Neuropeptide PEN and Its Receptor GPR83: Distribution, Signaling, and Regulation.

Neuropeptides are chemical messengers that act to regulate a number of physiological processes, including feeding, reward, pain, and memory, among others. PEN is one of the most abundant hypothalamic neuropeptides; however, until recently, its target receptor remained unknown. In this Review, we summarize recent developments in research focusing on PEN and its receptor GPR83. We describe the studies leading to the deorphanization of GPR83 as the receptor for PEN. We also describe the signaling mediated by the PEN-GPR83 system, as well as the physiological roles in which PEN-GPR83 has been implicated. As studies have suggested a role for the PEN-GPR83 system in food intake and body weight regulation, as well as in drug addiction and reward disorders, a thorough understanding of this novel neuropeptide-receptor system will help identify novel therapeutic targets to treat pathophysiological conditions involving PEN-GPR83.

Identification of GPR83 as the receptor for the neuroendocrine peptide PEN.

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.

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 ß-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 ß-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.

Orally active opioid µ/δ dual agonist MGM-16, a derivative of the indole alkaloid mitragynine, exhibits potent antiallodynic effect on neuropathic pain in mice.

(E)-Methyl 2-((2S,3S,7aS,12bS)-3-ethyl-7a-hydroxy-8-methoxy-1,2,3,4,6,7,7a,12b-octahydroindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate (7-hydroxymitragynine), a main active constituent of the traditional herbal medicine Mitragyna speciosa, is an indole alkaloid that is structurally different from morphine. 7-Hydroxymitragynine induces a potent antinociceptive effect on mouse acute pain through µ-opioid receptors. In this study, we developed dual-acting µ- and δ-opioid agonists MGM-15 and MGM-16 from 7-hydroxymitragynine for the treatment of acute and chronic pain. MGM-16 showed a higher potency than that of 7-hydroxymitragynine and MGM-15 in in vitro and in vivo assays. MGM-16 exhibited a high affinity for µ- and δ-opioid receptors, with K(i) values of 2.1 and 7.0 nM, respectively. MGM-16 showed µ- and δ-opioid full agonistic effects in a guanosine 5'-O-(3-[(35)S]thiotriphosphate) binding assay and in a functional test using electrically elicited guinea pig ileum and mouse vas deferens contractions. Systemic administration of MGM-16 produced antinociceptive effects in a mouse acute pain model and antiallodynic effects in a chronic pain model. The antinociceptive effect of MGM-16 was approximately 240 times more potent than that of morphine in a mouse tail-flick test, and its antiallodynic effect was approximately 100 times more potent than that of gabapentin in partial sciatic nerve-ligated mice, especially with oral administration. The antinociceptive effect of MGM-16 was completely and partially blocked by the µ-selective antagonist ß-funaltrexamine hydrochloride (ß-FNA) and by the δ-selective antagonist naltrindole, respectively, in a tail-flick test. The antiallodynic effect of MGM-16 was completely blocked by ß-FNA and naltrindole in a neuropathic pain model. These findings suggest that MGM-16 could become a class of a compound with potential therapeutic utility for treating neuropathic pain.

Antibodies against G-protein coupled receptors: novel uses in screening and drug development.

Antibodies are components of the body's humoral immune system that are generated in response to foreign pathogens. Modern biomedical research has employed these very specific and efficient molecules designed by nature in the diagnosis of diseases, localization of gene products as well as in the rapid screening of targets for drug discovery and testing. In addition, the introduction of antibodies with fluorescent or enzymatic tags has significantly contributed to advances in imaging and microarray technology, which are revolutionizing disease research and the search for effective therapeutics. More recently antibodies have been used in the isolation of dimeric G protein-coupled receptor (GPCR) complexes. In this review, we discuss antibodies as powerful research tools for studying GPCRs, and their potential to be developed as drugs themselves.

Peptidomics of Cpefat/fat mouse hypothalamus and striatum: effect of chronic morphine administration.

Chronic morphine administration is known to affect several neuropeptide systems, and this could contribute to the behavioral effects of opiates. To quantitate global changes in neuropeptide levels upon chronic morphine administration, we took advantage of a method that allows selective isolation of neuropeptides from brains of mice lacking carboxypeptidase E (Cpefat/fat mice), a critical enzyme in the generation of many neuroendocrine peptides. We used a differential labeling procedure with stable isotopic tags and mass spectrometry to quantitate the relative changes in a number of hypothalamic and striatal peptides in Cpefat/fat mice chronically treated with morphine. A total of 27 distinct peptides were detected in hypothalamus and striatum. Of these, 27 were identified by mass spectrometry-based sequencing, 1 was tentatively identified by the mass and charge, and 9 were not identified. The identified peptides included fragments of proenkephalin, prothyrotropin-releasing hormone, secretogranin II, chromogranin Aand B, protachykinin B, provasopressin, promelanin concentrating hormone, and pro-SAAS. Upon morphine administration, although the levels of most of the peptides were unaltered (within a factor of 1.3 to 0.7 compared with saline control), the levels of a small number of peptides did show consistent changes (increased or decreased by 1.3-fold or more) in hypothalamus and/or striatum. Taken together, these results provide interesting insights into endogenous neuropeptide systems that are modulated by morphine and suggest further experiments to link candidate peptides with long-term effects of morphine.

Quantification of VGF- and pro-SAAS-derived peptides in endocrine tissues and the brain, and their regulation by diet and cold stress.

Two novel granin-like polypeptides, VGF and pro-SAAS, which are stored in and released from secretory vesicles and are expressed widely in nervous, endocrine, and neuroendocrine tissues, play roles in the regulation of body weight, feeding, and energy expenditure. Both VGF and pro-SAAS are cleaved into peptide fragments, several of which are biologically active. We utilized a highly sensitive and specific radioimmunoassay (RIA) to immunoreactive, pro-SAAS-derived PEN peptides, developed another against immunoreactive, VGF-derived AQEE30 peptides, and quantified these peptides in various mouse tissues and brain regions. Immunoreactive AQEE30 was most abundant in the pituitary, while brain levels were highest in hypothalamus, striatum, and frontal cortex. Immunoreactive PEN levels were highest in the pancreas and spinal cord, and in brain, PEN was most abundant in striatum, hippocampus, pons and medulla, and cortex. Since both peptides were expressed in hypothalamus, a region of the brain that controls feeding and energy expenditure, double label immunofluorescence studies were employed. These demonstrated that 42% of hypothalamic arcuate neurons coexpress VGF and SAAS peptides, and that the intracellular distributions of these peptides in arcuate neurons differed. By RIA, cold stress increased immunoreactive AQEE30 and PEN peptide levels in female but not male hypothalamus, while a high fat diet increased AQEE30 and PEN peptide levels in female but not male hippocampus. VGF and SAAS-derived peptides are therefore widely expressed in endocrine, neuroendocrine, and neural tissues, can be accurately quantified by RIA, and are differentially regulated in the brain by diet and cold stress.

Targeting opioid receptor heterodimers: strategies for screening and drug development.

G-protein-coupled receptors are a major target for the development of new marketable drugs. A growing number of studies have shown that these receptors could bind to their ligands, signal, and be internalized as dimers. Most of the evidence comes from in vitro studies, but recent studies using animal models support an important role for dimerization in vivo and in human pathologies. It is therefore becoming highly relevant to include dimerization in screening campaigns: the increased complexity reached by the ability to target 2 receptors should lead to the identification of more specific hits that could be developed into drugs with fewer side effects. In this review, we have summarized results from a series of studies characterizing the properties of G-protein-coupled receptor dimers using both in vitro and in vivo systems. Since opioid receptors exist as dimers and heterodimerization modulates their pharmacology, we have used them as a model system to develop strategies for the identification of compounds that will specifically bind and activate opioid receptor heterodimers: such compounds could represent the next generation of pain relievers with decreased side effects, including reduced drug abuse liability.