CA2427763A1 - Cell lines expressing a mch receptor - Google Patents

Abstract

The present invention features neuroblastoma and skin cell carcinoma cell lines functionally expressing MCHR1 and the use of such cells to measure MCH R1 activity. Functional expression is preferably achieved in a neuroblastoma or skin cell carcinoma using a recombinant gene expressing MCHR1. The presence of a recombinant MCHR1 gene increases the level of MCHR1 expression facilitatin g the production and detection of MCHR1 activity.

Description

TITLE OF THE INVENTION
CELL LINES EXPRESSING A MCH RECEPTOR
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to provisional application U.S.
Serial No. 60/244,700, filed October 31, 2000, hereby incorporated by reference herein.
BACKGROUND OF THE INVENTION
The references cited herein are not admitted to be prior art to the claimed invention.
Neuropeptides present in the hypothalamus play a major role in mediating the control of body weight. (Flier, et al., 1998. Cell, 92, 437-440.) Melanin-concentrating hormone (MCH) is a cyclic 19-amino acid neuropeptide synthesized as pa~.-t of a larger pre-prohormone precursor in the hypothalamus which also encodes neuropeptides NEI and NGE. (Nahon, et al., 1990. Mol. Endocrinol.
4, 632-637.) MCH was first identified in salmon pituitary, and in fish MCH
affects melanin aggregation thus affecting skin pigmentation. In trout and in eels MCH
has also been shown to be involved in stress induced or CRF-stimulated ACTH
release.
(Kawauchi, et al., 1983. Nature 305, 321-323.) In humans two genes encoding MCH have been identified that are expressed in the brain. (Breton, et al., 1993. Mol. Brai~z Res. 18, 297-310.) In mammals MCH has been localized primarily to neuronal cell bodies of the hypothalamus which are implicated in the control of food intake, including perikarya of the lateral hypothalamus and zona inertia. (Knigge, et al., 1996. Peptides 17, 1063-1073.) Pharmacological and genetic evidence suggest that the primary mode of MCH action is to promote feeding (orexigenic). MCH mRNA is up regulated in fasted mice and rats, in the oblob mouse and in mice with targeted disruption in the gene for neuropeptide Y (NPY). (Qu, et al., 1996. Nature 380, 243-247, and Ericlcson, et al., 1996. Nature 381, 415-418.) Injection of MCH centrally (ICV) stimulates food intake and MCH antagonizes the hypophagic effects seen with a melanocyte stimulating hormone (aMSH). (Qu, et al., 1996. Nature 380, 243-247.) MCH deficient mice are lean, hypophagic and have increased metabolic rate.
(Shimada, et al., 1998. Nature 396, 670-673.) MCH action is not limited to modulation of food intake as effects on the hypothalamic-pituitary-axis have been reported. (Nahon, 1994. Critical Rev. in Neurobiol. 8, 221-262.) MCH may be involved in the body response to stress as MCH can modulate the stress-induced release of CRF from the hypothalamus and ACTH from the pituitary. In addition, MCH neuronal systems may be involved in reproductive or maternal function.
Several references describe a receptor that is indicated to bind MCH
(human MCHR1). (Chambers, et al., 1999. Nature 400, 261-265; Saito, et al., 1999.
Nature 400, 265-269; Bachner, et al., 1999. FEBS Letters 457, 522-524;
Shimomura, et al., 1999. Biochemical and Biophysical Research Communications 261, 622-626;
and Lembo, et al., 1999. Nat. Cell Biol. l, 267-271.) SUMMARY OF THE INVENTION
The present invention features neuroblastoma and skin cell carcinoma cell lines functionally expressing MCHRl and the use of such cells to measure MCHR1 activity. Functional expression is preferably achieved in a neuroblastoma or skin cell carcinoma using a recombinant gene expressing MCHR1. The presence of a recombinant MCHR1 gene increases the level of MCHR1 expression facilitating the production and detection of MCHR1 activity.
Thus, a first aspect of the present invention describes a neuroblastoma or skin cell carcinoma comprising a recombinant MCHR1 gene that expresses functional MCHR1. Functional MCHR1 produces a detectable signal upon MCH
stimulation. The recombinant MCHRl gene may be part of a genome or may be present outside of the genome.
An MCHR1 gene contains nucleic acid encoding for MCHR1 and regulatory elements needed for functional expression. Examples of regulatory elements useful for functional expression include a promoter, a terminator, a ribosome binding site, and a polyadenylation region. The nucleic acid encoding for can be contiguous or may contain one or more introns.
A recombinant MCHR1 gene encodes for MCHR1 and contains one or more regions not naturally associated with each other. Examples of recombinant MCHR1 genes include those containing a human nucleic acid sequence encoding for MCHR1 present with a regulatory sequence not naturally associated with the encoding nucleic acid; and those containing a non-naturally occurring encoding region.
A non-naturally encoding region contains one or more combinations of nucleotides not -2_ present in the naturally occurring encoding nucleic acid. Recombinant genes can be produced with, or without, intron(s).
Another aspect of the present invention describes a neuroblastoma or skin cell carcinoma having increased MCHR1 expression produced by a process comprising the step of coupling endogenous nucleic acid encoding for MCHR1 to an exogenous promoter. The process results in the production of a recombinant gene having the same chromosomal location as the native MCHR1 gene.
Another aspect of the present invention describes a method of measuring the ability of a compound to affect MCHR1 activity. The method comprises the steps of: (a) providing a compound to a neuroblastoma or skin cell carcinoma expressing MCHR1; and (b) measuring MCHR1 activity.
Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples.
The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention.
Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Human neuroblastoma and skin cell carcinoma cell lines able to produce MCHR1 transcripts and to provide a suitable environment for measuring MCHR1 activity are identified herein. Human cell lines expressing MCHR1 provide a human cellular environment that naturally expresses MCHRl.
Cells expressing functional MCHR1 provide a system for screening for compounds active at MCHRl, measuring the effect of a compound at MCHR1, and measuring the effect of MCHR1 activity. MCHR1 expression in a neuroblastoma or skin cell carcinoma is preferably increased using a recombinant MCHRl gene that either makes use of endogenous nucleic acid encoding for MCHR1 or provides exogenous nucleic acid encoding MCHR1.
Compounds modulating MCHR1 activity have a variety of different uses including utility as a tool to further study MCHR1 activity and as an agent to achieve a beneficial effect in a patient. Beneficial effects of modulating MCHRl activity include one or more of the following: weight loss, weight gain, cancer treatment (e.g., colon or breast), pain reduction, diabetes treatment, stress reduction and sexual dysfunction treatment.

Modulating MCHR1 activity includes evolving a response at the receptor and altering a response evolved by a MCHR1 agonist or antagonist.
Generally, MCH receptor antagonists and allosteric modulators negatively affecting activity may be used to achieve weight loss, treat cancer (e.g., colon or breast), reduce pain, reduce stress or treat sexual dysfunction; and MCH receptor agonists and allosteric modulators positively affecting activity may be used to produce a weight gain.
A patient is a mammal, preferably a human. Reference to patient does not necessarily indicate the presence of a disease or disorder. The term patient includes subjects treated prophylactically and subjects afflicted with a disease or disorder.
Preferably, MCHR1 activity is modulated to treat diabetes, to obtain a weight loss, or to obtain a weight gain. Diabetes mellitus can be treated by, for example, one or both of the following: enhancing glucose tolerance and decreasing insulin resistance.
Excessive weight is a contributing factor to different diseases including hypertension, diabetes, dyslipidemias, cardiovascular disease, gall stones, osteoarthritis and certain forms of cancers. Bringing about a weight loss can be used, for example, to reduce the likelihood of such diseases and as part of a treatment for such diseases. Weight reduction can be achieved by, for example, one or more of the following: reducing appetite, increasing metabolic rate, reducing fat intake or reducing carbohydrate craving.
Increasing weight is particularly useful for a patient having a disease or disorder, or under going a treatment, accompanied by weight loss. Examples of diseases or disorders accompanied by weight loss include anorexia, ASS, wasting, cachexia, and frail elderly. Examples of treatments accompanied by weight loss include chemotherapy and radiation therapy.

MCHR1 employed in the present invention includes naturally occurring human MCHRl having the amino acid sequence provided by SEQ.1D. NO.
1 and variants thereof. Variants of MCHR1 have a substantially identical amino acid sequence as SEQ. ID. NO. 1 and have MCH receptor activity. Examples of SEQ.
ID.
NO. 1 variants include naturally occurring allelic variants and artificially produced mutants.

SEQ. ID. NO. 1 and the naturally occurnng encoding nucleic acid sequence were initially identified as the somatostatin-like receptor "SLC-1."
(Lalcaye, et al., 1998. BiachirrZica et Biophysics ACTA 1401:216-220.) Subsequently, SLC-was shown to be MCHR1. cDNA and genomic sequences encoding for MCHR1 are provided by SEQ. D7. NOs. 2 and 3.
In general, SEQ. ID. NO. 1 variants have a sequence identity of at least about 90%, preferably, at least about 95%, with SEQ. ID. NO. l; and/or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications from SEQ. ID. NO. 1. Amino acid modifications are additions, deletions, and substitutions.
Sequence similarity for polypeptides can be determined by BLAST.
(Altschul, et al., 1997. Nueleic Acids Res. 25, 3389-3402, hereby incorporated by reference herein.) In one embodiment, sequence similarity is determined using tBLASTn search program with the following parameters: MATRIX:BLOSUM62, PER RESIDUE GAP COST: 11, and Lambda ratio: 1.
Artificial variants of MCHR1 can be produced in a cell by introducing nucleic acid encoding for the variant. Nucleic acid sequences encoding for variants can be obtained by altering the nucleic acid sequence encoding fox SEQ. ID. NO. 1: The translation of a particular codon into a particular amino acid is well known in the art (see, e.g., Lewin, GENES IV, p. 119, Oxford University Press, 1990):
A=Ala=Alanine: codons GCA, GCC, GCG, GCU
C=Cys=Cysteine: codons UGC, UGU
D=Asp=Aspartic acid: codons GAC, GAU
E=Glu=Glutamic acid: codons GAA, GAG
F=Phe=Phenylalanine: codons UUC, UUU
G=Gly=Glycine: codons GGA, GGC, GGG, GGU
H=His=Histidine: codons CAC, CAU
I=lle=Isoleucine: codons AUA, AUC, AUU
K=Lys=Lysine: codons AAA, AAG
L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU
M=Met=Methionine: codon AUG
N=Asn=Asparagine: codons AAC, AAU
F=Pro=Proline: codons CCA, CCC, CCG, CCU
Q=Gln=Glutamine: codons CAA, CAG
R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU

S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU
T=Thr=Threonine: codons ACA, ACC, ACG, ACU
V=Val=Valine: codons GUA, GUC, GUG, GUU
W=Trp=Tryptophan: codon UGG
Y=Tyr=Tyrosine: codons UAC, UAU
Changes to SEQ. m. NO. 1 to produce functional variants may be empirically determined. Techniques for measuring MCH receptor activity are well known in the art.
One method of producing functional variants of SEQ. m. NO. 1 expected to retain some MCH receptor activity takes into account differences in amino acid R groups. An R group effects different properties of an amino acid such as physical size, charge, and hydrophobicity. Amino acids can be divided into different groups as follows: neutral and hydrophobic (alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, and methionine); neutral and polar (glycine, serine, threonine, tyrosine, cysteine, asparagine, and glutamine);
basic (lysine, arginine, and histidine); and acidic (aspartic acid and glutamic acid).
Generally, in substituting different amino acids it is preferable to exchange amino acids having similar properties. Substituting different amino acids within a particular group, such as substituting valine for leucine, arginine for lysine, and asparagine for glutamine are good candidates for not causing a change in polypeptide functioning.
Changes outside of different amino acid groups can also be made.
Preferably, such changes are made taping into account the position of the amino acid to be substituted in the polypeptide. For example, arginine can substitute more freely for nonpolor amino acids in the interior of a polypeptide then glutamate because of its long aliphatic side chain. (See, Ausubel, Current Protocols iyZ Molecular Biology, John Wiley, 1987-1998, Supplement 33 Appendix 1C.) Recombinant MCHR1 Gene A recombinant MCHR1 gene can be used to increase the level of MCHR1 expression in a neuroblastoma or skin cell carcinoma thereby facilitating the production and detection of MCHR1 activity. Methods for producing a recombinant MCHR1 gene include those altering the endogenous MCHR1 gene and those introducing an MCHR1 gene or coding region into a host cell.

Alterations of an endogenous MCHR1 gene producing a recombinant gene include the use of regulatory elements such as a promoter or enhancer not naturally associated with the MCHR1 coding region; and using a non-naturally encoding region containing one or more combinations of nucleotides not present in the naturally occurring encoding nucleic acid. Examples of exogenous promoters include the human cytomegalovirus promoter ("CMV"), oc-MHC promoter, PrP
(piton promoter), potent neuronal promoter and Thy-1 promoter.
Non-naturally occurring encoding regions can be produced based on the degeneracy of the genetic code. If desired, the nucleic acid encoding for can be altered based on the genetic code to adjust codon frequency.
Techniques that can be used for creating a recombinant chromosomal gene are well known in the art. (See Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998). Exogenous nucleic acid can be targeted to the MCHR1 gene using homologous recombination targeting sequences. Homologous recombination targeting sequences for insertion into the MCHR1 gene include coding and non-coding regions.
An exogenous promoter, such as the CMV promoter, can be functionally coupled to MCHR1 nucleic acid using standard techniques. For example, the GOTO MCHRl genomic sequence can be cloned into a plasmid vector. The MCHR1 promoter region (2-3 kb) upstream of coding sequence can be replaced by a CMV promoter cassette containing a loxP-neomycin-loxP gene. The resulting promoter-exchange vector would have sufficient MCHR1 genomic sequences flanking the CMV promoter cassette for homologous recombination. The vector can then be electroporated into the GOTO cell. Neomycin resistant clones can be selected and verified by genomic Southern analysis for successful promoter exchange.
The neomycin gene can be removed by loxP mediated recombination to reduce its possible interference with promoter activity.
Introducing an MCHR1 gene or coding region into a host can be achieved by inserting a MCHR1 coding region or gene into the host genome or through the use of an independently replicating vector. Techniques for inserting nucleic acid into the host genome include those targeting and selecting for insertion in a particular region and those involving random insertion.
_7_ Functional Assays Techniques for measuring MCHR1 activity include detecting a change in the intracellular conformation of MCHR1, measuring G-protein activity, and measuring the level of intracellular messengers. Assays measuring different G-protein activities, such as Gi, Gs, and Gq can be carried out using techniques that are well known in the art. MCHRl activity is preferably assayed for by measuring either Gi or Gq activity.
Gi and Gs activity can be measured using techniques such as a melonaphore assay, assaying cAMP production, assaying inhibition of cAMP
accumulation, intracellular acidification, and assaying 35S-GTP binding. CAMP
can be measured using different techniques such as a radioimmunoassay and indirectly by cAMP responsive gene reporter proteins.
Gq and Gi activity can be measured using techniques such as those detecting intracellular Ca2+ and intracellular acidification. Examples of techniques well known in the art that can be employed to measure Ca2+ include the use of dyes such as Fura-2 and the use of Ca2+-bioluminescent sensitive reporter proteins such as aequorin. (Button, et al.,1993. Cell Calcium 14, 663-671, and Feighner, et al., 1999.
Science 254, 2184-2188, both of which are hereby incorporated by reference herein.) Functional assays can be performed using individual compounds or preparations containing different compounds. A preparation containing different compounds where one or more compounds affect MCHR1 activity can be divided into smaller groups of compounds to identify the compounds) affecting MCHR1 activity.
In an embodiment of the present invention a test preparation containing at least 10 compounds is used in a functional assay.
MCHR1 Expressing Cell Lines Human cell lines able to express MCHR1 transcripts include neuroblastoma and skin cell carcinoma. The ability of different neuroblastoma cell lines and a squamous cell carcinoma is illustrated in the Examples provided below.
The squamous cell carcinoma provides an example of a skin cell carcinoma. In different embodiments of the present invention concerning a shin cell carcinoma, the skin cell carcinoma is a squamous cell carcinoma or a kertinocyte cell carcinoma.
The ability of different neuroblastoma cells lines and a skin cell carcinoma to express MCHR1 transcripts points to these types of cell lines as containing members able to express MCHR1. Additional neuroblastoma and skin cell _g_ carcinoma cell lines able to express MCHR1 transcripts can be identified using routine experimentation, for example, by measuring the ability of neuroblastoma and slun cell carcinoma cell lines present in depositories such as American Type Culture Collection (Virginia, US) and Health Science Research Resources Bank (Osaka, Japan) to express MCHR1 transcripts.
Modulating MCHR1 Activity Using the present application as a guide compounds able to modulate MCHRl activity can be obtained and used as a research tool to further explore the affects of MCHRl activation or as a therapeutic to achieve a beneficial effect in a patient. Beneficial effects can be obtained, for example, by using a compound active at MCHR1 to achieve one or more of the following: weight loss, weight gain, treat cancer (e.g., colon or breast), reduce pain, treat diabetes, reduce stress or teat sexual dysfunction.
Altering weight is particularly useful for gaining weight in an under weight patient or losing weight in an over weight patient. In addition, for example, farm animals can be treated to gain weight. Under weight patients include those having a body weight about' 10% or less, 20% or less, or 30°70 or less, than the lower end of a "normal" weight range or Body Mass Index ("BMI"). Over weight patients include those having a body weight about 10% or more, 20°70 or more, 30°7o or more, or 50% or more, than the upper end of a "normal" weight range or BMI. "Normal"
weight ranges are well known in the art and take into account factors such as a patient age, height, and body type.
BMI measures your height/weight ratio. It is determined by calculating weight in kilograms divided by the square of height in meters. The BMI
"normal"
range is 19-22.
MCHR1 modulating compounds can be provided in lut. Such a kit typically contains an active compound in dosage form for administration. A
dosage form contains a sufficient amount of active compound such that a beneficial effect can be obtained when administered to a patient during regular intervals, such as 1 to 6 times a day, during the course of 1 or more days. Preferably, a kit contains instructions indicating the use of the dosage form to achieve a beneficial effect and the amount of dosage form to be taken over a specified time period.
_g_ Dosing For Therapeutic Applications Guidelines for pharmaceutical administration in general are provided in, for example, Remi~zgtofz's Pharmaceutical Sciences 1 ~tjt Edition, Ed.
Gennaro, Mack Publishing, 1990, and Modenz Pharmaceutics 2'Id Editiofz, Eds. Banlcer and Rhodes, Marcel Dekker, Inc., 1990, both of which are hereby incorporated by reference herein.
MCHR1 active compounds having appropriate functional groups can be prepared as acid or base salts. Pharmaceutically acceptable salts (in the form of water- or oil-soluble or dispersible products) include conventional non-toxic salts or the quaternary ammonium salts that are formed, e.g., from inorganic or organic acids or bases. Examples of such salts include acid addition salts such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate; and base salts such as ammonium salts, allcali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine and lysine.
MCHR1 active compounds can be administered using different routes including oral, nasal, by injection, and transmucosally. Active ingredients to be administered orally as a suspension can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants.
When administered by nasal aerosol or inhalation, compositions can be prepared according to techniques well known in the art of pharmaceutical formulation. Examples of formulation components include solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents.
The compounds may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form. When administered by injection, the injectable solutions or suspensions may be formulated using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
When rectally administered in the form of suppositories, compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify andlor dissolve in the rectal cavity to release the drug.
Suitable dosing regimens for the therapeutic applications can be selected taking into account factors well known in the art including age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound employed.
Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug. The daily dose for a patient is expected to be between 0.01 and 1,000 mg per adult patient per day.
EXAMPLES
Examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.
Example l: MCHR1 Expression in Different Cell Lines RT-PCR experiments were performed to determine whether different human cell lines expressed mRNA for the MCHR1. Purified poly (A)+ mRNA was isolated from cultured cells using an oligo-dT kit (Poly (A) Pure, Ambion, Austin, TX). RT-PCR using ~1 ~,g of isolated mRNA was performed using Superscript II
reverse transcriptase (Life Technologies, Gaithersberg, MD) essentially following the manufacturer's instructions. PCR cycling conditions were: 94°C for 1 minute (one cycle), 94°C for 30 seconds then 72°C for 4 minutes (four cycles); 94°C for 30 seconds then 70°C for 4 minutes (four cycles); 94°C for 30 seconds then 68°C for 4 minutes (25 cycles); and 68°C for 10 minutes (one cycle). PCR primers were chosen based on the human MCHR1 DNA sequence to flank the mRNA splice junction on either side of the single intron between exon 1 and exon 2. Forward sense primers had the following sequences: SEQ. m. NO. 4:
ATGGACCTGGAAGCCTCGCTGCTG, SEQ. m. NO. 5:
GCCAGCAACACCTCTGATGGC, and SEQ. m. NO. 6:
GGCCCCGATAACCTCACTTCGGC. Reverse (anti sense) primers had the following sequences: SEQ. ID. NO. 7: GAGGAGATCTACTACCGAGAGG, SEQ.
m. NO. 8: GCCCATGAGCTGGTGGATCATG and SEQ. m. NO. 9:
GTGGACAGTGGCCAGGTAGAGGTC.
Amplified products were electrophoresed on an agarose gel, and Southern blotted with a human MCHR1 radiolabeled probe. The probe was random prime labeled with 32P. Post-hybridization washing stringency was at 65°C, 1 X
SSC, after which the filters were dried and exposed to X-ray film for 3 hours at -70°C.
The results are shown in Table 1.
Table 1 Cell Line ATCC No. T a RT-PCR Results HGT-1 Gastric carcinoma-H4 Neuro lioma -TE671 Medulloblastoma -SK-N-BE2 CRL-2271 Neuroblastoma +

T-98G Glioblastoma -U-87MG Glioblastoma -CCF-DTTG Astroc oma -MC-IXC Neuroblastoma -SMS-KAN Neuroblastoma -Cell Line ATCC No. T a RT-PCR Results SMS-MSN Neuroblastoma -CHP-212 CRL-2273 Neuroblastoma +

CHP-243 CRL-2272 Neuroblastoma +

)ZVVIR32 Neuroblastoma -GT1-7 Hypothalamus -(SV-40 immortalized) SH-SYSY CRL-2266 Neuroblastoma +

SCC-25 CRL-1628 Squamous cell +
carcinoma GOTO 1 Adrenal +
Neuroblastoma 1- JCRB0612, 1 l, obtained from Health Science Research Resources Bank (Osaka, Japan), see Sekiguchi, et al.,1979. Japan J. Exp. Med. 49, 67-83).
Neuroblastoma cells lines GOTO, CHP-212, CHP-243, SK-N-BE(2), and SH-SYSY were found to produce mRNA encoding for human MCHR1. In addition, the squamous cell carcinoma cell line SCC-25 was found to produce mRNA
encoding for human MCHR1.
Example 2: MCH Receptor Activity in Neurobastoma Cells Lines To assess whether neuroblastoma cells provide an environment for functional MCHR1, MCH receptor activity was measured using GOTO cells employing an aequorin bioluminescence assay. The aequorin bioluminescence assay can be used to measure the activity of G protein-coupled receptors that couple through the Ga protein subunit family consisting of Gq, G11, and Gi leading to the activation of phospholipase C, mobilization of intracellular calcium and activation of protein kinase C. Measurement of functional MCHR1 expression in GOTO cells transiently expressing aequorin was performed using a Luminoskan RT luminometer (Labsystems Inc., Gaithersburg, MD) controlled by custom software written for a Macintosh PowerPC 6100.
GOTO cells (1.2 x 107 cells plated 18 hours before transfection in a T75 flask) were transfected with human MCHR1 plasmid DNA and aequorin cDNA
using the Lipofectamine 2000 procedure (Life Technologies, Gaithersburg, MD).
Human MCHR1 plasmid DNA contained the open reading frame cDNA (SEQ. m.

NO. 2) encoding the human MCHR1 receptor inserted in the mammalian expression vector pcDNA-3 (Invitrogen, Carlsbad,CA). An aequorin expressing plasmid contained the cDNA for aequorin (Button, et al., 1993. Cell Calcium 14, 663-671), inserted in pcDNA-3. Following approximately 40 hours of expression the apo-aequorin in the cells was charged for 1 hour with coelenterazine (10 p,M) under reducing conditions (300 ~,M reduced glutathione) in ECB buffer (140 mM NaCI, mM KCI, 20 mM HEPES-NaOH [pH=7.4], 5 mM glucose, 1 mM MgCl2, 1 mM
CaCl2, 0.1 mg/ml bovine serum albumin). The cells were harvested, washed once in ECB medium and resuspended to 500,000 cells/ml or 1,OOO,OOOcells/ml.
100 p,1 of MCH or, for control responses, lysophosphatidic acid were injected into a cell suspension (corresponding to 5 x 104 cells or 100,000 cells).
Lysophosphatidic acid triggers native edg receptors coupled to PLC activation present on GOTO cells. The integrated light emission was recorded over 30 seconds, in 0.5 second units. 20 p,L of lysis buffer (0.1% final Triton X-100 concentration) was then injected and the integrated light emission recorded over 10 seconds, in 0.5 second units. The "fractional response" values for each well were calculated by taking the ratio of the integrated response to the initial challenge to the total integrated luminescence including the Triton X-100 lysis response. The results are shown in Table 2.
Table 2 Li and Transfected Buffer LPA MCH MCH
cDNA (ECB) (1 M) 1 M (1 M) No Ae 0 0 0 0 AEQ 2.7 5.24 1.9 3.43 AEQ + 2.76 6.83 26.4 26.6 The results are in bioluminescence (cps). "No Aeq" indicates the absence of plasmids encoding for aequorin and MCHR1. "AEQ" indicates the presence of a plasmid encoding for aequorin, and the absence of a plasmid encoding for MCHR1. "AEQ +
MCHRl" indicates the presence of plasmids encoding for aequorin and MCHRl.
Transfection of the reporter gene aequorin into GOTO cells permitted the detection of functional MCHR1 when co-transfected with a cDNA encoding the human MCHRl using 1 ~.M MCH to evolve a bioluminescent response. This observation indicates that neuroblastoma cells expressing MCHR1 are appropriate host cells for expressing the MCHR1 gene. In the absence of exogenous MCHR1, no signal over background (buffer injection only, ECB) could be observed suggesting that the level of MCHRl naturally present in GOTO cells is insufficient to permit detection using the employed conditions. A control response evoked by the application of 1 ~,M lysophosphatic acid (LPA) suggests the presence of an edg receptor (Im, et al., 2000. J. Biol. Chem. 275, 14281-14286), on GOTO cells linked to calcium mobilization.
Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.

SEQUENCE LISTING
<110> Merck & Co., Inc.
Banyu Pharmaceutical Co., LTD.
<120> CELL LINES EXPRESSING A MCH RECEPTOR
<130> 20677 PCT
<150> 60/244,700 <151> 2000-10-31 <160> 9 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 353 <212> PRT
<213> Human <400> 1 Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys Asn Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Val Ala Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile Thr Ala Ala Tyr Va1 Arg Ile Leu Gln Arg Met Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr 260 ~ 265 270 Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Ala Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly Thr <210> 2 <211> 1062 <212> DNA
<213> Human <400>

atggacctggaagcctcgctgctgcccactggtcccaacgccagcaacacctctgatggc 60 cccgataacctcacttcggcaggatcacctcctcgcacggggagcatctcctacatcaac 120 atcatcatgccttcggtgttcggcaccatctgcctcctgggcatcatcgggaactccacg 180 gtcatcttcgcggtcgtgaagaagtccaagctgcactggtgcaacaacgtccccgacatc 240 ttcatcatcaacctctcggtagtagatctcctctttctcctgggcatgcccttcatgatc 300 caccagctcatgggcaatggggtgtggcactttggggagaccatgtgcaccctcatcacg 360 gccatggatgccaatagtcagttcaccagcacctacatcctgaccgccatggccattgac 420 cgctacctggccactgtccaccccatctcttccacgaagttccggaagccctctgtggcc 480 accctggtgatctgcctcctgtgggccctctccttcatcagcatcacccctgtgtggctg 540 tatgccagactcatccccttcccaggaggtgcagtgggctgcggcatacgcctgcccaac 600 ccagacactgacctctactggttcaccctgtaccagtttttcctggcctttgccctgcct 660 tttgtggtcatcacagccgcatacgtgaggatcctgcagcgcatgacgtcctcagtggcc 720 cccgcctcccagcgcagcatccggctgcggacaaagagggtgacccgcacagccatcgcc 780 atctgtctggtcttctttgtgtgctgggcaccctactatgtgctacagctgacccagttg 840 tccatcagccgcccgaccctcacctttgtctacttatacaatgcggccatcagcttgggc 900 tatgccaacagctgcctcaacccctttgtgtacatcgtgctctgtgagacgttccgcaaa 960 cgcttggtcctgtcggtgaagcctgcagcccaggggcagcttcgcgctgtcagcaacgct 1020 cagacggctgacgaggagaggacagaaagcaaaggcacctga 1062 <210>
' 3 <211>

<212>
DNA

< 2'13 > Human <220>

<221>
CDS

<222> )...(4289) (4208 <221>
CDS

<222> )...(6482) (5504 <400>

atctctgctaaaattagccgggcatggtggcacatgcttgtaatcccagctactctggag 60 gctgaagccagagaatcacttgaaccaggaggcagaggtttcagtgagctgagatcatac 120 cactgcactccagcctgggcgacagagcaagactctgtctcaaatgaaaaaatacataca 180 taaattaattaattaaaaaagaaaggagacagtttattgacaggggagaactctcctgca 240 acacaggatgtagctggagccagtcctaacatgcttctgggatagctccatgaaatggtt 300 tacaataataaggtggggatgctgggatggccattgcagagttttgggattttttgtgca 360 tagtaaaatacacataaaatttgcctcttcactttatatattgtggtaggatatacgtaa 420 cataaaatttaccattttaaccacttttaagtgtccaatttagtggcattaagtacgttc 480 acaatgttgtgcagccattatcactgtccatttctagaacttttttttttttaagatgga 540 ttttcgctattgttgcccaggctggagggtaatggcacaatctcagctcactgcaaccct 600 cacctcccgggttcaagcaattctcctgcctcagcctcctgagtagctgggactacaggc 660 acatgccaccatgcccggctaatttttgtatgcttagtagaggcagggtttcaccatgcc 720 agccaggctggtttcaagctcctgacctcaggtgatccgcctgccttggcctcccaaagt 780 gctgggattagaggcaagagtcaccacacctggccagctacacacttttaaacaacaaga 840 tctcatgagcactcactcattatcacaaaaacaggaagggggaaatctgttcccatggtc 900 caatcacctcccaccaaacatgcttcctcacttccttaaggacttttctggaagtgcttc960 tcagtgaggccttcctctactgcaaaaagcttcccctgccacaaagagcttctttttttt1020 tgagacagagtctcgttctgtcacccaggctggagtgcagtggcgtgatctcagctcact1080 gcaatctccacctcctgggttcaagcgattctcatgcttcagcctcccaagtagctggga1140 ttacaggtgcttgccaccacacctggctaatttttgtacatttagtagacacagggtttc1200 accatgttggccaggctggtctcaaactcctgacctcagtcaatccacccgcctcagcct1260 cccaaaatgttgggattacaggcgtgagccactgtgcccggccaagactgatcctttcaa1320 aacatgtctgatcatgtcttttctcagctgaaaaccccctggtgactcccatttccccta1380 aaggcaaagccaaagtgcttatactggctggcaaggcctggcagaatctgcccttcctgc1440 tccttcctctctgatgacagctcctgctaatctctactatagccacagtgctctccagag1500 tcacactgcacactcctgccacagggcctctgcactgacaatttccatggcagggaatgc1560 tggtcctccagaatatttcttcttcttttttttttttttgagacggcgtctcactctgtc1620 acccaggctggagtgcaatggcatgatctcggctcactgcaatttccacctcccgggttc1680 aagcgattctcctgcctcagcctcccaagtagctgggactacaggcgcccgccaccacgc1740 ccagctaatttttatatttttaatagagagggggtttcaccttgttggccaggattgtct1800 cgatctcttgacatcatgatccgcctgcctcggcctcccaaagtgctgggatttcaggcg1860 tgagccactgcgcccggccactccagaatatttctaagcatgcttcctcacttccttcag1920 gacttttctcagagaggccttccttgatgatcaatcctctataaaatggtaacctccacc1980 tctcttggtattcctgggccctctcaccctgctttaagttttttacagcaattgccaata2040 tcataagttatctttatgtggagtatatattttttcttttttttgagacgtagtctcgct2100 ctgtcacccaggctggagtgcagtggcgtggtctcggctcactgcaagctccgcctcccg2160 ggttcacgccattctcctgcctcagcctcccgagtagctgggactacaggcacccgccac2220 cacgcccggttaattttttgtattttttaatagagatggggtttcaccatgttagccagg2280 atggtctcgatctcctgacctcgtgatctgccagcctcggcctcccaaagtgctggtatt2340 acaggtgtgagccactgcgcccggccatattttttcttttttctgagacagggtctttct2400 gtgtcgcccaggctggagtgcagtggcccaatctcagctcacttcaatctctgcctcctg2460 gattcaggcgattctcctgcttcagctgcttaagtagctggaattacaggcagctgccaa2520 catgtccggctaatttttgtatttttgtagagacggggtttcaccatgttgtccaggctg2580 gtcttgaactcctgagctcaagcaatccacccaccttggcttcgcaaagtgctgggatca2640 caggcatgagccactgcacctggcccaaccctgttcttcagaatcaccctgcacacttcc2700 tgccgcacggcctttgcactggcgatttccacagccaggaatgctggtcctccagaatat2760 ttctaagcatggttcctcacttccttcaggacttttctggaagcgcttctcagtgaggtc2820 ttcctcgatgatcaatcctatataaaatagcaacctccacctctgctggtattcaggggc2880 cagctcaccctgctttaagtttttcatagtaattgccaataccatgaattgtcttttgag2940 gggttttctgtctccccgcaacttgaatgcaatcttcatgaggacagggactttatcccc3000 caccccacccccacttttttttcttttttttttactgcaaaacctagaacagtgcttggc3060 aggtagtaagtactcaatgaatgtttgttggatgaacccaataagtaaacaagatagagg3120 acacatgtagggatctgcccgtgaacctcgacccctggctcctgagtctggcagtgggtg3180 cagtggcagctcctgtctgtgagggccccagaggctggcagcaggatgcctccgtggaaa3240 attcccttaacgcctttgcctctgcagctgttcctccgggatgatctctttgggggatca3300 tgctcagatatttgtctcaaagagtcccaggccaaacctcagggacctcagagcgtttag3360 aaaaataacacctctgtgagcttggtccaggcagatcccatgcagagaggagtttgtccc3420 cttccagtccccgaggtcctggctattgccagcatggagtgacctgtgtcacctctgagt3480 gccaggcaagggttcagcagctgacgactcagcttctgcaggatgctggcagcatagcca3540 gcgagatagttggaagccgtcagggcacagggaaggggccgagggtgccctgagtgtgca3600 tggggggcagccctgctgcagtccaagcctttgattcccaagctatgtgcacagtttcct3660 ctggactctgccatgtggcccagccacccatacctggaataggggctaagccaagctgct3720 ctctcctccaaagggaggcagcctgtgtgctttgtccgtttgcctttgcagagacctcga3780 tcttcacgcaaggcaagcagcagcccctgtaagcacacgagacaatcccaagtgtcagtg3840 ggaaggagatccctttcctgatggggctgcctgtgtccagtccctcccagcttccccagg3900 gccctggggctctgcaggcattcagaagtggaagccagccacagcctgggactgaagagg3960 ttaatgtgcatctgcctccgaatgttaatgtgtctaggtgatgtcagtgggagccatgaa4020 gaagggagtggggagggcagttgggcttggaggcggcagcggctgccaggctacggagga4080 agacccccttcccaactgcggggcttgcgctccgggacaaggtggcaggcgctggaggct4140 gccgcagcctgcgtgggtggaggggagctcagctcggttgtgggagcaggcgaccggcac4200 tggctgg gac ctg act ggt aac gcc 4249 atg gaa gcc ccc tcg ctg ctg ccc Met Asp Leu Thr Gly Glu Ala Pro Asn Ser Leu Ala Leu Pro agc aac acc tct gat ggc ccc gat aac ctc act tcg gca g gtgagttgac 4299 Ser Asn Thr Ser Asp G1y Pro Asp Asn Leu Thr Ser Ala tgggagccctccctcctctg ggctgtgggt ggaaaatgggaaggtttcacccctgagcca4359 aactgcttgggaaactttat cacagttctt ggggacaagatctgtggtctgctttgctct4419 gaggggcaggagaaaagggg gcaatggtcc gcaggggcagacgggcaggagcagagcagg4479 gggcgaaggcatattcagaa tggcaaggaa ggggggccagccgtgagacagcaggggaag4539 gctcgctgctgggttccaaa gatgcttggc agaaaaaattccaggctggaaaagcaagcg4599 agagaagctggagggtggta tgtgggagac agctgggggctcactcctgcactgttagcc4659 tcagctttttactcccactt ggatgatgag gtctgagacatccttactgccacctgggag4719 aggccctgggaagggaagac ttcacagagc catgaggggattaacttttctggtgaatta4779 agcttcctgacatttccaga gctgcggtgc cctgggattccagctttgaaggagaaagga4839 aggaaggaaaagaggaaagg cttatgtaga taatttttccaggctgctgagctccaacag4899 acagtttctgtctctgcttc actcaagaag cccaggctcagaagataccaatcaaggaaa4959 tccccgctaggaagcctggg gtagggagag ctgctggcttgaccagggcacagccggcaa5019 aagcctctacaagacagtca cccacagata tgcccaagaatcagtacacagtttccaacc5079 agagatctccaaaatgaaac actcagggct acacataggaaaagcacgcacacacacaca5139 cacacacatacacagacact tacttttgtg tccttctggctatgctgacgagttttcctg5199 gtgaagcccggggctcacag agtaatctct gcagacaactgtggttcttgcctctggtgc5259 ctgcaggaggcaggcatgtt gtgtccttcc aagacagatggctcagggcactctggtagg5319 attcaccaggaaactcatgg agaagggaaa agggacaagattagcaacagtgaagggagg5379 gagaatggtgggagaggatt ccagatgaac ggtgggtcgctggaggctgagcatgccagc5439 aggatgtcagttctcagagc aaagcccatg tcaaacagccaacgcttgctccttctgtcc5499 ccag ga cct cct cgc acg ggg agc tac atc atc atc 5547 tca atc tcc aac Gly Ser Pro Pro Arg Thr Gly Ser Tyr Ile Ile Ile Ile Ser Asn atg cct gtg ttc ggc acc atc tgc ggc atc ggg aac 5595 tcg ctc ctg atc Met Pro Val Phe Gly Thr Ile Cys Gly Ile Gly Asn Ser Leu Leu Ile tcc acg atc ttc gcg gtc gtg aag aag ctg tgg tgc 5643 gtc aag tcc cac Ser Thr Ile Phe Ala Val Val Lys Lys Leu Trp Cys Val Lys Ser His aac aac ccc gac atc ttc atc atc tcg gta gat ctc 5691 gtc aac ctc gta Asn Asn Pro Asp Ile Phe Ile Ile Ser Val Asp Leu Val Asn Leu Val ctc ttt ctg ggc atg ccc ttc atg cag ctc ggc aat 5739 ctc atc cac atg Leu Phe Leu Gly Met Pro Phe Met Gln Leu Gly Asn Leu Ile His Met ggg gtg cac ttt ggg gag acc atg ctc atc gcc atg 5787 tgg tgc acc acg Gly Val His Phe Gly Glu Thr Met Leu Ile Ala Met Trp Cys Thr Thr gat gcc agt cag ttc acc agc acc ctg acc atg gcc 5835 aat tac atc gcc Asp Ala Ser Gln Phe Thr Ser Thr Leu Thr Met Ala Asn Tyr Ile Ala att gac tac ctg gcc act gtc cac tct tcc aag ttc 5883 cgc ccc atc acg Ile Asp Tyr Leu Ala Thr Val His Ser Ser Lys Phe Arg Pro Ile Thr cgg aag tct gtg gcc acc ctg gtg ctc ctg gcc ctc 5931 ccc atc tgc tgg Arg Lys Ser Val Ala Thr Leu Val Leu Leu A1a Leu Pro Ile Cys Trp tcc ttc agc atc acc cct gtg tgg gcc aga atc ccc 5979 atc ctg tat ctc Ser Phe Ser Ile Thr Pro Val Trp A1a Arg Ile Pro Ile Leu Tyr Leu ttc cca gga ggt gca gtg ggc ata ctg ccc aac cca 6027 ggc tgc cgc gac Phe Pro Gly Gly Ala Val Gly Ile Leu Pro Asn Pro Gly Cys Arg Asp act gac ctc tac tgg ttc tac cag ttc ctg gcc ttt 6075 acc ctg ttt gcc Thr Asp Leu Tyr Trp Phe Tyr Gln Phe Leu Ala Phe Thr Leu Phe Ala ctg cct ttt gtg gtc atc gca tac agg atc ctg cag 6123 aca gcc gtg cgc Leu Pro Phe Val Val Ile Ala Tyr Arg Ile Leu Gln Thr Ala Val Arg atg acg tcc tca gtg gcc tcc cag agc atc cgg ctg 6171 ccc gcc cgc cgg Met Thr Ser Ser Val Ala Ser Gln Ser Ile Arg Leu Pro Ala Arg Arg aca aag agg gtg acc cgc atc gcc tgt ctg gtc ttc 6219 aca gcc atc ttt Thr Lys Arg Val Thr Arg Ile Ala Cys Leu Val Phe Thr Ala Ile Phe gtg tgc tgg gca ccc tac cta cag acc cag ttg tcc 6267 tat gtg ctg atc Val Cys Trp Ala Pro Tyr Leu Gln Thr Gln Leu Ser Tyr Val Leu Ile agc cgc ccg acc ctc acc tac tta aat gcg gcc atc 6315 ttt gtc tac agc Ser Arg Pro Thr Leu Thr Tyr Leu Asn Ala Ala Ile Phe Val Tyr Ser ttg ggc tat gcc aac agc aac ccc gtg tac atc gtg 6363 tgc ctc ttt ctc Leu Gly Tyr Ala Asn Ser Asn Pro Val Tyr Ile Val Cys Leu Phe Leu tgt gag acg ttc cgc aaa gtc ctg gtg aag Cct gca 6411 cgc ttg tcg gcc Cys Glu Thr Phe Arg Lys Val Leu Val Lys Pro Ala Arg Leu Ser Ala cag ggg cag ctt cgc get aac get acg get gac gag 6459 gtc agc cag gag Gln Gly Gln Leu Arg Ala Asn Ala Thr Ala Asp Glu Val Ser Gln Glu agg aca gaa agc aaa ggc tacttcccc 6512 acc tg a tgccaccctg cacacctcca Arg Thr Glu Ser Lys Gly Thr agtcagggca ccacaacacg ccaccgggagagatgctgagaaaaacccaa gaccgctcgg6572 gaaatgcagg aaggccgggt tgtgaggggttgttgcaatgaaataaatac attccatggg6632 gctcacacgt tgctggggag gcctggagtcaggtttggggttttcagata tcagaaatcc6692 ccttggggga gcaggatgag acctttggatagaacagaagctgagcaaga gaacatgttg6752 gtttggataa ccggttgcac tatatctgtgagctctcaaatgtcttcttc ccaaggcaag6812 aggtggaagg gtactgactg ggtttgtttaaagtcaggcagggctggagt gagcagccag6872 ggccatgttg cacaaggcct gagagacgggaaagggcccgatcgctcttt cccgcctctc6932 actggtgcga tggaaggtgg cctttctcccaagctggtggataatgaaaa ataaagcatc6992 ccatctctcg gcgttccagc atcctgtcaatttcccttttgctctagagg atgcatgttt7052 atttgagggg atgtggcact gagcccacaggagtaaaagcccagtttgct aggaggtctg7112 cttactgaaa acaaggagac ctggggtgggtgtggttgggggtcttaaaa ctaataaaag7172 ctggggtcgg ggggcttttg cagctctggtgacattctctccacggggca catttgctca7232 gtcactaatc cagcttgagt gtccgtgtgttctgcatgtgcaggggtcat tctagtgccc7292 ggtgtgttgg catcatcttt ttgctctagcccttcctctccaaaataaaa tcaaataaag7352 gaaaatctcc acccacatca ctctggatgttcttgtggacttgggggtgg gtgtgggctg7412 gggcggggaa ggtgggcagc agaaaagagaaagaggggtcacttggttgt ggaatttaga7472 tcttggttca tgctgcattt ttaggaagcatgaagcagggatctgttttt ctgaacccac7532 agggaggatt cagtggcata aatggaattactggggattcattagatttt gcagttctgc7592 tgctgggcttgttcttgggactcagcttcctgtcttctgc acaaaatccc ctgggctttg7652 ttgtcatcagtgatgagtcctCaggCCCCaagtCCCaaCC CCCaCtCCCC CgCCtCaaCC7712 ctcacccccgctgagtcaccagccgcagagccagctctta gggcagctga agcctctctg7772 ctttctacagctgagatctggtgtgggcaccttgaacaga agattacagc cggggccact7832 gggaggcagccacgactgctgcttggctgctgcttcttgg tgtcttcact gagaggggac7892 tgggagccgtcagtgcagtgctcagcagaccttactgaga gagcgggaga aagctgcaag7952 catcctgaaagcaggggcagcagcacagcctgttcctctt cagagctgca gcagggagcc8012 tcttcagaaaacttctgggcagcttccttgggtcctgggg actttttttt tgagacagag8072 tatcgctctgtcgcccaggctgaagtgcagtggtgtgatc tctgctcact gcaagctccg8132 cctcccgggttcacaccattctcctgcctcagcctcccga gtagctggga ctgcaggcat8192 ccgccaccacgcccggctaatttttgtatttttagtaaag acggggtttc accatgttag8252 ccaggatggtctcgatctcctaacctcatgatctgcctgc ctcggcctcc caaggtgctg8312 agactgcaggcgtaagccaccgtgcccggctttttttttt tttttttttt ttggacacag8372 ggccttgccctgttgcccaggctgtagtgcaggtgacatg atcacagctc actgcagcct8432 cgacctcccagattcaagcaatcctcccacctcagcctcc caagtagctg g 8483 <210>

<211>

<212>
DNA

<213>
Artificial Sequence <220>

<223> Primer Oligonucleotide <400>

atggacctggaagcctcgctgctg 24 <210>

<211>

<212>
DNA

<213>
Artificial Sequence <220>

<223> Primer Oligonucleotide <400>

gccagcaacacctctgatggc 21 <210>

<211>

<212>
DNA

<213>
Artificial Sequence <220>

<223> Primer Oligonucleotide <400>

ggccccgataacctcacttcggc 23 <210>

<211>

<212>
DNA

<213>
Artificial Sequence <220>

<223> Primer Oligonucleotide <400>

gaggagatctactaccgagagg 22 <210>

<211>

<212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide Primer <400> 8 gcccatgagc tggtggatca tg <210> 9 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide Primer <400> 9 gtggacagtg gccaggtagc ggtc 24

Claims (15)

WHAT IS CLAIMED IS:

1. A neuroblastoma cell or a skin cell carcinoma comprising a recombinant melanin-concentrating hormone receptor 1 (MCHR1) gene that expresses functional MCHR1.

2. The cell of claim 1, wherein said cell is a human neuroblastoma cell.

3. The neuroblastoma cell of claim 2, wherein said recombinant MCHR1 gene is present in the neuroblastoma cell genome and comprises endogenous nucleic acid encoding for MCHR1 transcriptionally coupled to an exogenous promoter.

4. The neuroblastoma cell of claim 3, wherein said exogenous promoter is a CMV promoter.

5. The neuroblastoma of claim 3, wherein said cell further comprises a recombinant gene encoding for aequorin.

6. The neuroblastoma cell of claim 3, wherein said neuroblastoma cell is selected from the group consisting of: GOTO, CHP-212, CHP-243, SK-N-BE(2), and SH-SY5Y.

7. The cell of claim 1, wherein said cell is SCC-25.

8. A neuroblastoma cell or skin cell carcinoma having increased MCHR1 expression produced by a process comprising the step of coupling endogenous nucleic acid encoding for MCHR1 to an exogenous promoter.

9. The cell of claim 8, wherein said promoter is a CMV promoter.

10. The cell of claim 8, wherein said cell is a neuroblastoma cell selected from the group consisting of: GOTO, CHP-212, SK-N-BE(2), CHP-243,and SH-SY5Y.

11. The cell of claim 8, wherein said cell is a shin cell carcinoma.

12. The cell of claim 11, wherein said cell is SCC-25.

13. A method of measuring the ability of a compound to effect MCHR1 activity comprising the steps of:
a) providing said compound to the cell of any one of claims 1-12;
and b) measuring MCHR1 activity.

14. The method of claim 13, wherein said step (a) further comprises the presence of an MCHR1 agonist.

15. The method of claim 14, wherein said MCHR1 agonist is human melanin-concentrating hormone.