- Email: [email protected]
Melanin-concentrating Hormone Receptor: An Orphan Receptor Fits the Key
REVIEWS Melanin-concentrating Hormone Receptor: An Orphan Receptor Fits the Key Yumiko Saito, Hans-Peter Nothacker and Olivier Civelli
The melanin-concentrating hormone (MCH), a hypothalamic peptide, was identified initially in teleost fish as a regulator of pigmentary changes in background adaptation, and was later also found, in mammals, to be a regulator of feeding and energy homeostasis. Its specific receptor remained an enigma until very recently when it was identified as the orphan G-protein-coupled receptor SLC-1. This review focuses on the identification, structure and signaling of the MCH receptor and discusses some of the implications of its discovery. Melanin-concentrating hormone (MCH) (Ref. 1) is a cyclic peptide, isolated and sequenced originally from salmon pituitary in 1983. The rat homolog has been purified from hypothalamus by using antibodies directed against the salmon MCH, and its primary structure has been determined2. The rat MCH is 19 amino acids long, two amino acids longer than its salmon counterpart, and is identical to the human MCH, the sequence of which was deduced by cloning3. MCH has distinct physiological roles. In teleost fish, the peptide is released from the pituitary into the circulation to induce pigment aggregation within the melanophores, which results in fish scale paling4. In rodents, MCH is prominently expressed in the perikarya of the lateral hypothalamus and the zona incerta, and projects broadly Y. Saito is at the Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. H-P. Nothacker and O. Civelli are at the Department of Pharmacology, Department of Development and Cell Biology, University of California Irvine, 389 Med Surge II, Irvine, CA 92697-4625, USA. Tel: 11 949 824 2522, Fax: 11 949 824 4855, e-mail: [email protected]
TEM Vol. 11, No. 8, 2000
throughout the central nervous system (CNS)5,6. This extensive terminal distribution suggests that MCH may function as a neurotransmitter/neuromodulator in a broad array of behavioral responses. Among its potential functions, the regulatory role of MCH in feeding has been the most intensely studied in the past few years. First, by using a PCR differential display method, MCH transcripts were found to be upregulated in the hypothalamus of obese Lepob/Lepob mice, while fasting further increased this expression in both normal and Lepob/Lepob mice. Furthermore, injections of MCH in normal rats led to a significant increase in food intake7, an observation that was confirmed in several laboratories8–10. Finally, mice genetically engineered to be devoid of MCH (Ref. 11) displayed reduced body weight and leanness owing to hypophagia and increased metabolic rate. On the basis of these data, MCH appears to be a critical effector of feeding behavior and energy balance by acting downstream of the leptin and melanocortin systems. While these data have increased our knowledge about
the physiological actions of MCH, the nature of the MCH receptor (MCHR) remained enigmatic. However, recent publications have shown that, by applying the orphan receptor strategy12, the MCHR receptor is in fact SLC-1, an orphan G-protein-coupled receptor (GPCR)13–17. • Identification of MCH as the Natural Ligand for the SLC-1 Receptor The SLC-1 GPCR was discovered as an expressed sequence tag exhibiting about 40% homology in its hydrophobic domains to the five somatostatin receptors18. SLC-1 was cloned by PCR amplification of human genomic DNA and assigned to chromosome 22q13.3 as an intronless gene18 (Fig. 1a). A rat ortholog was later identified that shares 91% overall sequence identity to the human SLC-1 receptor but which is 49 amino acid residues shorter in its N-terminal segment (Fig. 1b)19. The existence of a similar shorter form was also reported in humans. The SLC-1 receptor was therefore recognized as one of the ‘orphan’ GPCRs, that is, cloned GPCRs recognizing undiscovered natural ligands. Two related but distinct approaches led to the discovery of MCH as the ligand of SLC-1 (Table 1)13–17. Both approaches are based on the so-called ‘orphan receptor strategy’, where orphan GPCRs are used as baits to identify their natural ligands12. The principle of this strategy is to express the cloned orphan GPCRs in host cells. Exposing the cells to biological extracts containing the matching orphan receptor ligand will induce changes in intracellular second messenger levels in response to the orphan receptor activation. These level changes serve as a monitoring system for the purification of the ligand. The first approach14,16,17 used peptides extracted from brain tissues as a source
1043-2760/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(00)00290-3
299
(a)
T402
M MMM
SLC-1
(b) M1M6 M70 Exon 1
Exon 2
Intron A
Human gene
M1
(c)
Exon 2
Intron A
Exon 1 M 1, 70
Rat gene
T353 Rat and/or human MCHR 20 aa/(60 bp) trends in Endocrinology and Metabolism
Figure 1. Structure of the MCHR gene and proteins. The putative transmembrane regions are indicated as gray boxes. (a) Genomic structure of the 402 amino acid-long human SLC-1 originally isolated in the human genome18. The sequence in the 59-region overlaps with the intron sequence. Although this sequence has all seven transmembrane regions, the protein is not active in either calcium mobilization assays or radioligand binding assays13,14. (b) Gene structure of the human and rat MCHR (Ref. 19). Both are composed of exon I and exon II, and start codons exist in exon I. The deduced amino acid sequence of the human receptor showed two longer open reading frames than that of the rat receptor16. The cDNA starting from Met70 in the human receptor is similar to the rat counterpart in size. (c) Structure of the 353 amino acid-long human and rat MCHR. These proteins are functionally active in the calcium mobilization and cAMP assays13–17. These proteins are referred to as the MCHRs. Abbreviations: M, methionine; MCH, melanin-concentrating hormone; MCHR, MCH receptor; SLC-1, somatostatin-like receptor; T, threonine.
of the natural ligand of SLC-1. SLC-1 reactivity was monitored in three different ways: (1) by intracellular changes in calcium influxes upon coexpression of a Gaq/i3 chimeric protein engineered to force SLC-1 coupling to the phospholipase C pathway14; (2) by inhibition of intracellular cAMP accumulation16; and (3) by G-protein-gated inwardly rectifying potassium channels (GIRKs) in Xenopus oocytes injected with SLC-1 cRNAs (Ref. 17). When brain extracts of peptides were subjected to a first HPLC fractionation, a single peak of activity was detected. Further separations led to the isolation of a homogeneous com-
ponent containing a peptide with a sequence identical to that of MCH. Synthetic MCH was then shown to activate the SLC-1 receptor with an affinity in the nanomolar range (Fig. 2). The second approach13,15 used screening of large batteries of synthetic peptides and putative receptor ligands as potential activators of SLC-1. SLC-1 reactivity was monitored by calcium influx measurements13,15. Because MCH was part of the compound libraries that were tested, it was immediately identified as one possible ligand for the SLC-1 receptor. The advantage of the first approach is its general applicability, yet
Table 1. Strategy for characterizing the MCHRa Cell line
Transfected cDNA
Assay
Source
Ref.
HEK HEK CHO CHO Xenopus oocytes
Human MCHR Rat MCHR Rat MCHR 1 Gaq/i3 Human MCHR Rat MCHR 1 GIRK
Calcium influx Calcium influx Calcium influx cAMP GIRK-mediated current
Surrogate compounds Surrogate compounds Natural peptides Natural peptides Natural peptides
13 15 14 16 17
a
Abbreviations: GIRK, G-protein-gated inwardly rectifying potassium channels, MCHR, melaninconcentrating hormone receptor.
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it is far more technically demanding, while the matching strategy of the second is simple but expensive and limited to established ligands (which are fewer than the orphan GPCRs). • Pharmacological Specificity and Diversity in Biological Activities of the MCHR The fact that some GPCRs bind more than one natural ligand from the same precursor [e.g. adrenocorticotropin and a-melanocyte-stimulating hormone (aMSH)], that the MCH precursor may generate more than one bioactive peptide, and that functional and sequential relationships exist between the MCH, a-MSH (Ref. 20) and somatostatin systems respectively, led to the possibility that the MCHR could bind natural ligands other than MCH. MCH is encoded by a precursor that could produce two other bioactive peptides, namely neuropeptide GE (NGE) and EI (NEI) (Ref. 21) (Fig. 2). Furthermore, alternative splicing of the MCH precursor gene can lead to the expression of a different precursor encoding two other potential bioactive peptides, termed MCH gene-overprinted peptides (MGOP) 14 and 27 (Ref. 22; Fig. 2). Whether these peptides have physiological role(s) is still to be determined, although NEI has been shown to affect grooming and locomotion23 and could exert such an effect via the MCHR. Also, MCH and a-MSH have opposite actions on skin coloration4 and antagonize a variety of physiological functions, including feeding behavior8,23–25 (Table 2). It was therefore possible that a-MSH may exert its antagonistic activity by competing with MCH for the MCHR, as does Agouti-related protein for the MSH receptor. Finally, the similarities in sequences between the somatostatin and MCH receptors18 suggested that somatostatin, or one of its analogs, might also act as a ligand to the MCHR. However, when tested in conditions where MCH activates the MCHR, somatostatin-14 and the somatostatin analog RC-160, cortistatin-14 or -29 (structurally related to somatostatin), NEI, NGE, MGOP-14 and -27 and a-MSH were unable to activate the MCH (Refs 13,14) (Fig. 2). Furthermore, TEM Vol. 11, No. 8, 2000
neither a-MSH nor NEI, even at high concentrations, were able to block the activation of the MCHR by MCH. Because MCH is the only peptide isolated from the extracts of all brain peptides that can activate the MCHR, it can be concluded that the MCHR is specific to MCH. It follows that the other peptides derived from the MCH precursor must bind to different receptors to be active. As it is known that MCH is not recognized by the melanocortin receptors8, and because it has now been ascertained that MSH does not bind the MCHR, the functional antagonistic effect of these two molecules must result from the convergence of signaling pathways mediated by distinct receptors. Whereas the MCHR seems unique to MCH, its activation can lead to diverse intracellular responses. In different cells transfected with the MCHR, MCH activates not only calcium mobilization13–15, but also inwardly rectifying potassium channels17, and inhibits forskolininduced cAMP accumulation13–16. Because the calcium mobilization was partially inhibited by pertussis toxin (PTX) (Saito, Y., unpublished), it is expected that the MCHR interacts both with PTX-sensitive Gai protein and with the PTX-insensitive Gaq protein. Furthermore, experiments involving the use of a G-protein chimera indicate that the MCHR couples to Gai3 with a higher affinity than to Gaq (Ref. 14). The fact that MCHR couples to at least two Ga proteins shows that it can activate different intracellular signaling pathways in distinct cellular environments, a characteristic it shares with most GPCRs. • Different Forms of the MCHR MCHR exhibits several different structures. First, under its original name of somatostatin-like receptor (SLC)-1 or GPR24, it has been described as a 402 amino acid-long human GPCR that was discovered by genomic cloning and delineated by an assigned start codon18 (Fig. 1a). When the SLC-1 gene organization was analysed, the assigned start codon was found to be part of an intron that pointed to the existence of an additional upstream exon containing the translational start site19 (Met70 in TEM Vol. 11, No. 8, 2000
(a)
NGE
NEI
MCH
ProMCH MGOP14 ProMGOP MGOP27
(b) Peptide
Sequence
EC50 (nM)
Rat/human MCH Salmon MCH NGE NEI MGOP14 MGOP27
DFDMLRCMLGRVYRPCWQV DTMRCMVGRVYRPCWEV GSVAFPAENGVQNAESTQE EIGDEENSAKFPI TIHCKWREKPLMLM SDTCWSTTSFQKKTIHCKWREKPLMLM Ac-SYSMEHFRWGLPV
4.8 18.6 No effect No effect No effect No effect
α-MSH
No effect
trends in Endocrinology and Metabolism
Figure 2. Pharmacology of the MCHR. (a) Schematic diagram of the possible peptides derived from the MCH gene. PreproMCH precursor is composed of three exons and includes additional peptide sequences designated NGE and NEI (Ref. 21). An alternative splicing variant, which contains exon I and exon III, encodes two putative peptides named MGOP-14 and -27 (Ref. 22). Ac, acetyl. (b) Activation of the MCH receptor by MCH and related peptides. Intracellular calcium mobilization in CHO cells cotransfected with MCHR and Gaq/i3 chimera cDNAs was measured by integrating the kinetic curve of intracellular calcium concentration following addition of peptide. Peptides were tested at concentrations up to 1 mM. Abbreviations: CHO, Chinese hamster ovary; MCH, melanin-concentrating hormone; MCHR, MCH receptor; MGOP, MCH gene-overprinted peptides; a-MSH, a-melanocyte-stimulating hormone; NEI, neuropeptide EI; NGE, neuropeptide GE.
human and Met1 in rat; Fig. 1c). The SLC-1 form of the receptor, however, does exist and has been isolated from a human cerebral cortex cDNA library13, but cells transfected with SLC-1 cDNA are not, or are only weakly activated by MCH (Refs 13,14). Thus, the SLC-1
form of the MCHR appears to be present at very low levels, might be physiologically inactive and might represent an inappropriately spliced variant. In contrast, the sequence called MCHR is 96% identical in the human and rat counterparts, is sequentially similar at
Table 2. Relationships between the a-MSH and MCH systemsa Ligand
Receptor
Function
Fish melanophore
a-MSH Salmon MCH
Not characterized Not characterized
Pigment dispersion Pigment aggregation Antagonizes a-MSH
Melanoma cells
a-MSH Rat MCH
MC-1 ?
Melanin synthesis No effect on melanin synthesis? Binding sites
Mammalian brain
[Phe13, Tyr19]-MCH
?
a-MSH Rat MCH
MC-4 MCHR
Inhibits feeding Increases feeding Antagonizes a-MSH
a Abbreviations: MCH, melanin-concentrating hormone; MCHR, melanin-concentrating hormone receptor; a-MSH, a-melanocyte-stimulating hormone.
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the N terminus in both species (Fig. 1b) and is well activated by MCH (Refs 13–17). In addition to the SLC-1 form of the MCHR, two other MCHR cDNAs were found in a human embryonic brain cDNA library16. Their deduced amino acid sequences extend N-terminally, giving rise to two alternatively used start sites (Met1 and Met6 in Fig. 1c). The first start site (Met1) was reported to generate a receptor 25-times less active than that starting at Met70 (Ref. 17), which corresponds to the start site of the rat receptor. At present, it is unclear whether these longer open reading frames also exist in the rat. Of particular interest is whether subtypes of MCHR exist. For example, the distinct actions of MCH on melanocytes and on energy metabolism could rely on the activation of different, but related, receptors (Table 2). In particular, the MCH analog [Phe13, Tyr19]-MCH is able to detect specific binding sites in several cell lines, such as the B16F1 melanoma and the SVK14 keratinocyte26,27. In mouse B16-F1C29 melanoma cells, however, MCH has no effect on pigmentation whereas a-MSH stimulates melanogenesis8. However, our preliminary analysis using reverse transcription PCR (RT-PCR) and northern blot showed no evidence for the existence of MCHR-related mRNA, and MCH did not induce any decrease in adenylyl cyclase activity or mobilization of intracellular calcium in B16F1 cells (Saito, Y., unpublished). These discrepancies could be explained by the existence of a yet unknown MCHR-like receptor in melanoma cells that would couple to G proteins other than Gai or Gaq. There are, however, arguments against the existence of MCHR subtypes. First, searches in sequence databanks have failed to discover novel sequences similar to the MCHR sequence. Second, Southern blot analyses of genomic DNAs from human, monkey, rabbit, cow, rat, mouse and dog using the MCHR coding region under low-stringency hybridization conditions have revealed only one hybridizing band13. Also, using the rat MCHR as a probe, a rat genomic library has been screened under low-stringency hybridization, conditions suitable for the
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detection of sequentially-related GPCRs (Ref. 28), but were unable to identify any MCHR subtype genes (Saito, Y., unpublished). While not eliminating the possible existence of MCHR subtypes, these data suggest that, for the time being, the MCHR gene is unique in mammals and that the principal form is the 353 amino acid-long type depicted in Fig. 1c. • Expression of the MCHR and its Functional Implications The availability of MCHR-specific probes permits a reanalysis of the regional distribution of the MCH system. By northern blot analyses of adult rat tissues, MCHR mRNA was detected at high levels in brain, at moderate levels in eye and skeletal muscle, and in low levels in tongue and pituitary14. However, nothing is known about possible roles for the MCH system in eye, skeletal muscle or tongue. By contrast, the existence of the MCHR in the pituitary supports an effect of MCH injection both on adrenocorticotropic hormone (ACTH) release29,30 and in the regulation of the hypothalamic–pituitary–adrenocortical axis following mild stress8. In the rat CNS, in situ hybridization experiments revealed a selective pattern of MCHR expression; no expression was detected in the cerebellum. High levels of MCHR mRNA were detected in the olfactory region, in the CA1–3 fields of the hippocampus and in the neocortex13–15,18. This distribution corresponds to the monosynaptic connections of the MCH neurons from the zona incerta and the lateral hypothalamus to the area of the brain involved in integrating inputs related to taste and olfaction6. MCHR localization in these areas suggests a possible role for MCH in olfactory learning. The MCHR could be involved in the establishment or recall of olfactory memories formed by, or associated with, specific events in feeding behavior. Another tissue of interest is the hypothalamus, which is generally regarded as the primary brain center for the regulation of feeding behavior and energy metabolism. Within this region, the MCHR mRNA is expressed at moderate levels in the ventromedial nucleus13–15, classically known
as the ‘satiety’ center. High and moderate expression of MCHR mRNA was also detected in the nucleus accumbens and the ventral tegmental area, respectively14,15. The nucleus accumbens is the major recipient of the mesolimbic dopaminergic projection from the ventral tegmental area and plays a key role in the reward system. MCH, by acting on the nucleus accumbens or the ventral tegmental area, might mediate the positive reinforcing effect of food, thereby providing an indirect control of feeding behavior. However, in view of the extensive MCHR expression in the brain, the MCH system can be expected to modulate other behaviors in addition to feeding8,23–25,31–34; for example, MCH is reported to be involved in anxiety25,33. High levels of expression of MCHR mRNA were also discovered in the locus coeruleus, the orbital cortex, the cingulate gyrus and the amygdala13–15, areas believed to be linked to arousal and the sensation of anxiety. • Conclusions The hypothalamus is generally regarded as the primary brain center for the regulation of feeding behavior and energy metabolism. A large number of hypothalamic neuropeptides have been implicated in the mediation of food intake. Amongst these peptides, neuropeptide Y (NPY) was the most compelling because of its powerful stimulation of food intake. In addition to the NPY system, the melanocortin system is emerging as having a major modulatory role in regulating energy balance20. The melanocortin system is known to impinge on the MCH system and, indeed, a-MSH effects on food intake are antagonized by MCH. The melanocortin receptor most responsible for regulating feeding is MC-4 (Table 2). However, the MCHR had not been cloned and could not be studied precisely until its recent identification. The discovery of the MCHR, while adding one more success to the orphan receptor strategy, had one immediate impact on our understanding of the role of MCH in energy homeostasis: it permits the definition of the sites of expression of the MCHR in the CNS. As expected, the MCHR is present in most TEM Vol. 11, No. 8, 2000
areas of the CNS, but if high levels of expression are a reflection of the pathways most regulated by MCH, then the MCH system may modulate olfactory memories associated with feeding behavior and the positive reinforcing effect of food. This would set the role of MCH at a motivational level other than one directly regulating energy homeostasis. But one should not forget that, according to the same principle, the MCH system may also be a powerful regulator of anxiety. The identification of the molecular structure of the MCHR might also make possible the designing of surrogate molecules that might mimic or block MCH activity. These molecules will ultimately be the keys to determining putative therapeutic indications of the MCH–MCHR system.
9
10
11 12 13
14 15
16
Acknowledgements We thank our colleagues R.K. Reinscheid, Z. Wang, S.H.S. Lin and S.D. Clark for discussion and critical review of the manuscript. The work carried out in the authors’ laboratory was supported by a grant from NIH MH60231 (to OC), from the BioStar program S97-107 (to OC) and from the Eric and Lila Nelson Chair in Neuropharmacology.
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18
19
References 1 Kawauchi, H. et al. (1983) Characterization of melanin-concentrating hormone in chum salmon pituitaries. Nature 305, 321–323 2 Vaughan, J.M. et al. (1989) Characterization of melanin-concentrating hormone from rat hypothalamus. Endocrinology 125, 1660–1665 3 Presse, F. et al. (1990) Structure of the human melanin-concentrating hormone mRNA. Mol. Endocrinol. 4, 632–637 4 Baker, B.I. (1991) Melanin-concentrating hormone: A general vertebrate neuropeptide. Intern. Rev. Cytol. 126, 1–47 5 Skofitsch, G. et al. (1985) Immunohistochemical localization of a melanin concentrating hormone-like peptide in the rat brain. Brain Res. Bull. 15, 635–649 6 Bittencourt, J.C. et al. (1992) The melaninconcentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization. J. Comp. Neurol. 319, 218–245 7 Qu, D. et al. (1996) A role for melanin concentrating hormone in the central regulation of feeding behavior. Nature 380, 243–247 8 Ludwig, D.S. et al. (1998) Melanin-concentrating hormone: a functional melano-
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cortin antagonist in the hypothalamus. Am. J. Physiol. 274, E627–E633 Tritos, A. et al. (1998) Functional interactions between melanin-concentrating hormone, neuropeptide Y, and anorectic neuropeptides in the rat hypothalamus. Diabetes 47, 1687–1692 Rossi, M. et al. (1997) Melanin-concentrating hormone acutely stimulates feeding, but chronic administration has no effect on body weight. Endocrinology 138, 351–355 Shimada, M. et al. (1998) Mice lacking melanin-concentrating hormone are hypophagic and lean. Nature 396, 670–674 Civelli, O. (1998) Functional genomics: the search for novel neurotransmitters and neuropeptides. FEBS Lett. 430, 55–58 Chambers, J. et al. (1999) Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1. Nature 400, 261–265 Saito, Y. et al. (1999) Molecular characterization of the melanin-concentrating hormone receptor. Nature 400, 265–269 Lembo, P.M.C. et al. (1999) The receptor for the orexigenic peptide melanin-concentrating hormone is a G-protein-coupled receptor. Nature Cell Biol. 1, 267–271 Shimomura, Y. et al. (1999) Isolation and identification of melanin-concentrating hormone as the endogenous ligand of the SLC-1 receptor. Biochem. Biophys. Res. Commun. 261, 622–626 Bachner, D. et al. (1999) Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1). FEBS Lett. 457, 522–524 Kolakowski, L.F. et al. (1996) Characterization of a human gene related to genes encoding somatostatin receptors. FEBS Lett. 398, 255–258 Lakaye, B. et al. (1997) Cloning of the rat brain cDNA encoding for the SLC-1 G-protein-coupled receptor reveals the presence of an intron in the gene. Biochim. Biophys. Acta 1401, 216–220 Tritos, N.A. and Maratos-Flier, E. (1999) Two important systems in energy homeostasis: melanocortins and melanin-concentrating hormone. Neuropeptides 33, 339–349 Nahon, J.L. et al. (1989) The rat melaninconcentrating hormone messenger ribonucleic acid encodes putative neuropeptides coexpressed in the dorsolateral hypothalamus. Endocrinology 125, 2056–2065 Toumaniantz, G. et al. (1996) The rat melanin-concentrating hormone encodes
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an additional putative protein in a different reading frame. Endocrinology 137, 4518–4521 Sanchez, M. et al. (1997) Melanin-concentrating hormone (MCH) antagonizes the effects of a-MSH and neuropeptide E-I on grooming and locomotor activities in the rat. Peptides 18, 393–396 Miller, C.L. et al. (1993) a-MSH and MCH are functional antagonists in a CNS auditory paradigm. Peptides 14, 1–10 Gonzalez, M.I. et al. (1996) Behavioral effects of a-MSH and MCH after central administration in the female rat. Peptides 17, 171–177 Drozdz, R. et al. (1995) Melanin-concentrating hormone binding to mouse melanoma cells in vitro. FEBS Lett. 359, 199–202 Burgaud, J.L. et al. (1997) Melanin-concentrating hormone binding sites in human SVK14 keratinocytes. Biochem. Biophys. Res. Commun. 241, 622–629 Bunzow, J.R. et al. (1992) Cloning of the dopamine receptors: The homology approach. In Methods in Neuroscience (Conn, P.M., ed.) pp. 441–453, Academic Press Jezova, D. et al. (1992) Rat melanin-concentrating hormone stimulates adrenocorticotropin secretion: evidence for a site of action in brain regions protected by the blood–brain barrier. Endocrinology 130, 1024–1029 Bluet-Pajot, M.T. et al. (1995) Neuropeptide-EI antagonizes the action of melaninconcentrating hormone on stress-induced release of adrenocorticotropin in the rat. J. Neuroendocrinol. 7, 297–303 Hervieu, G. et al. (1995) Similarities in cellular expression and function of melaninconcentrating hormone and atrial natriuretic factor in the rat digestive tract. Endocrinology 137, 561–571 Gonzalez, M.I. et al. (1997) Stimulatory effect of melanin-concentrating hormone on luteinising hormone release. Neuroendocrinology 66, 254–262 Monzon, M.E. and De Barioglio, S.R. (1999) Response to novelty after i.c.v. injection of melanin-concentrating hormone (MCH) in rats. Physiol. Behav. 67, 813–817 Viale, A. et al. (2000) 17b-estradiol regulation of melanin-concentrating hormone and neuropeptide-E-I contents in cynomolgus monkeys: a preliminary study. Peptides 20, 553–559
Special Diabetes Issue – Don’t Miss Out! The November issue of Trends in Endocrinology and Metabolism will be a special issue covering aspects of diabetes. To subscribe to TEM today, please use the bound-in card
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The melanin-concentrating hormone (MCH), a hypothalamic peptide, was identified initially in teleost fish as a regulator of pigmentary changes in background adaptation, and was later also found, in mammals, to be a regulator of feeding and energy homeostasis. Its specific receptor remained an enigma until very recently when it was identified as the orphan G-protein-coupled receptor SLC-1. This review focuses on the identification, structure and signaling of the MCH receptor and discusses some of the implications of its discovery. Melanin-concentrating hormone (MCH) (Ref. 1) is a cyclic peptide, isolated and sequenced originally from salmon pituitary in 1983. The rat homolog has been purified from hypothalamus by using antibodies directed against the salmon MCH, and its primary structure has been determined2. The rat MCH is 19 amino acids long, two amino acids longer than its salmon counterpart, and is identical to the human MCH, the sequence of which was deduced by cloning3. MCH has distinct physiological roles. In teleost fish, the peptide is released from the pituitary into the circulation to induce pigment aggregation within the melanophores, which results in fish scale paling4. In rodents, MCH is prominently expressed in the perikarya of the lateral hypothalamus and the zona incerta, and projects broadly Y. Saito is at the Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. H-P. Nothacker and O. Civelli are at the Department of Pharmacology, Department of Development and Cell Biology, University of California Irvine, 389 Med Surge II, Irvine, CA 92697-4625, USA. Tel: 11 949 824 2522, Fax: 11 949 824 4855, e-mail: [email protected]
TEM Vol. 11, No. 8, 2000
throughout the central nervous system (CNS)5,6. This extensive terminal distribution suggests that MCH may function as a neurotransmitter/neuromodulator in a broad array of behavioral responses. Among its potential functions, the regulatory role of MCH in feeding has been the most intensely studied in the past few years. First, by using a PCR differential display method, MCH transcripts were found to be upregulated in the hypothalamus of obese Lepob/Lepob mice, while fasting further increased this expression in both normal and Lepob/Lepob mice. Furthermore, injections of MCH in normal rats led to a significant increase in food intake7, an observation that was confirmed in several laboratories8–10. Finally, mice genetically engineered to be devoid of MCH (Ref. 11) displayed reduced body weight and leanness owing to hypophagia and increased metabolic rate. On the basis of these data, MCH appears to be a critical effector of feeding behavior and energy balance by acting downstream of the leptin and melanocortin systems. While these data have increased our knowledge about
the physiological actions of MCH, the nature of the MCH receptor (MCHR) remained enigmatic. However, recent publications have shown that, by applying the orphan receptor strategy12, the MCHR receptor is in fact SLC-1, an orphan G-protein-coupled receptor (GPCR)13–17. • Identification of MCH as the Natural Ligand for the SLC-1 Receptor The SLC-1 GPCR was discovered as an expressed sequence tag exhibiting about 40% homology in its hydrophobic domains to the five somatostatin receptors18. SLC-1 was cloned by PCR amplification of human genomic DNA and assigned to chromosome 22q13.3 as an intronless gene18 (Fig. 1a). A rat ortholog was later identified that shares 91% overall sequence identity to the human SLC-1 receptor but which is 49 amino acid residues shorter in its N-terminal segment (Fig. 1b)19. The existence of a similar shorter form was also reported in humans. The SLC-1 receptor was therefore recognized as one of the ‘orphan’ GPCRs, that is, cloned GPCRs recognizing undiscovered natural ligands. Two related but distinct approaches led to the discovery of MCH as the ligand of SLC-1 (Table 1)13–17. Both approaches are based on the so-called ‘orphan receptor strategy’, where orphan GPCRs are used as baits to identify their natural ligands12. The principle of this strategy is to express the cloned orphan GPCRs in host cells. Exposing the cells to biological extracts containing the matching orphan receptor ligand will induce changes in intracellular second messenger levels in response to the orphan receptor activation. These level changes serve as a monitoring system for the purification of the ligand. The first approach14,16,17 used peptides extracted from brain tissues as a source
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(a)
T402
M MMM
SLC-1
(b) M1M6 M70 Exon 1
Exon 2
Intron A
Human gene
M1
(c)
Exon 2
Intron A
Exon 1 M 1, 70
Rat gene
T353 Rat and/or human MCHR 20 aa/(60 bp) trends in Endocrinology and Metabolism
Figure 1. Structure of the MCHR gene and proteins. The putative transmembrane regions are indicated as gray boxes. (a) Genomic structure of the 402 amino acid-long human SLC-1 originally isolated in the human genome18. The sequence in the 59-region overlaps with the intron sequence. Although this sequence has all seven transmembrane regions, the protein is not active in either calcium mobilization assays or radioligand binding assays13,14. (b) Gene structure of the human and rat MCHR (Ref. 19). Both are composed of exon I and exon II, and start codons exist in exon I. The deduced amino acid sequence of the human receptor showed two longer open reading frames than that of the rat receptor16. The cDNA starting from Met70 in the human receptor is similar to the rat counterpart in size. (c) Structure of the 353 amino acid-long human and rat MCHR. These proteins are functionally active in the calcium mobilization and cAMP assays13–17. These proteins are referred to as the MCHRs. Abbreviations: M, methionine; MCH, melanin-concentrating hormone; MCHR, MCH receptor; SLC-1, somatostatin-like receptor; T, threonine.
of the natural ligand of SLC-1. SLC-1 reactivity was monitored in three different ways: (1) by intracellular changes in calcium influxes upon coexpression of a Gaq/i3 chimeric protein engineered to force SLC-1 coupling to the phospholipase C pathway14; (2) by inhibition of intracellular cAMP accumulation16; and (3) by G-protein-gated inwardly rectifying potassium channels (GIRKs) in Xenopus oocytes injected with SLC-1 cRNAs (Ref. 17). When brain extracts of peptides were subjected to a first HPLC fractionation, a single peak of activity was detected. Further separations led to the isolation of a homogeneous com-
ponent containing a peptide with a sequence identical to that of MCH. Synthetic MCH was then shown to activate the SLC-1 receptor with an affinity in the nanomolar range (Fig. 2). The second approach13,15 used screening of large batteries of synthetic peptides and putative receptor ligands as potential activators of SLC-1. SLC-1 reactivity was monitored by calcium influx measurements13,15. Because MCH was part of the compound libraries that were tested, it was immediately identified as one possible ligand for the SLC-1 receptor. The advantage of the first approach is its general applicability, yet
Table 1. Strategy for characterizing the MCHRa Cell line
Transfected cDNA
Assay
Source
Ref.
HEK HEK CHO CHO Xenopus oocytes
Human MCHR Rat MCHR Rat MCHR 1 Gaq/i3 Human MCHR Rat MCHR 1 GIRK
Calcium influx Calcium influx Calcium influx cAMP GIRK-mediated current
Surrogate compounds Surrogate compounds Natural peptides Natural peptides Natural peptides
13 15 14 16 17
a
Abbreviations: GIRK, G-protein-gated inwardly rectifying potassium channels, MCHR, melaninconcentrating hormone receptor.
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it is far more technically demanding, while the matching strategy of the second is simple but expensive and limited to established ligands (which are fewer than the orphan GPCRs). • Pharmacological Specificity and Diversity in Biological Activities of the MCHR The fact that some GPCRs bind more than one natural ligand from the same precursor [e.g. adrenocorticotropin and a-melanocyte-stimulating hormone (aMSH)], that the MCH precursor may generate more than one bioactive peptide, and that functional and sequential relationships exist between the MCH, a-MSH (Ref. 20) and somatostatin systems respectively, led to the possibility that the MCHR could bind natural ligands other than MCH. MCH is encoded by a precursor that could produce two other bioactive peptides, namely neuropeptide GE (NGE) and EI (NEI) (Ref. 21) (Fig. 2). Furthermore, alternative splicing of the MCH precursor gene can lead to the expression of a different precursor encoding two other potential bioactive peptides, termed MCH gene-overprinted peptides (MGOP) 14 and 27 (Ref. 22; Fig. 2). Whether these peptides have physiological role(s) is still to be determined, although NEI has been shown to affect grooming and locomotion23 and could exert such an effect via the MCHR. Also, MCH and a-MSH have opposite actions on skin coloration4 and antagonize a variety of physiological functions, including feeding behavior8,23–25 (Table 2). It was therefore possible that a-MSH may exert its antagonistic activity by competing with MCH for the MCHR, as does Agouti-related protein for the MSH receptor. Finally, the similarities in sequences between the somatostatin and MCH receptors18 suggested that somatostatin, or one of its analogs, might also act as a ligand to the MCHR. However, when tested in conditions where MCH activates the MCHR, somatostatin-14 and the somatostatin analog RC-160, cortistatin-14 or -29 (structurally related to somatostatin), NEI, NGE, MGOP-14 and -27 and a-MSH were unable to activate the MCH (Refs 13,14) (Fig. 2). Furthermore, TEM Vol. 11, No. 8, 2000
neither a-MSH nor NEI, even at high concentrations, were able to block the activation of the MCHR by MCH. Because MCH is the only peptide isolated from the extracts of all brain peptides that can activate the MCHR, it can be concluded that the MCHR is specific to MCH. It follows that the other peptides derived from the MCH precursor must bind to different receptors to be active. As it is known that MCH is not recognized by the melanocortin receptors8, and because it has now been ascertained that MSH does not bind the MCHR, the functional antagonistic effect of these two molecules must result from the convergence of signaling pathways mediated by distinct receptors. Whereas the MCHR seems unique to MCH, its activation can lead to diverse intracellular responses. In different cells transfected with the MCHR, MCH activates not only calcium mobilization13–15, but also inwardly rectifying potassium channels17, and inhibits forskolininduced cAMP accumulation13–16. Because the calcium mobilization was partially inhibited by pertussis toxin (PTX) (Saito, Y., unpublished), it is expected that the MCHR interacts both with PTX-sensitive Gai protein and with the PTX-insensitive Gaq protein. Furthermore, experiments involving the use of a G-protein chimera indicate that the MCHR couples to Gai3 with a higher affinity than to Gaq (Ref. 14). The fact that MCHR couples to at least two Ga proteins shows that it can activate different intracellular signaling pathways in distinct cellular environments, a characteristic it shares with most GPCRs. • Different Forms of the MCHR MCHR exhibits several different structures. First, under its original name of somatostatin-like receptor (SLC)-1 or GPR24, it has been described as a 402 amino acid-long human GPCR that was discovered by genomic cloning and delineated by an assigned start codon18 (Fig. 1a). When the SLC-1 gene organization was analysed, the assigned start codon was found to be part of an intron that pointed to the existence of an additional upstream exon containing the translational start site19 (Met70 in TEM Vol. 11, No. 8, 2000
(a)
NGE
NEI
MCH
ProMCH MGOP14 ProMGOP MGOP27
(b) Peptide
Sequence
EC50 (nM)
Rat/human MCH Salmon MCH NGE NEI MGOP14 MGOP27
DFDMLRCMLGRVYRPCWQV DTMRCMVGRVYRPCWEV GSVAFPAENGVQNAESTQE EIGDEENSAKFPI TIHCKWREKPLMLM SDTCWSTTSFQKKTIHCKWREKPLMLM Ac-SYSMEHFRWGLPV
4.8 18.6 No effect No effect No effect No effect
α-MSH
No effect
trends in Endocrinology and Metabolism
Figure 2. Pharmacology of the MCHR. (a) Schematic diagram of the possible peptides derived from the MCH gene. PreproMCH precursor is composed of three exons and includes additional peptide sequences designated NGE and NEI (Ref. 21). An alternative splicing variant, which contains exon I and exon III, encodes two putative peptides named MGOP-14 and -27 (Ref. 22). Ac, acetyl. (b) Activation of the MCH receptor by MCH and related peptides. Intracellular calcium mobilization in CHO cells cotransfected with MCHR and Gaq/i3 chimera cDNAs was measured by integrating the kinetic curve of intracellular calcium concentration following addition of peptide. Peptides were tested at concentrations up to 1 mM. Abbreviations: CHO, Chinese hamster ovary; MCH, melanin-concentrating hormone; MCHR, MCH receptor; MGOP, MCH gene-overprinted peptides; a-MSH, a-melanocyte-stimulating hormone; NEI, neuropeptide EI; NGE, neuropeptide GE.
human and Met1 in rat; Fig. 1c). The SLC-1 form of the receptor, however, does exist and has been isolated from a human cerebral cortex cDNA library13, but cells transfected with SLC-1 cDNA are not, or are only weakly activated by MCH (Refs 13,14). Thus, the SLC-1
form of the MCHR appears to be present at very low levels, might be physiologically inactive and might represent an inappropriately spliced variant. In contrast, the sequence called MCHR is 96% identical in the human and rat counterparts, is sequentially similar at
Table 2. Relationships between the a-MSH and MCH systemsa Ligand
Receptor
Function
Fish melanophore
a-MSH Salmon MCH
Not characterized Not characterized
Pigment dispersion Pigment aggregation Antagonizes a-MSH
Melanoma cells
a-MSH Rat MCH
MC-1 ?
Melanin synthesis No effect on melanin synthesis? Binding sites
Mammalian brain
[Phe13, Tyr19]-MCH
?
a-MSH Rat MCH
MC-4 MCHR
Inhibits feeding Increases feeding Antagonizes a-MSH
a Abbreviations: MCH, melanin-concentrating hormone; MCHR, melanin-concentrating hormone receptor; a-MSH, a-melanocyte-stimulating hormone.
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the N terminus in both species (Fig. 1b) and is well activated by MCH (Refs 13–17). In addition to the SLC-1 form of the MCHR, two other MCHR cDNAs were found in a human embryonic brain cDNA library16. Their deduced amino acid sequences extend N-terminally, giving rise to two alternatively used start sites (Met1 and Met6 in Fig. 1c). The first start site (Met1) was reported to generate a receptor 25-times less active than that starting at Met70 (Ref. 17), which corresponds to the start site of the rat receptor. At present, it is unclear whether these longer open reading frames also exist in the rat. Of particular interest is whether subtypes of MCHR exist. For example, the distinct actions of MCH on melanocytes and on energy metabolism could rely on the activation of different, but related, receptors (Table 2). In particular, the MCH analog [Phe13, Tyr19]-MCH is able to detect specific binding sites in several cell lines, such as the B16F1 melanoma and the SVK14 keratinocyte26,27. In mouse B16-F1C29 melanoma cells, however, MCH has no effect on pigmentation whereas a-MSH stimulates melanogenesis8. However, our preliminary analysis using reverse transcription PCR (RT-PCR) and northern blot showed no evidence for the existence of MCHR-related mRNA, and MCH did not induce any decrease in adenylyl cyclase activity or mobilization of intracellular calcium in B16F1 cells (Saito, Y., unpublished). These discrepancies could be explained by the existence of a yet unknown MCHR-like receptor in melanoma cells that would couple to G proteins other than Gai or Gaq. There are, however, arguments against the existence of MCHR subtypes. First, searches in sequence databanks have failed to discover novel sequences similar to the MCHR sequence. Second, Southern blot analyses of genomic DNAs from human, monkey, rabbit, cow, rat, mouse and dog using the MCHR coding region under low-stringency hybridization conditions have revealed only one hybridizing band13. Also, using the rat MCHR as a probe, a rat genomic library has been screened under low-stringency hybridization, conditions suitable for the
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detection of sequentially-related GPCRs (Ref. 28), but were unable to identify any MCHR subtype genes (Saito, Y., unpublished). While not eliminating the possible existence of MCHR subtypes, these data suggest that, for the time being, the MCHR gene is unique in mammals and that the principal form is the 353 amino acid-long type depicted in Fig. 1c. • Expression of the MCHR and its Functional Implications The availability of MCHR-specific probes permits a reanalysis of the regional distribution of the MCH system. By northern blot analyses of adult rat tissues, MCHR mRNA was detected at high levels in brain, at moderate levels in eye and skeletal muscle, and in low levels in tongue and pituitary14. However, nothing is known about possible roles for the MCH system in eye, skeletal muscle or tongue. By contrast, the existence of the MCHR in the pituitary supports an effect of MCH injection both on adrenocorticotropic hormone (ACTH) release29,30 and in the regulation of the hypothalamic–pituitary–adrenocortical axis following mild stress8. In the rat CNS, in situ hybridization experiments revealed a selective pattern of MCHR expression; no expression was detected in the cerebellum. High levels of MCHR mRNA were detected in the olfactory region, in the CA1–3 fields of the hippocampus and in the neocortex13–15,18. This distribution corresponds to the monosynaptic connections of the MCH neurons from the zona incerta and the lateral hypothalamus to the area of the brain involved in integrating inputs related to taste and olfaction6. MCHR localization in these areas suggests a possible role for MCH in olfactory learning. The MCHR could be involved in the establishment or recall of olfactory memories formed by, or associated with, specific events in feeding behavior. Another tissue of interest is the hypothalamus, which is generally regarded as the primary brain center for the regulation of feeding behavior and energy metabolism. Within this region, the MCHR mRNA is expressed at moderate levels in the ventromedial nucleus13–15, classically known
as the ‘satiety’ center. High and moderate expression of MCHR mRNA was also detected in the nucleus accumbens and the ventral tegmental area, respectively14,15. The nucleus accumbens is the major recipient of the mesolimbic dopaminergic projection from the ventral tegmental area and plays a key role in the reward system. MCH, by acting on the nucleus accumbens or the ventral tegmental area, might mediate the positive reinforcing effect of food, thereby providing an indirect control of feeding behavior. However, in view of the extensive MCHR expression in the brain, the MCH system can be expected to modulate other behaviors in addition to feeding8,23–25,31–34; for example, MCH is reported to be involved in anxiety25,33. High levels of expression of MCHR mRNA were also discovered in the locus coeruleus, the orbital cortex, the cingulate gyrus and the amygdala13–15, areas believed to be linked to arousal and the sensation of anxiety. • Conclusions The hypothalamus is generally regarded as the primary brain center for the regulation of feeding behavior and energy metabolism. A large number of hypothalamic neuropeptides have been implicated in the mediation of food intake. Amongst these peptides, neuropeptide Y (NPY) was the most compelling because of its powerful stimulation of food intake. In addition to the NPY system, the melanocortin system is emerging as having a major modulatory role in regulating energy balance20. The melanocortin system is known to impinge on the MCH system and, indeed, a-MSH effects on food intake are antagonized by MCH. The melanocortin receptor most responsible for regulating feeding is MC-4 (Table 2). However, the MCHR had not been cloned and could not be studied precisely until its recent identification. The discovery of the MCHR, while adding one more success to the orphan receptor strategy, had one immediate impact on our understanding of the role of MCH in energy homeostasis: it permits the definition of the sites of expression of the MCHR in the CNS. As expected, the MCHR is present in most TEM Vol. 11, No. 8, 2000
areas of the CNS, but if high levels of expression are a reflection of the pathways most regulated by MCH, then the MCH system may modulate olfactory memories associated with feeding behavior and the positive reinforcing effect of food. This would set the role of MCH at a motivational level other than one directly regulating energy homeostasis. But one should not forget that, according to the same principle, the MCH system may also be a powerful regulator of anxiety. The identification of the molecular structure of the MCHR might also make possible the designing of surrogate molecules that might mimic or block MCH activity. These molecules will ultimately be the keys to determining putative therapeutic indications of the MCH–MCHR system.
9
10
11 12 13
14 15
16
Acknowledgements We thank our colleagues R.K. Reinscheid, Z. Wang, S.H.S. Lin and S.D. Clark for discussion and critical review of the manuscript. The work carried out in the authors’ laboratory was supported by a grant from NIH MH60231 (to OC), from the BioStar program S97-107 (to OC) and from the Eric and Lila Nelson Chair in Neuropharmacology.
17
18
19
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cortin antagonist in the hypothalamus. Am. J. Physiol. 274, E627–E633 Tritos, A. et al. (1998) Functional interactions between melanin-concentrating hormone, neuropeptide Y, and anorectic neuropeptides in the rat hypothalamus. Diabetes 47, 1687–1692 Rossi, M. et al. (1997) Melanin-concentrating hormone acutely stimulates feeding, but chronic administration has no effect on body weight. Endocrinology 138, 351–355 Shimada, M. et al. (1998) Mice lacking melanin-concentrating hormone are hypophagic and lean. Nature 396, 670–674 Civelli, O. (1998) Functional genomics: the search for novel neurotransmitters and neuropeptides. FEBS Lett. 430, 55–58 Chambers, J. et al. (1999) Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1. Nature 400, 261–265 Saito, Y. et al. (1999) Molecular characterization of the melanin-concentrating hormone receptor. Nature 400, 265–269 Lembo, P.M.C. et al. (1999) The receptor for the orexigenic peptide melanin-concentrating hormone is a G-protein-coupled receptor. Nature Cell Biol. 1, 267–271 Shimomura, Y. et al. (1999) Isolation and identification of melanin-concentrating hormone as the endogenous ligand of the SLC-1 receptor. Biochem. Biophys. Res. Commun. 261, 622–626 Bachner, D. et al. (1999) Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1). FEBS Lett. 457, 522–524 Kolakowski, L.F. et al. (1996) Characterization of a human gene related to genes encoding somatostatin receptors. FEBS Lett. 398, 255–258 Lakaye, B. et al. (1997) Cloning of the rat brain cDNA encoding for the SLC-1 G-protein-coupled receptor reveals the presence of an intron in the gene. Biochim. Biophys. Acta 1401, 216–220 Tritos, N.A. and Maratos-Flier, E. (1999) Two important systems in energy homeostasis: melanocortins and melanin-concentrating hormone. Neuropeptides 33, 339–349 Nahon, J.L. et al. (1989) The rat melaninconcentrating hormone messenger ribonucleic acid encodes putative neuropeptides coexpressed in the dorsolateral hypothalamus. Endocrinology 125, 2056–2065 Toumaniantz, G. et al. (1996) The rat melanin-concentrating hormone encodes
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an additional putative protein in a different reading frame. Endocrinology 137, 4518–4521 Sanchez, M. et al. (1997) Melanin-concentrating hormone (MCH) antagonizes the effects of a-MSH and neuropeptide E-I on grooming and locomotor activities in the rat. Peptides 18, 393–396 Miller, C.L. et al. (1993) a-MSH and MCH are functional antagonists in a CNS auditory paradigm. Peptides 14, 1–10 Gonzalez, M.I. et al. (1996) Behavioral effects of a-MSH and MCH after central administration in the female rat. Peptides 17, 171–177 Drozdz, R. et al. (1995) Melanin-concentrating hormone binding to mouse melanoma cells in vitro. FEBS Lett. 359, 199–202 Burgaud, J.L. et al. (1997) Melanin-concentrating hormone binding sites in human SVK14 keratinocytes. Biochem. Biophys. Res. Commun. 241, 622–629 Bunzow, J.R. et al. (1992) Cloning of the dopamine receptors: The homology approach. In Methods in Neuroscience (Conn, P.M., ed.) pp. 441–453, Academic Press Jezova, D. et al. (1992) Rat melanin-concentrating hormone stimulates adrenocorticotropin secretion: evidence for a site of action in brain regions protected by the blood–brain barrier. Endocrinology 130, 1024–1029 Bluet-Pajot, M.T. et al. (1995) Neuropeptide-EI antagonizes the action of melaninconcentrating hormone on stress-induced release of adrenocorticotropin in the rat. J. Neuroendocrinol. 7, 297–303 Hervieu, G. et al. (1995) Similarities in cellular expression and function of melaninconcentrating hormone and atrial natriuretic factor in the rat digestive tract. Endocrinology 137, 561–571 Gonzalez, M.I. et al. (1997) Stimulatory effect of melanin-concentrating hormone on luteinising hormone release. Neuroendocrinology 66, 254–262 Monzon, M.E. and De Barioglio, S.R. (1999) Response to novelty after i.c.v. injection of melanin-concentrating hormone (MCH) in rats. Physiol. Behav. 67, 813–817 Viale, A. et al. (2000) 17b-estradiol regulation of melanin-concentrating hormone and neuropeptide-E-I contents in cynomolgus monkeys: a preliminary study. Peptides 20, 553–559
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