Cloning and characterization of rhesus monkey MCH-R1 and MCH-R2

Rhesus monkey MCH-R1 and MCH-R2 receptors were cloned. Amino acid homology is 98.8% between monkey and human MCH-R1, while monkey and human MCH-R2 are 98% homologous. Binding and intracellular signaling characteristics of the monkey receptors were compared with the human homologues. The results demonstrate that MCH binds to the monkey MCH-R1 receptor with a K(d) of 6.5 nM and monkey MCH-R2 with a K(d) of 2.2 nM similar to K(d) values for human MCH-R1 and MCH-R2. Additionally, monkey MCH-R1 couples through G(i)/G(o) and G(q)-type G proteins similar to human MCH-R1 whereas monkey and human MCH-R2 utilize the G(q) signaling pathway.     

Identification and pharmacological characterization of a novel human melanin-concentrating hormone receptor, mch-r2.

Melanin-concentrating hormone (MCH) is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G protein-coupled receptor family. Recently an orphan receptor, SLC-1, has been identified as an MCH receptor (MCH-R1). Herein we identify and characterize a novel receptor for human MCH (MCH-R2). The receptor is composed of 340 amino acids encoded by a 1023-base pair cDNA and is 35% homologous to SLC-1. (125)I-MCH specifically bound to Chinese hamster ovary cells stably expressing MCH-R2. MCH stimulated dose-dependent increases in intracellular free Ca(2+) and inositol phosphate production in these cells but did not affect cAMP production. The pharmacological profile for mammalian MCH, [Phe(13),Tyr(19)]MCH, and salmon MCH at MCH-R2 differed compared with MCH-R1 as assessed by intracellular signaling and radioligand binding assays. The EC(50) in signaling assays and the IC(50) in radioligand binding assays of salmon MCH was an order of magnitude higher than mammalian MCH at MCH-R2. By comparison, the EC(50) and IC(50) values of salmon MCH and mammalian MCH at MCH-R1 were relatively similar. Blot hybridization revealed exclusive expression of MCH-R2 mRNA in several distinct brain regions, particularly in the cortical area, suggesting the involvement of MCH-R2 in the central regulation of MCH-mediated functions.     

Endogenous receptor for melanin-concentrating hormone in human neuroblastoma Kelly cells

Melanin-concentrating hormone (MCH), a cyclic nonadecapeptide, is predominantly expressed in mammalian neurons located in the zona incerta and lateral hypothalamus. Current interest in MCH relates to its role in the control of feeding behaviour. Two receptors for MCH were recently found: MCH-R(1) and MCH-R(2). We show here by RT-PCR analysis and immunofluorescence studies that the human neuroblastoma cell line Kelly expresses MCH and MCH-R(1) but not MCH-R(2). In competition assays using 125I-labelled MCH an inhibitory concentration 50% (IC(50)) of 76nM was determined for MCH, indicating a high affinity of Kelly cells for MCH. MCH induces mitogen-activated protein kinase (MAPK) phosphorylation in Kelly cells but no increase in the intracellular free Ca(2+) concentration. This suggests that MCH signals via Galpha(i)/Galpha(0) in these cells. The presence and functionality of MCH-R(1) renders this neuronal cell a very useful model for future structure-activity studies in a physiological environment mimicking the human brain for the evaluation of potential appetite-regulating drugs.     

Expression and characterization of melanin-concentrating hormone receptors on mammalian cell lines

The neuropeptide melanin-concentrating hormone (MCH) is expressed in central and peripheral tissues where it participates in the complex network regulating energy homeostasis as well as in other physiologically important functions. Two MCH receptor subtypes, MCH-R1 and MCH-R2, have been cloned which signal through activation of Gi/o/q proteins and hence regulate different intracellular signals, such as inhibition of cAMP formation, stimulation of IP3 production, increase in intracellular free Ca2+ and/or activation of MAP kinases. Most of the data were obtained with cell systems heterologously expressing either of the MCH receptors. Fewer reports exist on studies with cell lines which endogenously express MCH receptors. Here, we describe human and other mammalian cell lines with which MCH receptor activation can be studied under "natural" conditions and we summarize the characteristics and signaling pathways of the MCH receptors in the different cell systems.     

Expression of receptors for melanin-concentrating hormone (MCH) in different tissues and cell lines

Melanin-concentrating hormone (MCH) is a potent orexigenic neuropeptide and a physiological antagonist of alpha-melanocyte-stimulating hormone (alpha-MSH) in the brain as well as at peripheral sites, including the pigmentary systems of specific vertebrates. Two receptor subtypes for MCH, MCH-R1 and MCH-R2, have been cloned, but other receptor subtypes are likely to exist. Based on our own data and the current literature, we have compared the expression of different receptors for MCH in various mammalian cell lines and tissues. Summarizing all data currently available, we conclude that the two cloned MCH receptors, MCH-R1 and MCH-R2, exhibit differences in their expression pattern, although MCH-R1 is generally colocalized in all tissues where MCH-R2 expression is found. It appears that MCH-R1 is more abundant and has a wider distribution pattern than MCH-R2. Other hypothetical MCH-R subtypes may be expressed in specific tissues, e.g., in the pigment cell system.     

Melanin-concentrating hormone and melanin-concentrating hormone receptors in mammalian skin physiopathology

To date, there is a dearth of evidence to support functions for melanin-concentrating hormone (MCH) and melanin-concentrating hormone receptors (MCH-R) in mammalian skin physiology including pigmentation, inflammation and immune responses and skin cell proliferation. Much research is therefore still needed to define the roles of the hormone and its receptors in mammalian skin. This will be a crucial step to identifying pathogenic mechanisms that may involve the MCH/MCH-R system in the context of inflammatory and autoimmune skin diseases as well as skin cancers. The following review summarizes the studies which have been carried out to examine the expression and function of MCH and MCH-R in mammalian skin. Recent findings with regard to humoral immune responses to the MCH-R1 in patients with the skin depigmenting disease vitiligo are also discussed.     

Regulation of food intake by melanin-concentrating hormone in goldfish

Melanin-concentrating hormone (MCH), originally discovered in the teleost pituitary, is a hypothalamic neuropeptide involved in the regulation of body color in fish. Although MCH is also present in the mammalian brain, it has no evident function in providing pigmentation. Instead, this peptide is now recognized to be one of the key neuropeptides that act as appetite enhancers in mammals such as rodents and primates. Although there has been little information about the central action of MCH on appetite in fish, recent studies have indicated that, in goldfish, MCH acts as an anorexigenic neuropeptide, modulating the α-melanocyte-stimulating hormone signaling pathway through neuronal interaction. These observations indicate that there may be major differences in the mode of action of MCH between fish and mammals. This paper reviews what is currently known about the regulation of food intake by MCH in fish, especially the goldfish.     

Melanin-concentrating hormone stimulates human growth hormone secretion: a novel effect of MCH on the hypothalamic-pituitary axis

Melanin-concentrating hormone (MCH), a 19-amino acid orexigenic (appetite-stimulating) hypothalamic peptide, is an important regulator of energy homeostasis. It is cleaved from its precursor prepro-MCH (ppMCH) along with several other neuropeptides whose roles are not fully defined. Because pituitary hormones such as growth hormone (GH), ACTH, and thyroid-stimulating hormone affect body weight and composition, appetite, insulin sensitivity, and lipoprotein metabolism, we investigated whether MCH exerts direct effects on the human pituitary to regulate energy balance using dispersed human fetal pituitaries (21-22 wk gestation) and cultured GH-secreting adenomas. We found that MCH receptor-1 (MCH-R1), but not MCH receptor-2, is expressed in both normal (fetal and adult) human pituitary tissues and in GH cell adenomas. MCH (10 nM) stimulated GH release from human fetal pituitary cultures by up to 62% during a 4-h incubation (P < 0.05). Interestingly, neuropeptide EI (10 nM), which is also cleaved from ppMCH, increased human GH secretion by up to 124% in fetal pituitaries. A milder, albeit significant, induction of GH secretion by MCH (20%) was seen in cultured GH-secreting pituitary adenomas. A comparable stimulation of GH secretion was seen when cultured mouse pituitary cells were treated with MCH. Treatment of cultured GH adenoma cells with MCH (100 nM) induced extracellular signal-regulated kinases 1 and 2 phosphorylation, suggesting activation of MCH-R1. In aggregate, these data suggest that MCH may regulate pituitary GH secretion and imply a potential cross-talk mechanism between appetite-regulating neuropeptides and pituitary hormones.     

Solid-phase synthesis and structure-activity relationships of novel biarylethers as melanin-concentrating hormone receptor-1 antagonists

Melanin-concentrating hormone (MCH) is a cyclic 19 amino acid orexigenic neuropeptide. The action of MCH on feeding is thought to involve the activation of its respective G protein-coupled receptor MCH-R1. Consequently, antagonists that block MCH regulated MCH-R1 activity may provide a viable approach to the treatment of diet-induced obesity. This communication reports the discovery of a novel MCH-R1 receptor antagonist, the biarylether 7, identified through high throughput screening. The solid-phase synthesis and structure-activity relationship of related analogs is described.     

Different structural requirements for melanin-concentrating hormone (MCH) interacting with rat MCH-R1 (SLC-1) and mouse B16 cell MCH-R

Melanin-concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food-intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH-R1 and MCH-R2, are thought to mediate mainly the central effects of MCH, the MCH-R on pigment cells has not yet been identified, although in some studies MCH-R1 was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure-activity study in which 12 MCH peptides were tested on rat MCH-R1 and mouse B16 melanoma cell MCH-R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK-293 cells expressing rat MCH-R1 (SLC-1), the radioligand was [125I]-[Tyr13]-MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH-R, the analog [125I]-[D-Phe13, Tyr19]-MCH served as radioligand. The bioassay used for MCH-R1 was intracellular Ca2+ mobilization quantified with the FLIPR instrument, whereas for B16 MCH-R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrease of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side-chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N-terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5- to 10-fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH-R1 and B16 MCH-R was however observed with modifications at position 13 of MCH: whereas L-Phe13 in [Phe13, Tyr19]-MCH was well tolerated by both MCH-R1 and B16 MCH-R, change of configuration to D-Phe13 in [D-Phe13, Tyr19]-MCH or [D-Phe13]-MCH led to a complete loss of biological activity and to a 5- to 10-fold lower binding activity with MCH-R1. By contrast, the D-Phe13 residue increased the affinity of [D-Phe13, Tyr19]-MCH to B16 MCH-R about 10-fold and elicited MAP kinase activation as observed with [Phe13, Tyr19]-MCH or MCH. These data demonstrate that ligand recognition by B16 MCH-R differs from that of MCH-R1 in several respects, indicating that the B16 MCH-R represents an MCH-R subtype different from MCH-R1.     

Cellular models for the study of the pharmacology and signaling of melanin-concentrating hormone receptors

Cellular models for the study of the neuropeptide melanin-concentrating hormone (MCH) have become indispensable tools for pharmacological profiling and signaling analysis of MCH and its synthetic analogues. Although expression of MCH receptors is most abundant in the brain, MCH-R(1) is also found in different peripheral tissues. Therefore, not only cell lines derived from nervous tissue but also from peripheral tissues that naturally express MCH receptors have been used to study receptor signaling and regulation. For screening of novel compounds, however, heterologous expression of MCH-R(1) or MCH-R(2) genes in HEK293, Chinese hamster ovary, COS-7, or 3T3-L1 cells, or amplified MCH-R(1) expression/signaling in IRM23 cells transfected with the G(q) protein gene are the preferred tools because of more distinct pharmacological effects induced by MCH, which include inhibition of cAMP formation, stimulation of inositol triphosphate production, increase in intracellular free Ca(2+) and/or activation of mitogen-activated protein kinases. Most of the published data originate from this type of model system, whereas data based on studies with cell lines endogenously expressing MCH receptors are more limited. This review presents an update on the different cellular models currently used for the analysis of MCH receptor interaction and signaling.     

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of μ-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the  26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout μ-calpain and calpastatin were only partly purified; identity of trout μ-calpain was confirmed by labeling with antibodies to bovine skeletal muscle μ-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle μ- and m-calpain. Trout μ-calpain requires 4.4 ± 2.8 μM and trout m-calpain requires 585 ± 51 μM Ca²⁺ for half-maximal activity, similar to the Ca²⁺ requirements of μ- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.     

EXPRESSION OF RECEPTORS FOR MELANIN-CONCENTRATING HORMONE (MCH) IN DIFFERENT TISSUES AND CELL LINES

ABSTRACT

Melanin-concentrating hormone (MCH) is a potent orexigenic neuropeptide and a physiological antagonist of α-melanocyte-stimulating hormone (α-MSH) in the brain as well as at peripheral sites, including the pigmentary systems of specific vertebrates. Two receptor subtypes for MCH, MCH-R₁ and MCH-R₂, have been cloned, but other receptor subtypes are likely to exist. Based on our own data and the current literature, we have compared the expression of different receptors for MCH in various mammalian cell lines and tissues. Summarizing all data currently available, we conclude that the two cloned MCH receptors, MCH-R₁ and MCH-R₂, exhibit differences in their expression pattern, although MCH-R₁ is generally colocalized in all tissues where MCH-R₂ expression is found. It appears that MCH-R₁ is more abundant and has a wider distribution pattern than MCH-R₂. Other hypothetical MCH-R subtypes may be expressed in specific tissues, e.g., in the pigment cell system.     

Comparative analyses of the pigment-aggregating and -dispersing actions of MCH on fish chromatophores

In melanophores of the peppered catfish and the Nile tilapia, melanin-concentrating hormone (MCH) at low doses (<1 μM) induced pigment aggregation, and the aggregated state was maintained in the presence of MCH. However, at higher MCH concentrations (such as 1 and 10 μM), pigment aggregation was immediately followed by some re-dispersion, even in the continued presence of MCH, which led to an apparent decrease in aggregation. This pigment-dispersing activity at higher concentrations of MCH required extracellular Ca²⁺ ions. By contrast, medaka melanophores responded to MCH only by pigment aggregation, even at the highest concentration employed (10 μM). Since it is known that medaka melanophores possess specific receptors for α-melanophore-stimulating hormone (α-MSH), the possibility that interaction between MSH receptors and MCH at high doses in the presence of Ca²⁺ might cause pigment dispersion is ruled out. Cyclic MCH analogs, MCH (1–14) and MCH (5–17), failed to induce pigment dispersion, whereas they induced aggregation of melanin granules. These results suggest that another type of MCH receptor that mediates pigment dispersion is present in catfish and tilapia melanophores, and that intact MCH may be the only molecule that can bind to these receptors. Determinations of cAMP content in melanophores, which were isolated from the skin of three fish species and treated with 10 nM or 10 μM MCH, indicate that MCH receptors mediating aggregation may be coupled with Gi protein, whereas MCH receptors that mediate dispersion may be linked to Gs. The response of erythrophores, xanthophores and leucophores to MCH at various concentrations was also examined, and the results suggest that the distribution patterns of the two types of MCH receptors may differ among fish species and among types of chromatophore in the same fish.     

Lead optimization of 4-(dimethylamino)quinazolines, potent and selective antagonists for the melanin-concentrating hormone receptor 1

The optimization of a series of 4-(dimethylamino)quinazoline antagonists of the melanin-concentrating hormone receptor 1 (MCH-R1) is described. The combination of the elaboration of both the linker portion and the terminal phenyl ring provided N-(cis-4-{[4-(dimethylamino)quinazolin-2-yl]amino}cyclohexyl)-3,4-difluorobenzamide hydrochloride 28 (ATC0175), which showed excellent antagonist activity at the MCH-R1 (IC50 = 3.4 nM) as well as good selectivity over the Y5 and the alpha2A receptors.     

Identification of a new small molecule chemotype of Melanin Concentrating Hormone Receptor-1 antagonists using pharmacophore-based virtual screening

MCH receptor is a G protein-coupled receptor with two subtypes R1 and R2. Many studies have demonstrated the role of MCH-R1 in feeding and energy homeostasis. It has been proven that oral administration of small molecule MCH-R1 antagonists significantly reduces food intake and causes a dose-dependent weight loss. In this study, two ligand-based pharmacophores were developed and validated based on recently published MCH-R1 antagonists with diverse structures. Successful pharmacophores had one hydrogen bond acceptor, one positive ionizable, one ring aromatic and two or three hydrophobic groups. These 3D-QSAR models were used for virtual screening of the ZINC chemical database resulting in the identification of nine compounds with more than 50% displacement of radiolabeled MCH at a 20 μM concentration. Moreover, four of these compounds showed antagonistic activities in Aequorin functional assay, including MH-3 which is the first MCH-R1 antagonist based on a diazaspiro[4.5]decane scaffold. The most active compounds were also docked into our previously published MCH-R1 homology model to gain insights into their binding determinants. These compounds could represent a viable starting scaffold for the design of potent MCH-R1 antagonists with improved pharmacokinetic properties as an effective treatment for obesity.     

Evidence for alternative splicing of a dopamine D2 receptor in a teleost

Previous attempts at identifying an alternatively spliced dopamine (DA) D2 receptor in teleosts have proven unsuccessful. We provide evidence of a splicing event of a goldfish D2 (gfD2b1) receptor in the neuroendocrine brain of adult goldfish that produces a spliced short isoform (gfD2b1S). We also identify an additional novel D2b paralog (gfD2b2) that does not appear to be alternatively spliced in adult fish during the reproductive cycle. Relatively high mRNA levels of gfD2b1S were observed in the neuroendocrine brain and pituitary of sexually immature fish compared with sexually regressing fish. Real-time RT-PCR revealed that intraperitoneal injection of either SCH 23390 or sulpiride-D1- or D2-specific antagonists, respectively-decreased mRNA levels of gfD2b1S by 3.9-fold without affecting the unspliced isoforms. We suggest that the expression of the spliced D2 receptor modulates the inhibitory tone of DA throughout the reproductive cycle. The deduced amino acid sequence of gfD2b1S lacks 29 amino acids in the same region as the short isoform of mammalian D2. We propose that the gfD2b1S splice variant is the teleost ortholog of mammalian D2S. The hypothesis that D2 receptor splicing is a relatively recent innovation in higher tetrapods is not supported by our results.