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J. Biol. Chem., Vol. 276, Issue 23, 20125-20129, June 8, 2001
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From the Departments of
Received for publication, March 7, 2001, and in revised form, March 23, 2001
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.
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.
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 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.
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.
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.
Molecular Cloning and Functional Characterization of
MCH2, a Novel Human MCH Receptor*
§,
,,,,,, 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
Department of Molecular Biology,
GlaxoSmithKline, King of Prussia, Pennsylvania 19406-0939
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (46K):
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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.
View larger version (50K):
[in a new window]
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.
View larger version (18K):
[in a new window]
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, 
-endorphin, and
-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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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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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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DISCUSSION |
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ABSTRACT
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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 -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
-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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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* 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
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ABBREVIATIONS |
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The abbreviations used are: MCH, melanin-concentrating hormone; GPCR, G protein-coupled receptor; RT-PCR, reverse transcription-polymerase chain reaction.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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