Molecular Cloning and Functional Characterization of MCH2, a Novel Human MCH Receptor*

Jeffrey HillDagger §, Malcolm Duckworth, Paul Murdock||, Gillian Rennie||, Cibele Sabido-David**, Robert S. AmesDagger Dagger , Philip Szekeres§§, Shelagh Wilson§§, Derk J. BergsmaDagger Dagger , Israel S. GlogerDagger , Dana S. LevyDagger Dagger , Jon K. Chambers§§, and Alison I. Muir§§

From the Departments of Dagger  Molecular Biology,  Bioinformatics, || Gene Expression Sciences, ** Computational and Structural Sciences, and §§ Vascular Biology, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, United Kingdom and the Dagger Dagger  Department of Molecular Biology, GlaxoSmithKline, King of Prussia, Pennsylvania 19406-0939

Received for publication, March 7, 2001, and in revised form, March 23, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Melanin-concentrating hormone (MCH) is involved in the regulation of feeding and energy homeostasis. Recently, a 353-amino acid splice variant form of the human orphan receptor SLC-1 (1) (hereafter referred to as MCH1) was identified as an MCH receptor. This report describes the cloning and functional characterization of a novel second human MCH receptor, which we designate MCH2, initially identified in a genomic survey sequence as being homologous to MCH1 receptors. Using this sequence, a full-length cDNA was generated with an open reading frame of 1023 base pairs, encoding a polypeptide of 340 amino acids, with 38% identity to MCH1 and with many of the structural features conserved in G protein-coupled receptors. This newly discovered receptor belongs to class 1 (rhodopsin-like) of the G protein-coupled receptor superfamily. HEK293 cells transfected with MCH2 receptors responded to nanomolar concentrations of MCH with an increase in intracellular Ca2+ levels and increased cellular extrusion of protons. In addition, fluorescently labeled MCH bound with nanomolar affinity to these cells. The tissue localization of MCH2 receptor mRNA, as determined by quantitative reverse transcription-polymerase chain reaction, was similar to that of MCH1 in that both receptors are expressed predominantly in the brain. The discovery of a novel MCH receptor represents a new potential drug target and will allow the further elucidation of MCH-mediated responses.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Melanin-concentrating hormone (MCH)1 is a cyclic neuropeptide that was first discovered in teleost fish, in which it acts as a skin color-regulating hormone (2). In rodents its tissue distribution in the perikarya of the lateral hypothalamus and the zona incerta suggests that MCH may be involved in a variety of behavioral responses (3). Similar tissue distributions have been reported in both bird (4) and monkey (5). Reports implicating MCH in the regulation of feeding behavior show that increased food intake occurs after direct administration of MCH into the brain (6) and that MCH is up-regulated after fasting and in obese leptin-deficient mice (7). There are also reports that suggest MCH may be involved in aggressive behavior, anxiety, and reproductive function (8, 9).

Recently, several groups independently identified a 353-amino acid splice variant of the orphan G protein-coupled receptor (GPCR) SLC-1 (1) as an MCH receptor (10-14). In view of the findings of the current study, we propose that this form of SLC-1 be hereafter referred to as MCH1. Southern blot and related studies have indicated the absence of additional MCH receptor subtypes that closely resemble MCH1 at the DNA level (3, 10, 15). However, because degeneracy in receptor-ligand pairings throughout the GPCR superfamily is common, we reasoned that other MCH receptors with low homology to the MCH1 receptor may exist. This suggestion is supported by reports of pharmacological differences between the MCH1 receptor and MCH binding sites in various cell lines and tissues (10).

Sequencing of the human genome resulted in the deposition of a vast amount of unannotated sequence in public data bases in recent years. In the present study, we first describe how we identified a sequence with low but significant homology to the MCH1 receptor from one of these data bases and cloned a full-length cDNA from this. We then demonstrate that the predicted 340-amino acid polypeptide product of this sequence, which we term MCH2, exhibits many of the structural features of the GPCR superfamily, is a member of the class 1 (rhodopsin-like) subfamily, and after heterologous expression in HEK293 cells is selectively activated by nanomolar concentrations of MCH. Last, to investigate the biological significance of this finding we compare the tissue distribution of MCH1 and MCH2 receptors by RT-PCR analysis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Receptor Cloning, Transient Expression, and Generation of Stable Cell Lines-- A 195-base pair genomic survey sequence (GenBankTM accession number AQ311725) was identified that when translated exhibited 42% identity at the amino acid level to the transmembrane-4 region of MCH1. The primers were designed to perform 5' (5'-CAGAGTACATCGTCAGGGGATGTCAAATCAAAA-3') and 3' (5'-TACTTTGCCCTCGTCCAACCATTT-3') rapid amplification of cDNA ends on a Marathon human fetal brain cDNA template (CLONTECH). Extension of the known sequence at both the 5' and 3' ends revealed a coding sequence of 1023 base pairs with an in-frame upstream stop codon at position -24 (Fig. 1). The full-length gene was amplified from the fetal brain cDNA template using forward (5'-ACAATGAATCCATTTCATGCATCTTGT-3') and reverse (5'-TGCTGCTAAGAGTCACAAGTACACAAGAAG-3') primers. The cDNA was cloned into pcDNA3.1/V5/His-TOPO (Invitrogen), and both strands were sequenced on an ABI sequencer. HEK293 cells were transfected with the recombinant plasmid using LipofectAMINE plus reagent (Life Technologies, Inc.) following the manufacturer's instructions. Stable cell lines were generated by selection in geneticin (16), and clones were screened by MCH-induced calcium mobilization on a fluorometric imaging plate reader (as described below).

Calcium Mobilization Assays-- Intracellular calcium assays were carried out essentially as described previously (17). The maximum change in fluorescence above baseline, measured on a fluorometric imaging plate reader (FLIPR, Molecular Devices), was used to determine the agonist response. For cross-screening studies, HEK293 cells were screened against a large library of over 1500 known and putative GPCR agonists including all known mammalian neuropeptides as described previously (17). Peptides in this library were tested at a final concentration of >100 nM, and other potential agonists were tested at a final concentration of >1 µM. For antagonist studies, test substances were added 30 min before the addition of an EC50 concentration of agonist. The data were analyzed using GraFit (Erithacus Software). Peptides were purchased and synthesized as described previously (10).

Microphysiometry-- Changes in the extracellular pH of HEK293 cells stably transfected with MCH2 receptors were monitored using the Cytosensor microphysiometer (Molecular Devices) (18). Cells were seeded into poly-L-lysine-coated Cytosensor capsules (0.2 million cells/capsule) and cultured overnight. The capsules were placed on the Cytosensor and equilibrated for 2 h with modified RPMI 1640 medium (Life Technologies, Inc.), pH 7.4. MCH was applied to the cells for 20 s prior to the "get rate," and the cells were then washed with running medium to remove ligand. Extracellular acidification rates were determined as the rate of change of sensor output during the periodic interruption of media flow. The medium was allowed to flow for 80 s and stopped for 38 s. Rates were measured over 30 s, starting 8 s after the flow was stopped.

Laser Scanning Cytometry Binding Assays-- HEK293 cells transiently expressing MCH2 receptors were seeded into 16-well chambers (Lab-Tek, Nalge Nunc International). The cells were grown in Eagle's minimum essential medium (including L-glutamine, 10% fetal calf serum, 1% nonessential amino acids, and 400 µg/l G418) for 24 h and then incubated at 37 °C for 30 min with indodicarbocyanine-labeled MCH (Amersham Pharmacia Biotech) at concentrations ranging from 0 to 40 nM in HEPES-buffered saline (including 2.5 mM MgCl2, 1.5 mM CaCl2, and 0.5% bovine serum albumin). The cells were then washed in HEPES-buffered saline minus the bovine serum albumin and fixed with 4% paraformaldehyde. Analysis was performed using a laser scanning cytometer (CompuCyte). Excitation of the indodicarbocyanine label by a 5-milliwatt helium/neon laser resulted in the emission of fluorescence that was collected through a 650-nm long path filter and measured by monitoring the red fluorescence maximal pixel intensity. Nonspecific binding was determined in the presence of 40 µM unlabeled MCH, and specific binding-derived fluorescence was determined by subtraction of this from total binding. The data were analyzed by using Sigma Plot (SPSS, Inc.).

TaqMan RT-PCR Tissue Localization-- Quantitative RT-PCR was performed using gene-specific primers to MCH2 and MCH1 receptors on mRNA from 20 body tissues and 19 brain regions as described previously (19). The mRNA from each tissue and brain region analyzed by TaqMan quantitative RT-PCR was derived from four individuals, two male and two female (except prostate tissue). The MCH2 receptor primers were forward (5'-TTGCCTGTAGTGCCATCATGA-3'), TaqMan probe (5'-ACGAGGGCAAAGTACCTGTCCACACTCATT-3'), and reverse (5'-AACGTGTCAGTCGAAATGGTTG-3'). The MCH1 receptor primers were forward (5'-GCCACCATGGACCTGGAAG-3'), TaqMan probe (5'-CAATGCCAGCAACACCTCTGAT GGC-3'), and reverse (5'-GGTGATCCTGCCGAAGTGA-3'). Forward (5'-CAAGGTCATCCATGACAACTTTG-3'), TaqMan probe (5'-ACCACAGTCCATGCCATCACTGCCA-3'), and reverse (5'-GGCCATCCACAGTCTTCTGG-3') primers designed to the housekeeping gene GAPDH were used to produce reference mRNA profiles.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The MCH2 receptor was cloned from human fetal brain tissue cDNA. The 1023-base pair open reading frame encodes a 340-amino acid protein that structurally resembles members of the GPCR superfamily (Fig. 1). The receptor contains a short N terminus, seven distinct hydrophobic membrane-spanning domains, and the highly conserved DRY motif located at the interface between the third transmembrane helix and the cytoplasm (20). The receptor has only one initiator methionine in the open reading frame, which contrasts with three such putative initiator methionines in MCH1. There are two putative N-linked glycosylation sites, and there is no characteristic signal peptide. BLAST analysis of public data bases revealed MCH1 to be its most homologous relative. The two GPCRs are 57% identical at the nucleotide level and 59% similar and 38% identical at the amino acid level (Fig. 2). Somatostatin receptors were the most similar receptors to these two putative paralogs, with ~26% identity at the amino acid level.


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Fig. 1.   Nucleotide and deduced peptide sequence of the MCH2 receptor. The seven hydrophobic transmembrane-spanning domains are indicated in bold, two cysteine residues available for disulphide bridge formation are marked with asterisks, and two potential N-linked glycosylation sites are indicated in bold italics and are underlined.


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Fig. 2.   Protein alignment of MCH1 and MCH2 receptors. The amino acid sequences were aligned with ClustalW (37) using the BLOSUM series substitution matrix. The alignment is viewed using GENEDOC. Identical amino acids are highlighted in black. Amino acid residues are numbered in the right-hand margin.

We transiently transfected HEK293 cells with MCH2 receptor cDNA and tested these cells for responsiveness to MCH. MCH (100 nM) induced a clear, robust, and transient increase in intracellular Ca2+ in MCH2 receptor-transfected cells but not in control cells transfected with the same vector containing a µ opiate receptor (Fig. 3A). The dose dependence of this response was investigated, and an EC50 value (± S.E., n = 3) of 8.57 ± 0.62 nM was determined. To confirm the specificity of MCH2 receptor activation by MCH, we also screened these cells against a large library of known and putative GPCR ligands including all known mammalian neuropeptides at final concentrations greater than 100 nM. MCH was the only substance in this library observed to elicit MCH2 receptor-mediated Ca2+ responses in these cells.


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Fig. 3.   MCH2 receptor-mediated intracellular Ca2+ responses to MCH in HEK293 cells. A, cells transiently transfected with MCH2 DNA responded to 100 nM MCH with a clear, robust, and transient increase in intracellular Ca2+ (filled circles). However, cells transiently transfected with the same vector containing a µ opiate receptor did not respond to MCH (open circles). B, cells stably expressing MCH2 receptors responded in a dose-dependent fashion to MCH and two related peptides with robust increases in intracellular Ca2+. The data shown are from a single representative experiment, and values are given as the means of triplicate determinations (± S.E.). MCH, filled circles; salmon MCH, open squares; [Phe13,Tyr19]MCH, open triangles; variant MCH, filled squares.

A HEK293 cell line stably expressing the MCH2 receptor was established and used in all further functional studies. The concentration dependence of MCH2 receptor activation by MCH and related peptides was investigated in the intracellular Ca2+ assay (Fig. 3B). MCH, salmon MCH, and [Phe13,Tyr19]MCH all behaved as agonists with similar potencies (EC50 values (± S.E., n = 3) of 5.65 ± 1.78, 7.14 ± 3.13, and 4.29 ± 0.48 nM, respectively). Variant MCH, the putative product of a second, variant form of the MCH gene (21), was a weak agonist (EC50 > 3000 nM). To determine the type of G protein mediating this response, we pretreated cells with pertussis toxin (100 ng/ml for 16 h). The toxin treatment had no effect on calcium mobilization by MCH in these cells (data not shown), suggesting that the MCH2 receptor is coupled to G proteins of the Gq/11 subfamily.

In a variety of native receptor studies, a number of peptides have been reported to either exhibit functional antagonism of MCH responses or inhibit binding to MCH binding sites (22-24). To investigate whether these effects are mediated via MCH2 receptors, we tested the following peptides in agonist and antagonist modes over a range of concentrations up to 10 µM in an intracellular Ca2+ assay: rat atrial natriuretic peptide (1-28), rat atrial natriuretic peptide (3-28), human C-type natriuretic peptide-22, human brain natriuretic peptide-32, gamma -endorphin, and alpha -melanocyte-stimulating hormone. We also tested somatostatin-14, somatostatin-28, and cortistatin-14 because of the similarity of MCH1 and MCH2 receptors to somatostatin receptors. We also tested a number of putative products of the authentic MCH precursor and of a second, variant form of the MCH gene: neuropeptide EI, neuropeptide GE, MCH gene-overprinted peptide-14, and variant neuropeptide EI. None of these peptides were active as agonists or antagonists at concentrations up to 10 µM.

To confirm further that the MCH2 receptor responded specifically to MCH, we monitored extracellular acidification rates in cells stably expressing the receptor using the technique of microphysiometry. 1 and 10 nM MCH caused robust increases in the rate of proton extrusion in transfected cells but not in nontransfected HEK293 cells (Fig. 4). Control studies demonstrated that 100 µM muscarine elicited similarly robust acidification responses in both MCH2 receptor-transfected and nontransfected cells (data not shown), indicating that both cell lines are capable of responding in this assay and thus confirming the specificity of the response to MCH. The dose dependence of the acidification response was also investigated, and an EC50 value (± S.E., n = 4) of 1.43 ± 0.44 nM was determined.


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Fig. 4.   MCH increases extracellular acidification rates in HEK293 cells stably transfected with MCH2 receptor (filled symbols) but not in nontransfected cells (open symbols). Cells were exposed to either 10 (squares or diamonds) or 1 nM (triangles) MCH over the time course indicated by the bar. Data are taken from a single experiment representative of a total of three such experiments.

We used laser scanning cytometry to measure the binding parameters of fluorescently labeled MCH to MCH2 receptors. Saturation analysis of indodicarbocyanine-labeled MCH binding to HEK293 cells transiently transfected with MCH2 receptors showed specific high affinity binding with a Kd (± S.E., n = 3) of 6.02 ± 0.46 nM (Fig. 5), although expression levels were apparently low with the fluorescence signal (specific binding) arising from ~5% of the population. This value was comparable with the potencies observed for nonlabeled forms of MCH obtained in the two functional assays.


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Fig. 5.   Saturation binding analysis of a fluorescently labeled analogue of MCH (indodicarbocyanine-MCH) to HEK293 cells transiently transfected with MCH2 receptors. Values are the means (± S.E.) of three normalized data points, each from three separate experiments.

We compared the tissue distributions of MCH1 and MCH2 receptors using quantitative TaqMan RT-PCR. The mRNA profiles of the two receptors were similar, showing predominant expression in the brain (Fig. 6). However, a major difference was the much higher relative levels of MCH1 compared with the MCH2 mRNA in pituitary. The distribution of the two receptors within individual regions of the brain is similar, but there are subtle differences. For example, the hypothalamus, locus coeruleus, medulla oblongata, and cerebellum seem to express higher relative levels of MCH1 compared with MCH2 mRNA.


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Fig. 6.   Tissue localization of MCH1 and MCH2 receptors using TaqMan RT-PCR methodology. The expression profile of the two receptors over a range of 20 different human tissues and 19 different brain regions is shown. The data are presented as mean (± S.E.) mRNA copies detected of four nondiseased individuals (two males and two females (except prostate)) for each tissue from 1 ng of Poly(A)+ RNA. Each of the four individual intestine samples represents a 50:50 mix of one person's small intestine and another person's colon (same sex). Each brain sample represents an equal part of the RNA pool of the 19 key brain regions used. The GAPDH distribution profiles for the same samples are shown for comparison.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This report describes the cloning and functional characterization of a second, novel MCH receptor. Several lines of evidence suggest that MCH is involved in the regulation of food intake and energy balance. Mice have been generated that carry a targeted deletion of the MCH gene (25). These knockout mice show reduced food intake, have reduced body weight, and are leaner. The localization of MCH in the lateral hypothalamus and the zona incerta, areas that are involved in the regulation of ingestive behavior, is consistent with this role in feeding. However, perikarya from these areas project widely throughout the central nervous system, suggesting an involvement of MCH in a wide range of behaviors. Thus MCH has also been shown to interact within the hypothalamo-pituitary-adrenal axis or stress axis (26, 27) and to be anxiogenic (28). Hyperactivity of the stress axis is known to occur in individuals suffering from depression. Thus MCH may also have a significant role to play in psychiatric disorders.

Despite the large amount of data concerning the physiological actions of MCH, a receptor had not been identified until recently. Within a short period of time several groups (10-14) discovered that a 353-amino acid splice variant form of SLC-1 (MCH1) is an MCH receptor. Within the GPCR superfamily, there are numerous examples of natural ligands that can activate more than one molecular species of GPCR. We therefore reasoned that additional MCH receptors may exist. However, low stringency Southern blot studies (10) and related studies (3, 15) suggest that additional receptors with high sequence identity to MCH1 are unlikely to exist. We therefore interrogated public data bases to identify sequences with low but significant levels of homology to MCH1. A short sequence was identified in a genomic survey sequence with homology to MCH1. Extension of this sequence revealed a full-length cDNA with many of the motifs characteristic of a GPCR, which we designated MCH2 in view of its sequence similarity to MCH1 and its subsequent characterization.

MCH2, similar to MCH1, shares low but significant homology to somatostatin receptors. We tested three naturally occurring ligands for somatostatin receptors against MCH2 receptors and did not observe any agonism or antagonism of MCH responses. Similar studies have demonstrated that somatostatin does not interact with MCH1 receptors (10, 11, 15).

Recent studies have investigated the molecular mechanism by which MCH interacts with MCH1 (29) and demonstrate that Asp123 in transmembrane-3 plays a key role in the formation of a complex between receptor and MCH, possibly by direct interaction with the Arg11 of MCH. Interestingly, this transmembrane-3 aspartate is conserved within MCH2 (Asp113) and may therefore play a similar role in both paralogs. An equivalent aspartate residue with functional significance for receptor activation has also been identified in the related families of opiate (30) and somatostatin (31) receptors, and the same residue has been recognized for many years to be conserved in all biogenic amine GPCRs, in which it acts as a counter-ion for the protonated amine moiety of the ligand (32).

Both functional and binding assays were used to confirm MCH as the cognate ligand for this receptor. Further characterization using an intracellular Ca2+ assay demonstrated that salmon MCH and the synthetic analogue [Phe13,Tyr19]MCH were equipotent with MCH and were full agonists at MCH2. However, variant MCH, the putative product of a second, variant form of the MCH gene (21), was ~500-fold less potent than MCH. This profile of agonist activity is essentially similar to that observed at MCH1 (10). These data also demonstrate that variant MCH is unlikely to act as a natural ligand for either MCH1 or MCH2 receptors.

To further characterize the MCH2 receptor we tested, as both agonists and antagonists, a number of peptides that have been reported to bind with low affinity to MCH binding sites in various cell lines and tissues. Thus, MCH binding sites in mouse melanoma cells (22), human keratinocytes (23), and human brain (24) are weakly displaced by a number of natriuretic peptides. Because these peptides do not bind to MCH1 receptors, it has been proposed that they may bind to novel MCH receptors (10). In the present study we demonstrate that the MCH2 receptor is not activated or antagonized by these peptides. This may therefore imply the existence of still further subtypes of the MCH receptor. However, recent studies (33) have demonstrated that the specific binding to some of these cell lines is unlikely to be caused by the presence of a receptor involved in signal transduction, because binding is largely localized to microsomal and not plasma membranes, and the internalization kinetics are not typical of a receptor-mediated event.

We also tested a number of peptides that have been reported either to functionally antagonize MCH responses or are putative products of either the authentic or variant MCH gene. Thus the alpha -melanocyte-stimulating hormone and MCH have mutually antagonistic effects on a number of different physiological functions including feeding behavior (34, 35). The lack of activity of alpha -melanocyte-stimulating hormone at MCH2 receptors parallels similar findings with the MCH1 receptor (10-12) and confirms that both peptides exert their effects via separate receptor families. Likewise, we observed that none of the additional putative products of the authentic and variant MCH precursor (neuropeptide GE, neuropeptide EI, variant neuropeptide EI, MCH gene-overprinted peptide-14) seem to interact with MCH2 receptors. Previous studies have also demonstrated that these peptides do not interact with MCH1 receptors (10, 11). Taken together, these data indicate that any biological effects of these peptides are most likely to be mediated by receptors other than the two known MCH receptors.

Both MCH1 and MCH2 receptors are expressed predominantly in the brain. Within the brain, the pattern of expression of the two paralogs is similar. Both paralogs are widely distributed, but the mRNA profiles show noticeably higher contributions from limbic areas such as amygdala, hippocampus, and parahippocampal gyrus and in a number of cortical regions (cingulate-, medial frontal-, and superior frontal-gyri). The widespread distribution of MCH1 is in agreement with that observed previously in the rat brain (11, 36). Because the patterns of expression of these paralogs are so similar, these data suggest that neither paralog can yet be selectively implicated in mediating any specific effect of MCH. The lower relative levels of MCH2 mRNA in pituitary and hypothalamus compared with MCH1 suggests that this newly discovered MCH receptor may be involved in physiological processes other than feeding or neuroendocrine modulation.

The identification of a second MCH receptor will help to further elucidate the role of MCH in energy homeostasis and feeding behavior as well as in disorders such as obesity and social anxiety disorder.

    ACKNOWLEDGEMENTS

We thank Allison Goddard, Lisa Spinage, Mark Tornetta, and Parvathi Nuthulaganti for excellent technical assistance and are grateful to Darren Moore, Piers Emson, and Guillaume Hervieu for valuable discussions. Human brain tissue was obtained from the Netherlands Brain Bank in Amsterdam (Coordinator Dr. R. Ravid).

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF347063.

§ To whom correspondence should be addressed. Tel.: 44-1279-627-259; Fax: 44-1279-627-266; E-mail: Jeffrey_2_Hill@gsk.com.

Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M102068200

    ABBREVIATIONS

The abbreviations used are: MCH, melanin-concentrating hormone; GPCR, G protein-coupled receptor; RT-PCR, reverse transcription-polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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