Different Structural Requirements for Melanin‐Concentrating Hormone (MCH) Interacting with Rat MCH‐R1 (SLC‐1) and Mouse B16 Cell MCH‐R

Abstract

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 decrase 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.     

Melanin concentrating hormone exhibits both MSH and MCH activities on individual melanophores

Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val (melanin concentrating hormone, MCH) and several fragment analogs (MCH1-14, MCH5-17, MCH5-14) were synthesized and their biological activities determined in a very sensitive fish skin bioassay. The potency ranking and minimum effective doses of the peptides were determined to be: MCH1-17 (10(-12)M) greater than less than MCH5-17 (10(-12)M) greater than MCH1-14 (10(-11)M) greater than MCH5-14 (2 X 10(-10)M). The melanosome aggregating activity of MCH could be completely reversed by a 100-fold higher concentration of pounds-MSH. MCH was self-antagonized in a dose-related manner by higher concentrations of the peptide as was the activity of the MCH1-14 fragment analog. The MCH activities of the MCH5-17 and MCH5-14 analogs were not compromised by even the highest concentrations of the peptides employed. The MSH-like activity of MCH appears to relate to the N-terminus of the peptide whereas MCH activity is more a function of the C-terminus of the hormone. Self-antagonism of MCH at high concentrations appears to relate to the N-terminal tetrapeptide, which is responsible for the intrinsic MSH-like activity of the hormone.     

Ionic requirements for melanin concentrating hormone (MCH) actions on teleost Poecilia reticulata melanophores

Melanin concentrating hormone (MCH) is a cyclic heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the hypothalamus and released by the neurohypophysis of teleost fish. This hormone is a potent lightening agent of fish skin. This lightening results from the stimulation of a centripetal melanosome (melanin granule) migration to a perinuclear position within integumental melanophores. MCH and related fragment analogues, MCH5-17 and MCH1-14 were used to investigate the ionic requirements for receptor activation by MCH on dermal melanophores of the fish Poecilia reticulata. In calcium-free saline, the sensitivity of the melanophores to MCH and MCH1-14 increased, whereas the sensitivity of the cells to MCH5-17 decreased. Verapamil diminished the sensitivity to MCH5-17, but did not affect melanophore responses to MCH or MCH1-14. The melanosome aggregating response to MCH was not affected in the presence of tetrodotoxin or in sodium- or potassium-free (choline-substituted) saline. These results suggest that neither TTX-sensitive sodium channels nor extracellular sodium or potassium ions play a role in MCH-induced melanosome aggregation. It is known that MCH and MCH1-14 also exhibit MSH-like melanosome dispersion within melanophores, skin darkening activity on fish melanophores whereas MCH5-17 lacks this characteristic. Since the darkening activity of MCH and MCH1-14 requires calcium, these analogues exhibited a diminished lightening (MCH-like) activity in the presence of the divalent cation. In the absence of the N-terminal tetrapeptide sequence (necessary for the expression of MSH-like activity), a role for calcium on melanosome aggregation became evident. These results demonstrate a bifunctional role of calcium on melanosome movements.     

Expression of melanin-concentrating hormone receptors in insulin-producing cells: MCH stimulates insulin release in RINm5F and CRI-G1 cell-lines

Melanin-concentrating hormone (MCH) is a hypothalamic orexigenic peptide. Recently, an orphan G-protein-coupled receptor (SLC-1) was identified that binds MCH with high affinity. Here, we demonstrate the mRNA expression of this receptor in insulin-producing cells including CRI-G1 and RINm5F cells, and in rat islets of Langerhans. Immunofluorescence studies in CRI-G1 and RINm5F cell-lines demonstrated cell-surface expression of the receptor. Rat MCH significantly stimulated insulin secretion in both cell-lines. The potency and the efficacy of MCH were significantly increased in the simultaneous presence of forskolin, suggesting that MCH may amplify the insulinotropic effect of cyclic AMP elevating stimuli. Salmon MCH, which differs from rat/human MCH by six amino acids, was less efficacious than rat/human MCH in stimulating insulin release. The data provide evidence for the expression of MCH receptors in insulin producing cells. The insulinotropic effect of MCH may contribute to the regulation of metabolism and energy balance by this peptide.     

Structure-activity relationships of melanin-concentrating hormone

Melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide (H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-O H) that induces aggregation of melanin granules within the melanophores of teleost fishes. Chemical and enzymatic modifications of MCH were conducted in order to deduce the structure-activity relationship using an in vitro bioassay with fish scales, and a radioimmunoassay using a specific antiserum to synthetic MCH. Micro-modification of MCH was employed with the natural peptide, and the modified form was purified by reverse-phase HPLC. MCH1-14 and NPS-Trp15-MCH were equipotent to MCH. Reduction and carboxamidomethylation of MCH caused complete loss of biological activity. Modification of the Tyr residue with tetranitromethane and Arg residues with 1,2-cyclohexadione significantly reduced activity, while oxidation with hydrogen peroxide caused only partial loss (10%) of activity. These results suggest that the configuration of the S-S loop is essential for activity, and Arg and Tyr may play an important role in the biological activity. In the radioimmunoassay, MCH1-14, MCH5-14 and CAM-Cys5,14-MCH showed no cross-reactivity, whereas MCH5-17 and other derivatives gave inhibition slopes parallel to the MCH standard, suggesting that the antigenic determinant of the antiserum is located in the carboxy-terminal.     

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.     

SVK14 cells express an MCH binding site different from the MCH1 or MCH2 receptor

Melanin-concentrating hormone (MCH) is a cyclic peptide, mainly involved in the regulation of skin pigmentation in teleosts and feeding behavior in mammals. The human keratinocyte SVK14 cell line has been previously shown to express binding sites for the MCH analog [125I]-[Phe13,3-iodo-Tyr19]MCH. We report here that: (1) this binding site similarly recognized [125I]-[3-iodo-Tyr13]MCH; (2) its pharmacological profile clearly differed from those observed at the two human MCH receptor subtypes, MCH1-R and MCH2-R; (3) MCH did not induce any effect on second messenger systems (including cAMP, calcium, and MAP kinase signaling pathways), and (4) no mRNAs corresponding to the MCH receptors were found. In conclusion, the binding site characterized in the SVK14 cell line is distinct from the MCH1 and MCH2 receptors and deserves therefore further investigation.     

The structure-based 3D-QSAR study of MCH1 receptor antagonists

Melanin-concentrating hormone 1 receptor (MCH1-R) mediates the orexigenic effects of melanin-concentrating hormone and its antagonists are considered as potential targets for the treatment of obesity. To design more potent and selective MCH1-R antagonists, at first, we built up the homology structure of MCH1-R. Then, we carried out the receptor based three dimensional Quantitative Structure Activity Relationship (3D-QSAR) using comparative molecular field analysis and Comparative Molecular Similarity Indices Analysis (CoMSIA) for a series of scaffold of MCH1-R antagonists and the docking study for MCH1-R. These models are proved as statistically valid models with a good correlative and predictive power. Based on these models, we are going to develop more potent and selective MCH1-R antagonists.     

Structure-activity studies with fragments and analogues of salmonid melanin-concentrating hormone

A number of cyclic and linear fragments and analogues of MCH were synthesized and their biological potencies tested using the isolated carp scale melanophore assay. In this system, the cyclic portion MCH(5–14) exhibited only 0.1% bioactivity, which was markedly enhanced by the addition of the exocyclic sequences MCH(15–17) and MCH(1–4). The exocyclic sequence itself, MCH(1–4, 15–17), had minimal activity, however. Substitution of Tyr¹¹ with phenylalanine reduced the potency of the ring structure MCH(5–14) by about 4-fold. Substitution of Gly⁸ with D-alanine reduced the potency of MCH(5–14) 16-fold, while both substitutions together caused a still more marked reduction (200-fold) in bioactivity. Linearized fragments of MCH, extending from MCH(15–17) to [Cys(Acm)^5,14]MCH(1–17), showed a progressive increase in potency. The linearized forms of MCH, MCH(5–17) and MCH(5–14), were approximately 100-fold or less potent than their cyclic forms. The significant increases in bioactivity produced by the addition of the C- and N-terminal exocyclic sequence even to these linearized forms further emphasizes the importance of these regions for interaction at the receptor site.     

Beyond skin color: emerging roles of melanin-concentrating hormone in energy homeostasis and other physiological functions

Melanin-concentrating hormone (MCH) is a cyclic peptide that mediates its effects by the activation of two G-protein-coupled seven transmembrane receptors (MCHR1 and MCHR2) in humans. In contrast to its primary role in regulating skin color in fish, MCH has evolved in mammals to regulate dynamic physiological functions, from food intake and energy expenditure to behavior and emotion. Chronic infusion or transgenic expression of MCH stimulates feeding and increases adipocity, whereas targeted deletion of MCH or its receptor (MCHR1) leads to resistance to diet-induced obesity with increased energy expenditure and thermogenesis. The involvement of MCH in energy homeostasis and in brain activity has also been validated in mice treated with non-peptide antagonists, suggesting that blockade of MCHR1 could provide a viable approach for treatment of obesity and certain neurological disorders. This review focuses on emerging roles of MCH in regulating central and peripheral mechanisms.     

Properties of rat melanin-concentrating hormone receptor 1 internalization

Melanin-concentrating hormone (MCH) is a neuropeptide that plays an important role in several physiological processes. It activates two G protein-coupled receptors (GPCRs), MCH1R and MCH2R, of which MCH1R seems to be a key regulator of food intake. By using HEK293T cells stably transfected with Flag-tagged rat MCH1R, we investigated the mechanism underlying the MCH-induced internalization pathway, which is important for the desensitization or regulation of the receptor response. Quantitative analysis by flow cytometry indicated that the rate of MCH1R internalization progressed in a rapid and time-dependent manner during the first 30 min, and was partly inhibited by pretreatment with the selective protein kinase C (PKC) inhibitor Go6850. Overexpression of dominant-negative beta-arrestin-2 (284-409) or dynamin I-K44A significantly prevented MCH-induced internalization of MCH1R, while overexpression of dominant-negative beta-arrestin-1-V53D had no effect. A triple-substituted mutant at Thr317, Ser325 and Thr342 to Ala residue in the C-terminus significantly prevented MCH-induced receptor internalization. Similar extents of internalization prevention were noted with the deletion mutants DeltaThr342 and DeltaGlu346, lacking 11 and 7 residues in the C-terminal tail, respectively. Our data suggest that MCH1R undergoes rapid MCH-induced internalization through a PKC-, beta-arrestin-2- and dynamin I-dependent pathway and that a portion of the C-terminal tail plays an important role in the internalization process.     

Novel benzimidazole-based MCH R1 antagonists

The identification of an MCH R1 antagonist screening hit led to the optimization of a class of benzimidazole-based MCH R1 antagonists. Structure-activity relationships and efforts to optimize pharmacokinetic properties are detailed along with the demonstration of the effectiveness of an MCH R1 antagonist in an animal model of obesity.     

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.     

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.     

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.     

Identification and Characterization of Single‐Nucleotide Polymorphisms in MCH‐R1 and MCH‐R2*

Objective: To identify and functionally characterize single‐nucleotide polymorphisms (SNPs) in melanin‐concentrating hormone (MCH)‐R1 and ‐R2. Research Methods and Procedures: The entire coding regions and intron/exon splice junction regions of MCH‐R1 and MCH‐R2 were sequenced from anonymous white (n = 45) and African‐American (n = 46) individuals. DNA was analyzed, and SNPs were identified using Phred, Phrap, and Consed software. DNA constructs containing MCH‐R1 and MCH‐R2 SNPs were generated and expressed in CHO cells. The effect of the SNPs in MCH‐R1 and MCH‐R2 were assessed in receptor binding assays and functional assays measuring changes in intracellular cAMP and Ca²⁺ levels. Results: We identified 12 SNPs in the MCH‐R1 gene. Two of these SNPs are in coding regions, and one produces an arginine‐for‐glycine substitution at residue 34 in the MCH‐R1 sequence. This SNP is present at a minor allele frequency of 15% in the African‐American population tested in this study. We identified eight SNPs in the MCH‐R2 gene. Four of these SNPs are in coding regions, and two produce amino acid substitutions. Lysine substitutes for arginine at residue 63 of the African‐American population, and glutamine substitutes for arginine at residue 152 in whites (minor allele frequency of 2% for both SNPs). No changes in receptor binding or functional signaling were observed with the SNP mutations in MCH‐R1 or MCH‐R2. Discussion: These data indicate that potential therapeutics designed to act at the MCH receptor are unlikely to have altered effects in subpopulations that express variant forms of MCH‐R1 or MCH‐R2.     

The receptor for the orexigenic peptide melanin-concentrating hormone is a G-protein-coupled receptor

Gene-knockout studies of melanin-concentrating hormone (MCH) and its effect on feeding and energy balance have firmly established MCH as an orexigenic (appetite-stimulating) peptide hormone. Here we identify MCH as the ligand for the orphan receptor SLC-1. The rat SLC-1 is activated by nanomolar concentrations of MCH and is coupled to the G protein G alpha i/o. The pattern of SLC-1 messenger RNA expression coincides with the distribution of MCH-containing nerve terminals and is consistent with the known central effects of MCH. Our identification of an MCH receptor could have implications for the development of new anti-obesity therapies.     

Melanin-concentrating hormone activates signaling pathways in 3T3-L1 adipocytes

Energy homeostasis is regulated by peripheral signals, such as leptin, and by several orexigenic and anorectic neuropeptides. Recently, we reported that the orexigenic neuropeptide melanin-concentrating hormone (MCH) stimulates leptin production by rat adipocytes and that the MCH receptor (MCH-R1) is present on these cells. Here, we show that MCH-R1 is present on murine 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with 1 micromolar MCH for up to 2 h acutely downregulated MCH-R1, indicating a mechanism of ligand-induced receptor downregulation. Potential signaling pathways mediating MCH-R1 action in adipocytes were investigated. Treatment of 3T3-L1 adipocytes with 1 micromolar MCH rapidly induced a threefold and a fivefold increase in p44/42 MAPK and pp70 S6 kinase activities, respectively. In addition, 3T3-L1 adipocytes transiently transfected with a murine leptin-luciferase promoter construct showed a fourfold and a sixfold increase in leptin promoter-reporter gene expression at 1 h and 4 h, respectively, in response to MCH. Activity decreased to basal levels at 8 h. Furthermore, MCH-stimulated leptin promoter-driven luciferase activity was diminished in the presence of the MAP/ERK kinase inhibitor PD-98059 and in the presence of rapamycin, an inhibitor of pp70 S6 kinase activation. These results provide further evidence for a functional MCH signaling pathway in adipocytes.