Melanin-Concentrating Hormone Receptor 1 (MCHR1)
MCHR1 is a receptor for melanin-concentrating hormone (MCH). MCH was originally discovered from salmon pituitary as a 17-amino acid cyclic peptide (Kawauchi et al. 1983); in teleost fish, MCH causes the aggregation of melanosomes in the scales, which lightens skin color. The mammalian form of MCH is highly homologous to that of the teleost, but its expression is detected at high levels in the lateral hypothalamus, which is classically a feeding center (Bittencourt et al. 1992). Based on physiological and genetic experiments, MCH is strongly implicated in the regulation of feeding behavior and energy metabolism (Shimada et al. 1998).
Initial efforts to identify the receptor for MCH were based on binding assays by using radiolabeled MCH. MCH binding was detected in mouse melanoma cells in vitro and in rat brain, as well as in some peripheral tissues. However, the identity of MCH receptor was unknown until it was revealed to be an orphan G-protein-coupled receptor (GPCR), SLC-1. SLC-1 was cloned from rat DNA by PCR amplification and characterized as a GPCR with 40% amino acid identity in the transmembrane (TM) regions of the five known human somatostatin receptors. SLC-1 gene is intron-less in its open reading frame, encodes a receptor of 353 amino acids in the rat, and maps to chromosome 22, q13.3 in humans. The MCH receptor was discovered by two approaches. The first used an orphan receptor strategy (Civelli 2012). In principle, an orphan GPCR is transfected into cell lines, which are then exposed to biological extracts to provoke receptor-induced changes in intracellular signaling, such as second-messenger activation. An active component can be isolated by monitoring changes in messenger systems such as calcium mobilization. Three groups used rat whole brain extract as material to identify the endogenous agonist for SLC-1 by measuring three second-messenger responses: (1) calcium mobilization in CHO cells transiently transfected with a chimeric Gα protein (Gαq/i3) and rat SLC-1, (2) cyclic AMP (cAMP) inhibition assays in CHO cells stably expressing human SLC-1, and (3) G-protein-coupled inwardly-rectifying potassium channel (GIRK)-mediated current in Xenopus oocytes expressing rat SLC-1 and GIRK. These groups independently reached to the same conclusion that MCH is the only ligand from rat brain extract to activate either rat or human SLC-1 receptor. Two other groups used the reverse pharmacology approach to identify a ligand for SLC-1 receptor. A compound library containing more than 500 known bioactive substances was used to search for any known ligand that has activity at the human SLC-1 receptor. Among those, MCH was the only compound to activate the SLC-1 receptor with nanomolar affinity. Finally, the conclusion from all five groups was that SLC-1 is a receptor for MCH (Chambers et al. 1999; Saito et al. 1999). A second MCH receptor subtype, designated MCHR2, was identified by six independent groups using human genomic sequence searches (Mori et al. 2001). It shares 31.5% amino acid identity with MCHR1 and shows high-affinity MCH binding. In contrast to MCHR1, MCHR2 couples to Gαq but not to Gαi/o proteins. The human MCHR2 gene is mapped to the long arm of chromosome 6 at band 6q16.2–16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. MCHR2 is a pseudogene in rodent species, but is functional in dogs, ferrets, rhesus monkeys, and humans (Tan et al. 2002). Thus, MCH acts solely through the MCHR1 in rodents.
Physiological Functions in the Central Nervous System
While MCHR1 is present in several peripheral tissues, such as adipose and islets of Langerhans, MCHR1 mRNA is detected at very high levels in the brain (Hervieu et al. 2000; Saito et al. 2001). There were clear mRNA signals in the olfactory nucleus, olfactory tubercle, and piriform cortex. Strong labeling is detected in several limbic structures, including the hippocampal formation, septum, and amygdala. Distinct MCHR1 signals have also been detected in the locus coeruleus, the major noradrenergic nucleus. Furthermore, the nucleus accumbens shell, which plays a role in the regulation of motivation and reward, contains very high levels of MCHR1 mRNA. Moderate mRNA expression is found in various hypothalamic regions including the arcuate nucleus, ventromedial hypothalamic nucleus, and paraventricular nucleus that are involved in feeding and energy homeostasis. Autoradiographic analysis using a MCHR1-specific radioligand also showed intense binding to the rat nucleus accumbens, caudate-putamen, and piriform cortex (Able et al. 2009). This diffuse localization indicates the diversity of physiological functions of the MCH-MCHR1 system.
Various rodent genetic models have implicated the MCH-MCHR1 system as an important factor in the control of feeding and energy metabolism (MacNeil 2013). For example, mice and rats lacking prepro-MCH are hypophagic and lean, with increased resting energy expenditure. Furthermore, the selective loss of MCH neurons results in late-onset leanness of body weight in mice (Alon and Friedman 2006), and the deletion of the MCHR1 gene in mice produces increased locomotor activity and metabolic rate, such that the mice display mild overeating but remain lean with lower body fat (Chen et al. 2002; Marsh et al. 2002). Such hyperactivity could be due to an increase in dopaminergic sensitivity in the nucleus accumbens. A series of in vivo pharmacological studies used MCHR1-selective antagonists provided further evidence for the role of MCHR1 in energy homeostasis, while several non-peptide MCHR1 antagonists have shown efficacy in a variety of rodent models for acute food intake (Takekawa et al. 2002; Luthin 2007). A number of MCHR1 antagonists caused sustained and dose-dependent efficacy in chronic models of obesity. However, there are few reports confirming the specificity of these MCHR1 antagonists in MCHR1 knockout mice, and further research using MCHR1-specific models is required to fully understand the efficacy of MCHR1 antagonists in food intake systems.
Some MCHR1 antagonists exhibit anxiolytic- and antidepressant-like behavior when tested in a variety of preclinical behavioral models such as the forced-swim test, elevated plus-maze, stress-induced hyperthermia paradigm, and social interaction test (Borowsky et al. 2002; Antal-Zimanyi and Khawaja 2009). Consistent with this, MCHR1 knockout mice display reduced anxiety responses. Recently, a central action of the MCHR1 has also been revealed a role in arousal and sleep. Both optogenetic and pharmacological studies suggested that MCHR1-mediated signaling promotes paradoxical (REM) sleep. By contrast, MCHR1-deficient mice exhibit a hypersomnia-like phenotype, characterized by higher amounts of REM sleep during the active dark phase, especially after sleep deprivation. These conflicting reports could highlight genetic differences in sleep mechanisms, or alternatively that MCHR1 may mediate auto-inhibition of MCH neurons localized in the lateral hypothalamus (Richter et al. 2014). Such loss of this inhibitory feedback loop in MCHR1 knockout mice would enable MCH neurons to release its co-transmitter, GABA, which could initiate REM sleep episodes and prolong their duration.
Regulation of MCHR1 Activity
The extracellular N-terminal domain of rat MCHR1 has three N-linked glycosylation sites (Asn13, Asn16, and Asn23). These residues, particularly Asn23, are necessary for receptor expression at the cell surface, ligand binding, and signal transduction. Molecular modeling of the MCH-MCHR1 identified that Asp123 in the third transmembrane domain plays a crucial role in ligand binding and receptor activation (Macdonald et al. 2000). Indeed, substitution of Asp123 resulted in a loss of detectable MCH binding and of MCH-stimulated calcium mobilization, despite having no effect on cell surface expression. In the second intracellular loop, mutation of Arg155 (R155Q) led to a drastic loss of Gαi/o- and Gαq-mediated signaling, although no significant change was observed in ligand-binding activity (Saito et al. 2005). A T255A mutant, which substitutes alanine for a threonine residue located at the junction of the third intracellular loop and the sixth transmembrane domain, was largely retained in the endoplasmic reticulum causing a dramatic loss of cell surface expression (Fan et al. 2005). In helix 8 – a short cytoplasmic amphiphilic helical domain in the proximal C-terminal tail – two dibasic amino acids (Arg319 and Lys320) are essential for Gαi/o- and Gαq-mediated signaling (Tetsuka et al. 2004), while the distal part of the C-terminal tail is necessary for receptor internalization. Indeed, triple substitution of T317A/S325A/T342A partially prevents MCH-induced receptor internalization through PKC and β-arrestin 2-dependent processes without affecting signal transduction.
Mammalian MCHR1 couples to both Gαi/o and Gαq, whereas goldfish MCHR1 exclusively couples to Gαq. By analyzing sequence alignments between those two MCHR1 variants, two mutants with multiple substitutions in either the second intracellular loop or in the third intracellular loop and fifth transmembrane domain, respectively (Fig. 3b), were found to exhibit reduced Gαi/o activity without altering Gαq-mediated signaling (Hamamoto et al. 2015). Thus, two regions (S150/S151/T152/K153/K156/S158 and Y228/V229/R234/A242/S243/T257) appear to independently affect Gαi/o-protein preference. Meanwhile, point mutation of F318K, located in the conserved NPxxY(x)5,6F motif, produces a receptor that efficiently and selectively signal via Gαq, with no concomitant Gαi/o-mediated signaling (Hamamoto et al. 2012).
The signaling properties of MCHR1 are depending on the cellular context. Such fine-tuning of receptor function can be attributed to receptor-selective partners. Regulator of G-protein signaling (RGS) proteins are direct modulators of G-protein activity, binding to the activated form of Gα and enhancing its GTPase activity, which inhibits GPCR-mediated signaling. Among 30 different RGS proteins, RGS8 significantly suppresses MCHR1-induced calcium mobilization (Miyamoto-Matsubara et al. 2008). Arg253 and Arg256, which are located at the distal end of the third intracellular loop, are structurally essential residues for direct interaction with the N-terminus of RGS8 (Fig. 3a). Moreover, the actin- and intermediate filament-binding protein, periplakin, and the neurite outgrowth-related factor, neurochondrin, both interact with the proximal C-terminus of MCHR1 (but not with the third intracellular loop), resulting in reduced MCH-mediated signaling (Murdoch et al. 2005). Interactions of MCHR1 with neurochondrin and periplakin were competitive, indicating that these proteins bind to overlapping regions of MCHR1.
MCHR1 Localized in Primary Cilia
Primary cilia are specialized microtubule-based organelles that project from the cell surface in most vertebrate cells. These cilia convert extracellular signals into a cellular response, thereby functioning as sensory antennae. The physiological importance of neuronal cilia is highlighted by rare human genetic diseases involving primary cilia (ciliopathies), such as autosomal recessive Bardet-Biedl syndrome (BBS) and Alstrom syndrome, in which patients exhibit truncal obesity, renal dysfunction, and behavioral dysfunction (Hildebrandt et al. 2011). Primary cilia also serve as signaling hubs through selective interactions with ion channels and conventional (nonolfactory) GPCRs (Schou et al. 2015). Among the many GPCRs, only a small subset (that includes MCHR1) is highly enriched in neuronal cilia. MCHR1 fails to accumulate in the ciliary membrane in distinct brain regions including hippocampus and nucleus accumbens in both BBS2 and BBS4 knockout mice (Berbari et al. 2008). Altered MCHR1 signaling in the ciliary membrane lacking this receptor could lead to irregular activation or downregulation of signaling in these mutant mice. Because BBS is associated with hyperphagia-induced obesity, specific signaling via ciliary MCHR1 may be functionally important in the regulation of feeding and energy homeostasis. Furthermore, a recent report describes that MCHR1-mediated signaling effectively induces shortening of the ciliary length via a Gαi/o-dependent Akt pathway in hRPE1 cells (Hamamoto et al. 2016). This may suggest that the MCH-MCHR1 axis can modulate the sensitivity of cells to external environments – including satiety signals – by controlling cilia length in a non-synaptic-dependent manner. Neuronal cilia were significantly shortened in the hypothalamus of diet-induced obese mice and ob/ob leptin-deficient obese mice and in the hippocampus and amygdala of BBS4 knockout mice. Further characterization of MCHR1 as a ciliary GPCR may reveal further evidence of a potential molecular mechanism link ciliary length control with obesity.
Considerable literature has accumulated to support the involvement of the hypothalamic neuropeptide, MCH, in the regulation of food intake. The identification of a specific receptor for MCH, found to be the previously identified orphan GPCR, SLC-1 (MCHR1), had an immediate impact, and the importance of the MCH-MCHR1 axis in feeding and energy expenditure in vivo was confirmed through the publication of extensive studies using MCHR1-selective antagonists and genetically engineered animals. MCHR1 mRNA is widely expressed in the brain, suggesting a role for the MCH-MCHR1 axis in diverse physiological functions and behavioral outcomes such as anxiety, stress, and sleep/wakefulness responses. When MCHR1 is transfected into mammalian cells, MCHR1 activates second messengers by coupling to Gαi/o and Gαq proteins. Using such heterologous expression systems, site-directed mutagenesis studies have been performed which have provided deep insight into the structure-function relationships of the MCHR1. These studies may aid the development of a novel antagonist or allosteric modulator of MCHR1 signaling, which could revolutionize our understanding of the physiological roles of the endogenous MCHR1.
- Able SL, Ivarsson M, Fish RL, Clarke TL, McCourt C, Duckworth JM, Napier C, Katugampola SD. Localisation of melanin-concentrating hormone receptor 1 in rat brain and evidence that sleep parameters are not altered despite high central receptor occupancy. Eur J Pharmacol. 2009;616:101–6.PubMedCrossRefGoogle Scholar
- Borowsky B, Durkin MM, Ogozalek K, Marzabadi MR, DeLeon J, Lagu B, Heurich R, Lichtblau H, Shaposhnik Z, Daniewska I, Blackburn TP, Branchek TA, Gerald C, Vaysse PJ, Forray C. Antidepressant, anxiolytic and anorectic effects of a melanin-concentrating hormone-1 receptor antagonist. Nat Med. 2002;8:825–30.PubMedCrossRefGoogle Scholar
- Chambers J, Ames RS, Bergsma D, Muir A, Fitzgerald LR, Hervieu G, Dytko GM, Foley JJ, Martin J, Liu WS, Park J, Ellis JC, Ganguly S, Konchar S, Cluderay J, Leslie R, Wilson S, Sarau HM. Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1. Nature. 1999;400:261–5.PubMedCrossRefGoogle Scholar
- Hamamoto A, Horikawa M, Saho T, Saito Y. Mutation of Phe318 within the NPxxY(x)(5,6)F motif in melanin-concentrating hormone receptor 1 results in an efficient signaling activity. Front Endocrinol (Lausanne). 2012;26:147.Google Scholar
- Macneil DJ. The role of melanin-concentrating hormone and its receptors in energy homeostasis. Front Endocrinol (Lausanne). 2013;4:49.Google Scholar
- Marsh DJ, Weingarth DT, Novi DE, Chen HY, Trumbauer ME, Chen AS, Guan XM, Jiang MM, Feng Y, Camacho RE, Shen Z, Frazier EZ, Yu H, Metzger JM, Kuca SJ, Shearman LP, Gopal-Truter S, MacNeil DJ, Strack AM, MacIntyre DE, Van der Ploeg LH, Qian S. Melanin-concentrating hormone 1 receptor-deficient mice are lean, hyperactive, and hyperphagic and have altered metabolism. Proc Natl Acad Sci U S A. 2002;99:3240–5.Google Scholar
- Tan CP, Sano H, Iwaasa H, Pan J, Sailer AW, Hreniuk DL, Feighner SD, Palyha OC, Pong SS, Figueroa DJ, Austin CP, Jiang MM, Yu H, Ito J, Ito M, Ito M, Guan XM, MacNeil DJ, Kanatani A, Van der Ploeg LH, Howard AD. Melanin-concentrating hormone receptor subtypes 1 and 2: species-specific gene expression. Genomics. 2002;79:785–92.PubMedCrossRefGoogle Scholar