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

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

Melanin-concentrating hormone (MCH) 1 is a cyclic neuropeptide originally identified in salmon pituitaries responsible for skin color changes in teleost fish (1). This peptide is composed of 17 amino acids (DTMRCMVGRVYRPCWEV), whereas the mammalian form of MCH is a 19-amino acid peptide (DFDMLRCMLGRVYRPCWQV) expressed throughout the brain with the highest concentration in the lateral hypothalamic and zona incerta areas (2)(3)(4).
MCH plays a role in several important physiologic processes.
The most active area of research on MCH has focused on the role of MCH in the regulation of feeding behavior and energy balance. Several studies provide evidence for a role for MCH in regulation of body weight.1) Mice that lack MCH are hypophagic and exhibit an increased metabolic rate, resulting in decreased body weight and body fat content (5). 2) Intracerebroventricular injection of MCH increases feeding in rats (6 -8).
Other physiological roles of MCH include regulation of the hypothalamopituitary adrenal gland axis (11), modulation of water and electrolyte fluxes in the gut (12), stimulation of oxytocin secretion from isolated rat neurohypophysis (13), regulation of sensory processing (14), and modulation of monoaminergic activity in the medial preoptic area (15). In addition, several lines of evidence also suggest a role for MCH in modification of memory retention (16), modulation of other hormones regulating food intake (17) and sexual behavior (18) and involvement in seizure (19). MCH is also localized in neurons functionally involved in circuits of the extrapyramidal motor systems from striatal centers to the thalamus and cerebral cortex and to the midbrain and spinal cord (20). The complex central nervous system expression pattern of MCH together with the many diverse functions mediated by MCH suggest the existence of more than one MCH receptor.
The molecular identities of MCH receptors were unknown until recent reports of the first MCH receptor (MCH-R1) (21)(22)(23)(24)(25). MCH-R1 is composed of seven transmembrane domains typical of G protein-coupled receptors and binds to MCH with high affinity. MCH-R1 is coupled to G i , G o , and G q -type G proteins and mediates decreases in intracellular cAMP and increases in intracellular Ca 2ϩ levels, inositol phosphate production, and mitogen-activate protein kinase activity (26). In this report, we describe a novel MCH receptor identified through bioinformatic and molecular cloning approaches. The receptor is similar (35% identical) to the previously published MCH receptor, is able to bind 125 I-labeled MCH, and can be activated by MCH leading to intracellular signaling. In addition, the expression of the receptor is localized to distinct regions of the brain.

MATERIALS AND METHODS
Human Marathon-Ready cDNAs and the rapid amplification of cDNA ends (RACE) kit were from CLONTECH (Palo Alto, CA). Oligonucleotides were custom-synthesized by Life Technologies, Inc. The 293-EBNA cell line (293 cells) was obtained from Invitrogen. MCH and other peptides were purchased from Sigma or BACHEM. 125 I-MCH was from PerkinElmer Life Sciences.
Isolation of cDNA for MCH-R2-The amino acid sequences of known G protein-coupled receptors were used to conduct a BLAST search of the public data base GenBank TM . The search identified a BAC clone (accession number AQ492353) with a sequence of 429 bp. The fragment * The costs of publication of this article were defrayed in part by the payment of page charges. This 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 GenBank TM  contained an ϳ120-bp coding sequence belonging to the transmembrane regions 2 and 3 of a putative G protein-coupled receptor, and the rest was a portion of an intron. A BLAST analysis revealed high homology of the 120-bp coding sequence to the recently published MCH receptor (MCH-R1) (21)(22)(23)(24)(25). RACE was used to obtain the full-length cDNA for this putative G protein-coupled receptor (MCH-R2). The 5Јfragment containing the translation start codon and the 3Ј-fragment containing the stop codon were generated using the Marathon RACE kit for PCR reactions and human brain and brain cerebral cortex Marathon-Ready cDNA (CLONTECH) as the PCR template (see Fig. 1A). PCR conditions for this reaction were 94°C, 30 s; 35 cycles at 94°C, 30 s and 68°C, 3 min; and 68°C 7 min. For each RACE, nested PCR primers specific to the MCH-R2 cDNA sequence and primers AP1 and AP2 that bind to Marathon-Ready cDNA adapter (CLONTECH) were used. Multiple RACE reactions were run to extend the sequence to the start codon of the MCH-R2 full-length cDNA. Similar conditions were used to obtain the 3Ј-RACE products to obtain the stop codon of the open reading frame (ORF).
To clone the full length of MCH-R2 cDNA, a new PCR primer pair (oligoJ634 and oligoJ636) was designed to amplify in a PCR by using CLONTECH Marathon-Ready cDNA as template (see Fig. 1A). The PCR product containing the full-length cDNA was cloned into expression vector pCR3.1 (Invitrogen) to form an expression construct pCR3. The arrows below denote multiple RACE reactions to extend the sequence toward the 5Ј and 3Ј directions. Primers oligoJ634 and oligoJ636 were used in PCR to obtain the full-length ORF of MCH-R2. The ORF is indicated by the long bar with the nucleotide sequence shown below (GenBank TM accession number AF399937). B, amino acid sequence alignment of MCH-R2 with those of MCH-R1 (27). Residues that match exactly are shaded black.
min. Cells were then treated with an additional 300 l of binding buffer containing 125 I-MCH at concentrations described in the figures for 45 min at room temperature. Cells were then placed on ice and washed four times with cold binding buffer. Cells were scraped into 12 ϫ 75-mm test tubes and centrifuged at 3000 ϫ g for 5 min. Excess fluid was aspirated, and the radioactivity bound to the cell pellet was counted in a ␥ counter.
Measurement of Intracellular Ca 2ϩ Levels-CHO/MCH-R2 and CHO/ MCH-R1 cells were split into 96-well plates and incubated overnight. Subsequently the medium was removed, and 100 l of FLIPR buffer (Hanks' balanced salt solution, 20 mM Hepes, 2.5 mM probenecid, 0.1% bovine serum albumin) containing 4 M Fluo-3/AM was added to the wells. Cells were incubated at 37°C in 5% CO 2 for 1 h. Cells were then washed two times with 150 l of FLIPR buffer. Subsequently 150 l of FLIPR buffer was added to each well, and changes in intracellular Ca 2ϩ Messenger RNA Expression Analysis of MCH-R2-Tissue expression of MCH-R2 was examined using dot blots and Northern blots obtained from a commercial source (CLONTECH). Hybridization to blots was carried out using a PCR-generated DNA fragment encompassing 800 bp at the 3Ј-end of the MCH-R2 cDNA. The primer sequences were as follows: forward primer, 5Ј-ttggaacacctctgccgaactt; reverse primer, 5Ј-aacccacatagaaggccagtgt. The DNA fragments were random primelabeled with [ 32 P]dCTP, and the blots were hybridized for 14 h in ExpressHyb (CLONTECH) containing ϳ2 million cpm/ml of radiolabeled probe. The following day the blots were washed and exposed to Kodak Biomax MS film for 3 days at Ϫ70°C. The films were analyzed for relative expression levels using the MCID M4 image analysis system (Imaging Research, Ontario, Canada).

RESULTS AND DISCUSSION
The 120-bp coding sequence (Fig. 1A) was used as a starting sequence for the 5Ј-and 3Ј-RACE extensions. Multiple RACE reactions were performed to obtain the 5Ј translation initiation codon and the 3Ј translation termination codon by using human brain cDNA preparations. Based on the 5Ј-and 3Ј-end sequences, a full-length ORF was generated by PCR. The ORF is 1023 bp long (Fig. 1A) and encodes a protein of 340 amino acids (Fig. 1B). Immediately upstream of the ATG initiation codon is a short stretch of untranslated sequence with an in-frame termination codon (TAA tcc ctg gaa agt cca cga aca ATG), suggesting a correct assignment of the Met as the N terminus of the receptor.
Comparison with other G protein-coupled receptor amino acid sequences and hydrophobicity analysis of the 340-amino acid protein suggests that there are seven transmembranespanning regions within the protein, a feature shared by the G protein-coupled receptor superfamily. BLAST analysis with the amino acid sequence of MCH-R2 against the GenBank TM data base revealed moderately high homology to known receptors, i.e. the MCH-R1 receptor, somatostatin receptor types 5, 3, 2, and 1, and the and ␦ opioid receptor subtypes. The alignment analysis of the protein sequences by the J. Hein method (27) showed that the highest homology of MCH-R2 is to the human MCH-R1 receptor (35%). Fig. 1B illustrates the alignment of the MCH-R2 amino acid sequence with human MCH-R1. These analyses suggest that the protein encoded by the ORF sequence may be a novel MCH receptor. The amino acid sequence is identical to that in recent reports that also concluded it to be MCH-R2 (28,29).
Radioligand binding assays were performed to test the ability of MCH-R2 to bind 125 I-labeled MCH. MCH-R2 ORF cDNA was cloned into the expression vector pME18. A CHO cell line was generated that stably expressed MCH-R2 (CHO/MCH-R2). Subsequent studies were performed using this cell line. Specific binding of 125 I-MCH to the CHO/MCH-R2 cells was detected ( Fig. 2A). No specific binding was observed with wild type CHO cells. Nonradiolabeled MCH competitively inhibited 125 I-MCH (0.5 nM) binding to CHO/MCH-R2 with an IC 50 of 5.0 Ϯ 1.0 nM (Fig. 2B). This data demonstrates that MCH specifically binds to MCH-R2 with high affinity.
The ability of MCH to activate MCH-R2 and regulate intracellular signaling is shown in Fig. 3. MCH dose dependently stimulated an increase in intracellular free Ca 2ϩ levels (Fig.  3A) and IP production (Fig. 3B) in CHO/MCH-R2 cells and in CHO cells expressing MCH-R1 (CHO/MCH-R1). The EC 50 values of MCH at MCH-R2 (0.54 Ϯ 0.05 nM in the Ca 2ϩ assay and 3.4 Ϯ 0.2 nM in the IP assay) were lower in both functional assays compared with the EC 50 values of MCH at MCH-R1 (5.0 Ϯ 0.6 nM in the Ca 2ϩ assay and 27 Ϯ 9 nM in the IP assay). MCH stimulation of CHO/MCH-R2 cells had no effect on basal (data not shown) or forskolin-stimulated (Fig. 3C) cAMP production. By contrast MCH stimulation of CHO/MCH-R1 cells inhibited forskolin-provoked increases in cAMP production (Fig. 3C). Cells expressing MCH-R2 were not responsive to stimulation by somatostatin, dynorphin A, dynorphin B, neuropeptide EI, or neuropeptide GE at concentrations up to 1 M (data not shown). These data demonstrate that MCH specifically activates MCH-R2 and suggest that MCH-R2 couples to a G q -type G protein to mediate intracellular effects. By comparison, MCH-R1 couples to G i /G o (as demonstrated by inhibition of cAMP production) and G q -type G proteins in CHO cells (26).
To compare the pharmacological profile of MCH-R2 to MCH-R1, the abilities of mammalian MCH, [Phe 13 ,Tyr 19 ]MCH, and salmon MCH to stimulate increases in intracellular free Ca 2ϩ levels and IP production in CHO/MCH-R1 and CHO/MCH-R2 were assessed. As shown in Fig. 4A, the EC 50 for mammalian MCH-stimulated increases in intracellular free Ca 2ϩ levels in CHO/MCH-R2 cells was 0.54 Ϯ 0.05 nM. By comparison, the EC 50 of [Phe 13 ,Tyr 19 ]MCH was higher (2.26 Ϯ 0.43 nM). The EC 50 for salmon MCH (4.0 Ϯ 0.4 nM) was an order of magnitude higher than that for mammalian MCH. In contrast, in CHO/ MCH-R1 cells (Fig. 4B) Fig. 4 illustrate different pharmacological profiles between the two cloned MCH receptors. The EC 50 of mammalian MCH at MCH-R2 is an order of magnitude lower than that at MCH-R1 in both functional assays. The potency of salmon MCH is much less (10 times in the Ca 2ϩ assay and 30 times in the IP assay) than mammalian MCH at MCH-R2. In contrast, the potencies of salmon MCH and mammalian MCH at MCH-R1 are relatively similar.
To determine whether the binding affinity of mammalian MCH, [Phe 13 ,Tyr 19 ]MCH, and salmon MCH for MCH-R2 differed, whole cell binding experiments were performed. Binding of 125 I-MCH to CHO/MCH-R2 cells was competitively inhibited by increasing concentrations of mammalian MCH, [Phe 13 ,Tyr 19 ]MCH, and salmon MCH (Fig. 5A) 19 ]MCH, and 100 Ϯ 20 nM for salmon MCH. By comparison, the affinities for the three MCH peptides for MCH-R1 appear to be relatively similar (Fig. 5B). The IC 50 values were 3.0 Ϯ 1.0 nM for mammalian MCH, 5.0 Ϯ 1.0 nM for [Phe 13 ,Tyr 19 ]MCH, and 4.0 Ϯ 1.0 nM for salmon MCH at MCH-R1. These data are consistent with the functional data in Fig. 4 and with recent reports characterizing MCH-R2 (28,29).
The interaction of a ligand with its receptor is a major determinant of affinity. Comparison of mammalian MCH (DFDMLRCMLGRVYRPCWQV) with salmon MCH (DTMRC-MVGRVYRPCWEV) reveals that the primary difference between the two ligands is near the N terminus (FDML) outside of the cyclic structure formed by the disulfide bond between Cys 7 and Cys 16 . Thus the lower affinity of salmon MCH for MCH-R2 suggests that the 4-amino acid stretch FDML as well as Leu 9 and Gln 18 may play a role in ligand binding of mam-malian MCH with MCH-R2.
The expression of MCH-R2 mRNA was determined with Northern and dot blot hybridization with an 800-bp MCH-R2 cDNA fragment in the ORF as probe. The MCH-R2 mRNA was localized primarily to telencephalic regions of the human brain (Fig. 6A). As shown by Northern blot, there is a prominent 4.4-kilobase pair band in total brain and all of the cortical RNAs (Fig. 6A). The multiple tissue expression array shows strong signals in the amygdala, hippocampus, and nucleus accumbens as well as in the fetal brain (Fig. 6B). Previous studies in rat and human brain have defined the expression of MCH-R1. Northern blot and in situ hybridization studies have shown MCH-R1 to be expressed in brain, eye, skeletal muscle, and tongue (21,22,25,30). In rat brain, MCH-R1 mRNA is broadly expressed and appears to be most abundant in neurons in the olfactory system, nucleus accumbens, amygdala, hippocampus, and hypothalamus (25). In contrast, the MCH-R2 mRNA is expressed almost exclusively in cortical regions of human brain (Fig. 6). Major areas of overlap between the two subtypes appear to be the nucleus accumbens, amygdala, and hippocampus. MCH-R2 also appears to be neuronal in nature because there is no signal in the corpus callosum RNA.
The differential expression of the two receptor subtypes in brain suggests that the two receptors serve different functions. Much of the functionality attributed to MCH revolves around homeostatic mechanisms such as food intake and energy balance as well as neuroendocrine processes. These functions and the MCH peptide appear to reside in subcortical regions like the hypothalamus in rodents, and thus the presence of MCH-R1 in many of these same regions suggests MCH-R1 is the receptor responsible for mediating homeostatic mechanisms. We note that MCH mRNA is expressed in neocortex in human (31) but not in rodents. The presence of MCH-R2 in the cortical regions suggests that either known MCH functions, such as the cognitive and emotional representation of food intake, or unknown MCH functions in which cortex is involved may be mediated by MCH-R2 only in human.
In summary, we have cloned and characterized a novel human MCH receptor subtype (MCH-R2). The receptor shares homology to the previously cloned MCH-R1 receptor SLC-1. The pharmacology of MCH-R2 is distinguished from that of MCH-R1 by having a lower relative affinity for salmon MCH. The distribution of MCH-R2 mRNA expression is highly restricted to cortical regions of the brain. Unlike MCH-R1, MCH-R2 is not detected in peripheral tissues. The identification of this new MCH receptor subtype is essential to understanding the complex physiological roles that MCH plays and will aid in dissecting the specific roles of MCH receptors in mediating these functions.