agonist δ opioid receptor (δOR) antagonist developed for the treatment of diarrhea-predominant irritable bowel syndrome, normalizes gastrointestinal (GI) transit and defecation under conditions of novel environment stress or post-inflammatory altered GI function. Furthermore, compared to loperamide, which is used to treat non-specific diarrhea, the effects of eluxadoline on GI transit occur over a wider dosage range. However, the mechanisms of action of eluxadoline are unclear. In this study, we compared the ability of eluxadoline and loperamide to activate G-protein- and β-arrestin-mediated signaling at μOR homomers or μOR-δOR heteromers in heterologous cells. We also examined the ability of both compounds to reduce castor oil induced diarrhea in wild type (WT) and mice lacking δOR. We find that eluxadoline is more potent than loperamide in eliciting G-protein activity and β-arrestin recruitment in μOR expressing cells. However, in cells expressing μOR-δOR heteromers, the potency of eluxadoline is higher, but its maximal effect is lower than that of loperamide. Moreover, in these cells the signaling mediated by eluxadoline but not loperamide is reduced by μOR-δOR heteromer-selective antibodies. We find that in castor oil-induced diarrhea eluxadoline is more efficacious compared to loperamide in WT mice, and δOR appears to play a role in this process. Taken together these results indicate that eluxadoline behaves as a potent μOR agonist in the absence of δOR, while in the presence of δOR eluxadoline’s effects are mediated through the μOR-δOR heteromer.

Disease-Specific Heteromerization of G-Protein-Coupled Receptors That Target Drugs of Abuse

Drugs of abuse such as morphine or marijuana exert their effects through the activation of G-protein-coupled receptors (GPCRs), the opioid and cannabinoid receptors, respectively. Moreover, interactions between either of these receptors have been shown to be involved in the rewarding effects of drugs of abuse. Recent advances in the field, using a variety of approaches, have demonstrated that many GPCRs, including opioid, cannabinoid, and dopamine receptors, can form associations between different receptor subtypes or with other GPCRs to form heteromeric complexes. The formation of these complexes, in turn, leads to the modulation of the properties of individual protomers. The development of tools that can selectively disrupt GPCR heteromers as well as monoclonal antibodies that can selectively block signaling by specific heteromer pairs has indicated that heteromers involving opioid, cannabinoid, or dopamine receptors may play a role in various disease states. In this review, we describe evidence for opioid, cannabinoid, and dopamine receptor heteromerization and the potential role of GPCR heteromers in pathophysiological conditions.

Identification of GPR83 as the receptor for the neuroendocrine peptide PEN

Functional coupling occurs between PEN-GPR83 and bigLEN-GPR171, ligand-receptor pairs implicated in feeding. PEN adopts GPR83 Neuropeptides produced by proteolytic processing of the protein proSAAS include PEN and bigLEN and are implicated in the regulation of appetite. The receptor for bigLEN is GPR171, which was an orphan G protein–coupled receptor (GPCR) until this ligand was identified. Here, Gomes et al. identify the receptor for PEN as the orphan GPCR GPR83, a receptor implicated in behavior, learning, and metabolic regulation. These two ligand-receptor pairs altered each other’s pharmacological properties. Proximity ligation assays and immunofluorescence colocalization assays in mouse brain indicated that the two receptors were present in some of the same cells in the region of the brain that controls feeding. Because PEN and bigLEN are produced from the same precursor, they are sometimes packaged in the same vesicles and are released together. GPR83 also colocalizes and functionally interacts with the receptor for ghrelin, another peptide involved in regulation of organismal metabolism by the central nervous system. Knowing the ligand for GPR83 will enable the investigation of how GPR83, GPR171, and other receptors interact to control body weight, which has implications for treating metabolic disorders associated with being underweight or overweight. PEN is an abundant peptide in the brain that has been implicated in the regulation of feeding. We identified a receptor for PEN in mouse hypothalamus and Neuro2A cells. PEN bound to and activated GPR83, a G protein (heterotrimeric guanine nucleotide)–binding protein)–coupled receptor (GPCR). Reduction of GPR83 expression in mouse brain and Neuro2A cells reduced PEN binding and signaling, consistent with GPR83 functioning as the major receptor for PEN. In some brain regions, GPR83 colocalized with GPR171, a GPCR that binds the neuropeptide bigLEN, another neuropeptide that is involved in feeding and is generated from the same precursor protein as is PEN. Coexpression of these two receptors in cell lines altered the signaling properties of each receptor, suggesting a functional interaction. Our data established PEN as a neuropeptide that binds GPR83 and suggested that these two ligand-receptor systems—PEN-GPR83 and bigLEN-GPR171—may be functionally coupled in the regulation of feeding.

Endothelin‐converting enzyme 2 differentially regulates opioid receptor activity

Opioid receptor function is modulated by post‐activation events such as receptor endocytosis, recycling and/or degradation. While it is generally understood that the peptide ligand gets co‐endocytosed with the receptor, relatively few studies have investigated the role of the endocytosed peptide and peptide processing enzymes in regulating receptor function. In this study, we focused on endothelin‐converting enzyme 2 (ECE2), a member of the neprilysin family of metallopeptidases that exhibits an acidic pH optimum, localizes to an intracellular compartment and selectively processes neuropeptides including opioid peptides in vitro, and examined its role in modulating μ receptor recycling and resensitization.

Two delta opioid receptor subtypes are functional in single ventral tegmental area neurons, and can interact with the mu opioid receptor

&NA; The mu and delta opioid receptors (MOR and DOR) are highly homologous members of the opioid family of GPCRs. There is evidence that MOR and DOR interact, however the extent to which these interactions occur in vivo and affect synaptic function is unknown. There are two stable DOR subtypes: DPDPE sensitive (DOR1) and deltorphin II sensitive (DOR2); both agonists are blocked by DOR selective antagonists. Robust motivational effects are produced by local actions of both MOR and DOR ligands in the ventral tegmental area (VTA). Here we demonstrate that a majority of both dopaminergic and non‐dopaminergic VTA neurons express combinations of functional DOR1, DOR2, and/or MOR, and that within a single VTA neuron, DOR1, DOR2, and MOR agonists can differentially couple to downstream signaling pathways. As reported for the MOR agonist DAMGO, DPDPE and deltorphin II produced either a predominant K+ dependent hyperpolarization or a Cav2.1 mediated depolarization in different neurons. In some neurons DPDPE and deltorphin II produced opposite responses. Excitation, inhibition, or no effect by DAMGO did not predict the response to DPDPE or deltorphin II, arguing against a MOR‐DOR interaction generating DOR subtypes. However, in a subset of VTA neurons the DOR antagonist TIPP‐&PSgr; augmented DAMGO responses; we also observed DPDPE or deltorphin II responses augmented by the MOR selective antagonist CTAP. These findings directly support the existence of two independent, stable forms of the DOR, and show that MOR and DOR can interact in some neurons to alter downstream signaling. HighlightsIn VTA neurons, DOR agonists can postsynaptically activate a hyperpolarizing K+ channel and/or a depolarizing Cav2.1 current.Individual VTA neurons can respond similarly or differentially to DOR1, DOR2, and MOR activation.Direct opioid receptor agonist responses are observed in both dopamine and non‐dopamine neurons.In a subset of VTA neurons we provide evidence for MOR‐DOR physiological interactions.

Regulation of a opioid receptor chaperone protein, RTP4, by morphine

Signaling by classic analgesics, such as morphine, is governed primarily by the relative abundance of opioid receptors at the cell surface, and this is regulated by receptor delivery to, and retrieval from, the plasma membrane. Although retrieval mechanisms, such as receptor endocytosis, have been extensively investigated, fewer studies have explored mechanisms of receptor maturation and delivery to the plasma membrane. A previous study implicated receptor transporter proteins (RTPs) in the latter process. Since not much is known about regulation of RTP expression, we initiated studies examining the effect of chronic morphine administration on the levels of RTPs in the brain. Among the four RTPs, we detected selective and region-specific changes in RTP4 expression; RTP4 mRNA is significantly upregulated in the hypothalamus compared with other brain regions. We examined whether increased RTP4 expression impacted receptor protein levels and found a significant increase in the abundance of mu opioid receptors (MOPrs) but not other related G protein–coupled receptors (GPCRs, such as delta opioid, CB1 cannabinoid, or D2 dopamine receptors) in hypothalamic membranes from animals chronically treated with morphine. Next, we used a cell culture system to show that RTP4 expression is necessary and sufficient for regulating opioid receptor abundance at the cell surface. Interestingly, selective MOPr-mediated increase in RTP4 expression leads to increases in cell surface levels of MOPr–delta opioid receptor heteromers, and this increase is significantly attenuated by RTP4 small interfering RNA. Together, these results suggest that RTP4 expression is regulated by chronic morphine administration, and this, in turn, regulates opioid receptor cell surface levels and function.