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WO2000075166A1 - Recepteur de l'hormone concentratrice de la melanine - Google Patents

Recepteur de l'hormone concentratrice de la melanine Download PDF

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Publication number
WO2000075166A1
WO2000075166A1 PCT/US2000/015503 US0015503W WO0075166A1 WO 2000075166 A1 WO2000075166 A1 WO 2000075166A1 US 0015503 W US0015503 W US 0015503W WO 0075166 A1 WO0075166 A1 WO 0075166A1
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Prior art keywords
mch receptor
mch
receptor
seq
identifying
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Inventor
Olivier Civelli
Yumiko Saito
Hans-Peter Nothacker
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Priority to AU58691/00A priority Critical patent/AU5869100A/en
Publication of WO2000075166A1 publication Critical patent/WO2000075166A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones

Definitions

  • the present invention relates generally to the field of medicine and, more specifically, to therapeutic and diagnostic methods and compositions related to melanin concentrating hormone receptor.
  • Obesity or excess deposition of body fat, represents a primary health concern m industrialized countries. Obesity correlates with and may trigger the onset of serious medical conditions, including hypertension, diabetes, cardiovascular disease and psychological malad ustments. Whereas diet, exercise and appetite suppressants can produce modest results in the reduction of body fat deposits, no consistently effective or practical treatment has been found for controlling obesity and its physiological and psychological consequences .
  • cachexia which commonly occurs m chronic diseases such as cancer and AIDS
  • the weight loss characteristic of cachexia has been associated with several contributing factors, including food aversion due to altered sensitivity to taste and smell, malfunction of the gastrointestinal tract, insufficient nutrient intake, and metabolic disturbances.
  • MCH Melanm-concentratmg hormone
  • MCH is a small, cyclic neuropeptide that plays an important role n regulating body weight, metabolism, and feeding behavior.
  • MCH was first isolated from the pituitary gland of salmon, where it functions to regulate scale color. Intracerebral administration of MCH peptide m mammals has been shown to produce a dose-dependent stimulation of food intake, whereas mice deficient in MCH exhibit decreased body weight due to reduced feeding behavior and an inappropriately increased metabolic rate. Expression of MCH is increased in the ob mouse model of obesity as well as in normal animals following fasting. Thus, it is clear that MCH plays a critical role in regulating body weight, metabolism and appetite.
  • MCH ⁇ -melanocyte stimulating hormone
  • the invention provides a method of identifying an MCH receptor agonist or antagonist.
  • the method consists of contacting an MCH receptor with one or more candidate compounds under conditions wherein the MCH receptor produces a predetermined signal in response to an MCH receptor agonist.
  • a candidate compound that alters production of the predetermined signal is identified.
  • the compound is characterized as an MCH receptor agonist or antagonist.
  • the invention also provides a method of identifying an MCH receptor ligand.
  • the method consists of contacting an MCH receptor with one or more candidate compounds under conditions that allow selective binding between the MCH receptor and an MCH receptor ligand.
  • a compound that selectively binds the MCH receptor is identified.
  • the compound is characterized as an MCH receptor ligand.
  • the method consists of detecting MCH receptor nucleic acid molecule in a sample from the individual. Abnormal structure or expression of the MCH receptor nucleic acid molecule in the sample indicates that the individual has or is susceptible to an MCH receptor-associated condition. In another embodiment, the method consists of detecting MCH receptor polypeptide in a sample from the individual. Abnormal expression or activity of the MCH receptor polypeptide in the sample indicates that the individual has or is susceptible to an MCH receptor-associated condition.
  • MCH receptor-associated conditions include disorders of body weight, mood, memory, learning, sleep, dopaminergic system function, reproduction or growth.
  • the invention also provides signaling compositions.
  • the signaling composition contains a recombinantly expressed MCH receptor and a recombinantly expressed G ⁇ subunit of a G protein.
  • the signaling composition contains a recombinantly expressed MCH receptor, a G protein, and a calcium indicator.
  • Figure 1 shows the nucleotide sequence (SEQ ID NO:l) and deduced amino acid sequence (SEQ ID NO: 2) of human melanin-concentrating hormone receptor (GPR24 or SLC-1) (Kolakowski et al., FEBS Letters 398:253-258 (1996), GenBank accession number U71092).
  • Figure 2 shows the nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQ ID NO: 4) of rat melanin concentrating hormone receptor (SLC-1)
  • Figure 3 shows purification of SLC-1 endogenous ligand from rat brain extracts.
  • Figure 3A shows a C18 reverse-phase HPLC elution profile.
  • Figure 3B shows a kinetic of the [Ca 2+ ] ⁇ changes evoked by fraction 57, with or without trypsin treatment.
  • Figure 3C shows final purification of active peptide by Sephasil C8 SC2.1/10 using SMART system. The inset panel shows the peak increments in [Ca 2+ ] : induced by designated HPLC fractions .
  • Figure 4 shows the specificity of interaction between MCH and SLC-1.
  • Figure 4A shows the alignment of rat/human MCH sequence with salmon MCH, somatostatin 14 and cortistatin 14.
  • Figure 4B shows [Ca 2* ] ! changes in CHO cells transfected with SLC-1 and G ⁇ q/i3 induced by MCH, salmon MCH, somatostatin-14 (SST-14), cortistatin-14 (CS-14), ⁇ -MSH, NEI, and MGOP-1 .
  • Figure 4C left, shows dose-response curves for changes in [Ca 2+ ]j induced by SLC-1 alone or SLC-1 coexpressed with G ⁇ q/i3.
  • Figure 4C right, shows inhibition of forskolin-stimulated cAMP accumulation in CHO cells transfected with SLC-1 alone.
  • Figure 5 shows the distribution of SLC-1 mRNA.
  • Figure 5A shows Northern blot analysis from indicated rat tissues using an SLC-1 cDNA probe (top panel) and G3PDH control probe (bottom panel) .
  • Figure 5B shows localization of SLC-1 transcripts in rat brain sections by in si tu hybridization.
  • Ctx cortex; AON: anterior olfactory nucleus; TT : taenia tecta; Tu: olfactory tubercle; Acb: nucleus accumbens; Pir: piroform cortex; Hpx: hippocampus; Th: thalamus; Hyp: hypothalamus; Amy: amygdala; LC: locus coeruleus .
  • the present invention relates to the identification of the receptor for melanin concentrating hormone (MCH) and its signal transduction pathway.
  • MCH melanin concentrating hormone
  • the invention thus provides novel compositions and methods that can be used to identify compounds that specifically bind to or modulate signaling through the MCH receptor. Such compounds can be used therapeutically to prevent or ameliorate MCH receptor-associated conditions, including disorders of body weight, behavior, memory, learning, mood, sleep, or movement.
  • the invention also provides methods of identifying an individual having or susceptible to an MCH receptor-associated condition. Such knowledge allows optimal medical care for the individual, including appropriate genetic counseling and prophylactic and therapeutic intervention.
  • the invention provides a method of identifying an MCH receptor agonist or antagonist.
  • the method consists of contacting an MCH receptor with a candidate compound under conditions wherein the MCH receptor produces a predetermined signal in response to an MCH receptor agonist, and identifying a compound that alters production of the predetermined signal.
  • a compound that alters production of the predetermined signal is characterized as an MCH receptor agonist or antagonist.
  • MCH receptor refers to a heptahelical membrane-spanning G-protem coupled polypeptide, previously designated SLC-1 or GPR24, which, as disclosed herein, is the endogenous receptor for melanin-concentrating hormone.
  • MCH receptor encompasses native MCH receptor polypeptides from all vertebrate species including but not limited to human, non-human primate, rat, mouse, rabbit, bovine, porcine, ovine, canine, feline, avian, reptile, amphibian or fish.
  • the numan MCH receptor nucleotide sequence (SEQ ID NO:l) and encoded ammo acid sequence (SEQ ID NO: 2) are described m Kolakowski et al., FEBS Letters 398:253-258 (1996), and are shown in Figure 1.
  • the rat MCH receptor nucleotide sequence (SEQ ID NO: 3) and encoded ammo acid sequence (SEQ ID NO: ) are described Lakaye et al., Bioc. Biophvs. Acta 1401:216-220 (1998), and are shown Figure 2. Based on the high degree of identity between rat and human MCH receptor nucleotide and ammo acid sequences, it is predicted that MCH receptor from other species will be highly homologous to the rat and human MCH receptor.
  • MCH receptor also encompasses polypeptides containing minor modifications with respect to a native MCH receptor sequence, and fragments of full- length MCH receptor, so long as the modified polypeptide or fragment retains one or more of the biological activities of a native MCH receptor, such as the ability to selectively bind MCH, or the ability to couple to and signal through a G protein in response to an MCH receptor ligand.
  • a modified polypeptide can have, for example, one or more additions, deletions, or substitutions of natural or non-natural ammo acids relative to the native polypeptide, so long as a biological activity of a native MCH receptor is retained.
  • MCH receptor encompasses MCH receptor polypeptides as they are found m vertebrate host cells or tissues which express MCH receptor, including but not limited to brain, eye, skeletal muscle, tongue and pituitary, or as they are present in membrane extracts or substantially pure preparations derived from these tissues by standard biochemical fractionation procedures. Additionally, the term “MCH receptor” encompasses recombinantly expressed MCH receptor polypeptides, modifications or fragments, such as recombmant polypeptides expressed in cells or cell lysates that support transcription and translation. Methods of producing recombmant polypeptides in cells and lysates are well known m the art. Likewise, the term “MCH receptor” includes chemically synthesized MCH receptor polypeptides, which can be prepared by standard peptide synthesis methods.
  • the method of identifying an MCH receptor agonist or antagonist is practiced by contacting an MCH receptor with a candidate compound under appropriate conditions in which MCH receptor produces a predetermined signal response to a known MCH receptor agonist.
  • the term “candidate compound” refers to any molecule that potentially acts as an MCH receptor agonist, antagonist or ligand m the screening methods disclosed herein.
  • a candidate compound can be a naturally occurring macromolecule, such as a polypeptide, nucleic acid, carbohydrate, lipid, or any combination thereof.
  • a candidate compound also can be a partially or completely synthetic derivative, analog or mimetic of such a macromolecule or, a small organic molecule prepared by combinatorial chemistry methods. If desired m a particular assay format, a candidate compound can be detectably labeled or attached to a solid support.
  • candidate compounds to test in the methods of the invention will depend on the application of the method. For example, one or a small number of candidate compounds can be advantageous in manual screening procedures, or when it is desired to compare efficacy among several identified ligands, agonists or antagonists. However, it is generally understood that the larger the number of candidate compounds, the greater the likelihood of identifying a compound having the desired activity in a screening assay. Additionally, large numbers of compounds can be processed in high-throughput automated screening assays. Therefore, "one or more candidate compounds" can contain, for example, greater than about 10 3 different compounds, preferably greater than about 10 5 different compounds, more preferably, greater than about 10 7 different compounds.
  • MCH receptor agonist refers to a compound that selectively promotes or enhances normal signal transduction through the MCH receptor.
  • An MCH receptor agonist can act by any agonistic mechanism, such as by binding an MCH receptor at the normal MCH binding site, thereby promoting MCH receptor signaling.
  • An MCH receptor agonist can also act, for example, by potentiating the binding activity of MCH or signaling activity of MCH receptor. The methods of the invention can advantageously be used to identify an MCH receptor agonist that acts through any agonistic mechanism.
  • an example of an MCH receptor agonist is the 19 am o acid MCH cyclic peptide from rat or human having the ammo acid sequence shown in Figure 4A (SEQ ID NO: 5).
  • a further example of an MCH receptor agonist is the 17 ammo acid MCH cyclic peptide from salmon shown in Figure 4A (SEQ ID NO: 6) .
  • somatostatm-14 ( Figure 4A, SEQ ID NO: 7), the somatostatm analog RC-160, cort ⁇ statm-14 ( Figure 4A, SEQ ID NO: 8), cort ⁇ statm-29, MCH-precursor-de ⁇ ved peptide NEI, MCH-gene-overpnnted-polypeptide, MGOP-14, MGOP-27, and ⁇ -melanotropm (MSH) , as disclosed herein, are not MCH receptor agonists, as they do not promote signaling through the MCH receptor under conditions which MCH receptor produces a predetermined signal m response to an MCH receptor agonist.
  • MCH receptor antagonist refers to a compound that selectively inhibits or decreases normal signal transduction through the MCH receptor.
  • An MCH receptor antagonist can act by any antagonistic mechanism, such as by binding an MCH receptor or MCH, thereby inhibiting binding between MCH and MCH receptor.
  • An MCH receptor antagonist can also act, for example, by inhibiting the binding activity of MCH or signaling activity of MCH receptor.
  • an MCH receptor antagonist can act by altering the state of phosphorylation or glycosylation of MCH receptor. The methods of the invention can advantageously be used to identify an MCH receptor antagonist that acts through any antagonistic mechanism.
  • an MCH receptor antagonist is a pepti ⁇ e or peptidomimetic derived from a portion of MCH receptor that binds MCH.
  • the ligand binding pocket is predicted to include residues Tyr230, of the fourth transmembrane domain, Phe266, of the fifth transmembrane domain, and Trp318, Tyr322 and Gln325, positioned in the sixth transmembrane domain (Kolakowski et al., FEBS Letters 398:253-258 (1996)).
  • a peptide or peptidomimetic that includes an MCH receptor ammo acid sequence spanning one or more of these residues that constitute the binding pocket of MCH receptor can act as an MCH receptor antagonist.
  • Suitable assay conditions under which MCH receptor produces a predetermined signal m response to an MCH receptor agonist can be determined by those skilled m the art, and will depend on the particular predetermined signal one intends to detect.
  • the term "predetermined signal” refers to a readout, detectable by any analytical means, that is a qualitative or quantitative indication of activation of signal transduction through the MCH receptor.
  • MCH receptor couples to G proteins in response to the MCH receptor agonist MCH. Therefore, any known or predicted G protem-coupled cellular event, such as elicitation of second messengers, induction of gene expression or altered cell proliferation, differentiation or viability, can be a "predetermined signal” that is an indication of activation of signal transduction through the MCH receptor.
  • G protein refers to a class of heterotrime ⁇ c GTP binding proteins, with subunits designated G ⁇ , G ⁇ and Gy, that couple to seven- transmembrane cell surface receptors to transduce a variety of extracellular stimuli, including light, neurotransmitters, hormones and odorants to various intracellular effector proteins.
  • G protein encompasses endogenous and recombinantly expressed G proteins from all eukaryotic and prokaryotic organisms, including mammals, other vertebrate organisms, Drosophila and yeast. Also encompassed within the term “G protein” are modifications and fragments of native G proteins that maintain MCH receptor binding activity, signal transduction activity, or both, of a native G protein.
  • G proteins Four major classes of G proteins have been identified, which are defined by their G ⁇ subunits, G ⁇ i, Gas, Gaq and G ⁇ l2. As disclosed herein, MCH receptor couples to G proteins containing either G ⁇ i and G ⁇ q subunits, and potentially couples to G proteins containing other G ⁇ subunits. Signaling through G ⁇ i- contam g G proteins inhibits adenylyl cyclase activity, which can be determined, for example, in an assay that measures increased or decreased forskolin-stimulated cAMP accumulation as the predetermined signal (see Example III, below) . Signaling through G ⁇ q-contam g G proteins promotes calcium ion influx, which can be determined, for example, m an assay that measures an increase or decrease intracellular Ca ⁇ as the predetermined signal (see Examples I-III, below).
  • G ⁇ subunits for cell-surface receptors is determined by the C-termmal five ammo acids of the G ⁇ .
  • any convenient G-protem mediated signal transduction pathway can be assayed by constructing a chimeric G ⁇ containing the C-termmal residues of a G ⁇ known or predicted to couple to MCH receptor, with the remainder of the protein corresponding to a G ⁇ that couples to the signal transduction pathway it is desired to assay.
  • the term "chimeric G ⁇ " refers to any functional G ⁇ polypeptide that contains at least the five C-termmal ammo acids of one G ⁇ , with the remainder of the polypeptide including ammo acid sequences corresponding to one or more different G ⁇ subunits.
  • nucleotide sequences and signal transduction pathways of different classes and subclasses of G ⁇ subunits in a variety of eukaryotic and prokaryotic organisms are well known in the art.
  • one skilled the art can readily construct any desired chimeric G ⁇ by methods known in the art and described, for example, in Conklm et al . , Nature 363:274-276 (1993), and Komatsuzaki et al., FEBS Letters 406:165-170 (1995).
  • a chimeric G ⁇ that contains ammo acids 1-354 of a G ⁇ q and the C- terminal 5 residues of a G ⁇ 3 can be constructed by PCR, and used to couple MCH receptor to signaling through the G ⁇ q pathway.
  • a chimeric G ⁇ useful m the methods of the invention can include the C-termmal 5 residues of a G ⁇ i and the N terminal residues of a different G ⁇ i, a G s or a G ⁇ l2.
  • a chimeric G ⁇ useful in the methods of the invention can alternatively include, for example, the C-termmal 5 residues of a G ⁇ q and the N terminal residues of a G ⁇ i, a Gas or a G ⁇ l2.
  • G proteins containing various G ⁇ subunits can lead to increased or decreased production or liberation of second messengers, including, for example, aracmdonic acid, acetylcholme, diacylglycerol, cGMP, cAMP, mositol phosphate and ions; altered cell memorane potential; GTP hydrolysis; influx or efflux of ammo acids; increased or decreased phosphorylation of mtracellular proteins; or activation of transcription.
  • second messengers including, for example, aracmdonic acid, acetylcholme, diacylglycerol, cGMP, cAMP, mositol phosphate and ions; altered cell memorane potential; GTP hydrolysis; influx or efflux of ammo acids; increased or decreased phosphorylation of mtracellular proteins; or activation of transcription.
  • Those skilled m the art can determine an appropriate assay for detecting alterations in any desired signal transduction pathway response to a candidate compound.
  • Exemplary assays including high throughput automated screening assays, to identify alterations m signal transduction pathways and gene expression are described, for example, in Gonzalez et al., Curr. Opm. m Biotech. 9:624-631 (1998) and Jayawickreme et al., Curr. Opm. Biotech. 8:629-634 (1997), and m references reviewed therein.
  • Yeast cell-based bioassays for hign-throughput screening of drug targets for G protein coupled receptors are described, for example, Pausch, Trends m Biotech. 15:487-494 (1997).
  • a variety of cell-based expression systems including bacterial, yeast, baculovirus/msect systems and mammalian cells, useful for detecting G protein coupled receptor agonists and antagonists are described, for example, m Tate et al., Trends in Biotech. 14:426-430 (1996).
  • Assays to detect and measure signal transduction can involve first contacting the cell, extract or artificial assay system with a detectable indicator. Calcium indicators, pH indicators, and meta__ ion indicators, and assays for using these indicators to detect and measure selected signal transduction pathways are described, for example, in Haugland, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals ,
  • Assays to determine changes m gene expression can involve transducing cells with a promoter-reporter nucleic acid construct such that, for example, ⁇ -lactamase, luciferase, green fluorescent protein or ⁇ -gaiactosidase will be expressed in response to contacting MCH receptor with an agonist or antagonist.
  • a promoter-reporter nucleic acid construct such that, for example, ⁇ -lactamase, luciferase, green fluorescent protein or ⁇ -gaiactosidase will be expressed in response to contacting MCH receptor with an agonist or antagonist.
  • Such assays and reporter systems are well known m the art and are described, for example, at http : //www . aurorabio . com/tech_platform- assay_technolog ⁇ es . html .
  • An assay to determine whether a candidate compound is an MCH receptor agonist or antagonist can be performed either in the presence or absence of a known MCH receptor agonist, such as MCH.
  • a known MCH receptor agonist such as MCH.
  • the invention also provides compositions useful for identifying MCH receptor agonists and antagonists.
  • the invention provides a signaling composition containing a recombinantly expressed MCH receptor and a recombinantly expressed G ⁇ subunit of a G protein.
  • a signaling composition containing a recombinantly expressed MCH receptor and a recombinantly expressed G ⁇ subunit of a G protein.
  • An example of such a composition is the CHO cell line or HEK 293-T cell line expressing recombmant MCH receptor and recombmant G ⁇ q/ ⁇ 3, described in Example II, below.
  • the term "signaling composition” refers to any composition m which contacting MCH receptor with an MCH receptor agonist will elicit a predetermined signal.
  • the invention provides a signaling composition containing a recombinantly expressed MCH receptor, a G protein, and a calcium indicator.
  • Calcium indicators and their use are well known in the art, and include compounds like Fluo-3 AM, Fura-2, Indo-1, FURA RED, CALCIUM GREEN, CALCIUM ORANGE, CALCIUM CRIMSON, BTC, OREGON GREEN BAPTA, which are available from Molecular Probes, Inc., Eugene Oreg., and described, for example, in U.S. Patent Nos . 5,453,517, 5,501,980 and 4,849,362.
  • An example of a signaling composition containing a recombinantly expressed MCH receptor, endogenous G protein, and a calcium indicator is the CHO cell line expressing recombmant MCH receptor loaded with the calcium indicator Fluo-3 AM, described Example III, below.
  • the G ⁇ subunit of the G protein in the signaling composition containing a recombinantly expressed MCH receptor, a G protein, and a calcium indicator can be recombinantly expressed.
  • An example of such a composition is the CHO cell line expressing recombmant MCH receptor, recombmant G ⁇ q/ ⁇ 3, and loaded with the calcium indicator Fluo-3 AM, described in Example I, below.
  • m reference to an MCH receptor or G ⁇ subunit of a G protein refers to a polypeptide that is transiently or stably expressed from a non-natural nucleic acid molecule. Recombmant expression is advantageous in providing a higher level of expression of the polypeptide than is found endogenously, and also allows expression in cells or systems which the polypeptide is not normally found.
  • a "non-natural" nucleic acid molecule is one that has been constructed, at least m part, by molecular biological methods, such as PCR, restriction digestion and ligation.
  • a non-natural nucleic acid expression construct generally will contain a constitutive or mducible promoter of RNA transcription appropriate for the host cell or transcription-translation system, operatively linked to a nucleotide sequence that encodes the polypeptide of interest.
  • the expression construct can be DNA or RNA, and optionally can be contained m a vector, such as a plasmid or viral vector.
  • the signaling compositions of the invention include, for example, cells, cell extracts and reconstituted artificial signaling systems.
  • the cell compositions of the invention include any cell type m which MCH receptor can couple to a G protein and induce a detectable signal in response to an agonist, such as a vertebrate cell, insect cell (e.g. Drosophila) , yeast cell (e.g. S . cerevisiae, S . pombe, or Pichia pasto ⁇ s) or prokaryotic cell (e.g. E. coll ) .
  • Exemplary vertebrate cells include, but are not limited to, mammalian primary cells and established cell lines, such as COS, CHO, HeLa, NIH3T3, HEK 293-T, PC12, and amphibian cells, such as Xenopus embryos and oocytes . Also included are cells from transgenic animals, such as transgenic mice, that have been engineered by known methods to express recombmant MCH receptor or G ⁇ subunit.
  • the signaling compositions of the invention also include crude or partially purified lysates or extracts of the cell compositions of the invention, and reconstituted artificial signaling systems.
  • Artificial signaling systems can include, for example, a natural or artificial lipid bilayer, such as a liposome, to maintain MCH receptor in a natural configuration, and cellular fractions or isolated components necessary for transducing and detecting the desired predetermined signal .
  • the invention also provides a method of identifying an MCH receptor ligand.
  • the method consists of contacting an MCH receptor with one or more candidate compounds under conditions that allow selective binding between MCH receptor and an MCH receptor ligand.
  • a compound that selectively binds MCH receptor is identified, and the compound is characterized as an MCH receptor ligand.
  • MCH receptor ligand refers to any biological or chemical compound that selectively binds an MCH receptor polypeptide.
  • An “MCH receptor ligand” can be an agonist or antagonist of MCH receptor, as described above, or can be a compound having little or no effect on MCH receptor signaling, so long as the compound selectively binds an MCH receptor polypeptide.
  • An MCH receptor ligand can be used to specifically target a diagnostic or therapeutic moiety to a region of the brain, or other organ or tissue of the body, that expresses MCH receptor.
  • an MCH receptor ligand can be labeled with a detectable moiety, such as a radiolabel, fluorochrome, ferromagnetic substance, or luminescent substance, and used to detect expression of MCH receptor polypeptide m an isolated sample or in m vivo diagnostic imaging procedures.
  • a detectable moiety such as a radiolabel, fluorochrome, ferromagnetic substance, or luminescent substance
  • an MCH receptor ligand can be labeled with a therapeutic moiety, such as a cytotoxic or cytostatic agent or radioisotope, and administered in an effective amount to arrest proliferation or kill a cell or tissue that expresses MCH receptor.
  • an MCH receptor ligand labeled with a therapeutic moiety can be used to treat proliferative diseases, including cancer and inflammatory diseases, that affect MCH receptor-expressing tissues, or as an alternative to neurosurgery to ablate regions of the brain responsible for MCH receptor-associated conditions, such as the conditions described below.
  • An MCH receptor ligand that selectively binds MCH receptor will bind MCH receptor with high affinity, but will not bind, or bind with low affinity, to a structurally related receptor that is not an MCH receptor, such as a somatostatm receptor.
  • High affinity binding refers to a dissociation constant (Kd) of less than about 10 " ° M, preferably less than about 10 "7 M, such as less than about 10 "8 M.
  • low affinity binding refers to a Kd of about 10 "4 M or more.
  • MCH receptor ligand is mammalian or salmon MCH which, as disclosed herein, binds and activates MCH receptor with a half-maximal response at nanomolar concentration.
  • MCH receptor ligand is an antibody specific for MCH receptor, such as an antibody specific for an extracellular region of an MCH receptor.
  • a peptide containing substantially the sequence of one of the three extracellular loops of MCH receptor such as substantially the sequence HQLMGNGVWHFGETMCT (SEQ ID NO: 9), RLIPFPGGAVGCGIRLPNPDTDL (SEQ ID NO: 10), QLISISRPTLTFVY (SEQ ID NO: 11), or immunogenic fragment therefrom, or substantially the N-termmal sequence MLCPSKTDGSGHSGRIHQETHGEGKRDKISNSEGRENGGRGFQMNGGSLEAEHASRM SVLRAKPMSNSQRLLLLSP (SEQ ID NO: 12), or immunogenic fragment therefrom, can be produced, either by direct synthesis, by recombmant means, or by enzymatic digestion of MCH receptor.
  • the peptide can formulated in an immunogenic composition, such as conjugated to a carrier protein or formulated with an adjuvant, to generate an MCH receptor specific polyclonal or monoclonal antibody using methods well known in the art and described, for example, Harlow and Lane,
  • Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989). Methods of preparing fragments of antibodies with specific binding activity, such as Fab fragments, and methods of preparing recombmant, chimeric or humanized antibodies directed against a peptide sequence, are also well known in the art, and such antibodies and fragments directed against MCH receptor are also contemplated as MCH receptor ligands .
  • FCS fluorescence correlation spectroscopy
  • SPA scintillation proximity assays
  • assays for detecting binding interactions include, for example, ELISA assays, FACS analysis, and affinity separation methods, which are described, for example, m Harlow and Lane, Eds., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) .
  • Such assays can be performed, for example, with whole cells that express MCH receptor, membrane fractions therefrom or artificial systems, as described previously, or with substantially purified MCH receptor polypeptide, either in solution or bound to a solid support.
  • the MCH receptor ligands, agonists and antagonists identified using the methods and compositions of the invention can be isolated and administered to an individual, such as a human or other mammal, in an effective amount to prevent or ameliorate the severity of an MCH receptor-associated condition.
  • MCH receptor-associated condition refers to any pathological condition associated with a tissue or cell in which MCH receptor is expressed.
  • MCH receptor-associated condition includes any abnormal physiological or psychological condition in which a quantitative or qualitative alteration m signaling through the MCH receptor contributes to the symptoms of the condition.
  • An MCH receptor-associated condition also includes any physiological or psychological condition in which altering signaling through the MCH receptor has a beneficial effect in the individual .
  • An MCH receptor-associated condition can have any of a variety of causes, including genetic, environmental and pathological causes.
  • an MCH receptor-associated condition can be caused by a mutation in MCH receptor nucleic acid that alters its expression or structure, a mutation in MCH, or a mutation in a molecule that normally regulates expression or bioavailability of MCH or MCH receptor.
  • An MCH receptor-associated condition can also be caused by environmental factors, such as exposure to toxins, therapeutic drugs or hormones that alter signaling through the MCH receptor, or affect the viability or function of cells or tissues that express the MCH receptor, MCH agonists and antagonists, or their regulatory molecules.
  • An MCH receptor-associated condition can also be due to a patnc-ogical condition that affects the viability or function of cells or tissues that express the MCH receptor, MCH agonists and antagonists, or their regulatory molecules, such as neurodegenerative diseases, infectious diseases, endocrine disorders, and benign and malignant tumors and tumor metastases.
  • MCH receptor is expressed in regions of the brain involved m taste, olfaction, feeding behavior and metabolism. Furthermore, central administration of MCH promotes feeding (Ludwig et al., Am. J. Phvsiol. 274 : E627-E633 (1998)), and MCH mRNA amounts rise as a result of starvation and leptm deficiency (Qu et al., Nature 380:243-247 ( 1996)). In contrast, MCH deficiency results m reduced body weight and leanness due to reduced feeding and inappropriately increased metabolic rate (Shimada et al., Nature 396:670- 674 (1998)).
  • MCH receptor-associated conditions can include disorders of body weight and metabolism, including disorders involving increased body weight, such as moderate or severe obesity due to endocrine dysfunction or overfeeding, and disorders involving decreased body weight, such as moderate underweight or cachexia.
  • cachexia refers to a general weight loss and wasting occurring m the course of a chronic disease, such as cancer or AIDS, or as a result of emotional disturbance, such as anorexia.
  • an MCH receptor agonist, antagonist or ligand can be used as a drug to restore more normal weight, metabolism and feeding behavior.
  • MCH receptor is expressed in regions of the brain involved dopammergic-modulated responses. Therefore, MCH receptor-associated conditions include pathologies associated with dopamme insufficiency or excess, including, but not limited to, Parkinson's disease and parkmsonian syndromes, Huntmgton's disease, and drug- and toxm-mduced movement disorders caused by altered availability or activity of dopamme . Thus, MCH receptor agonists and antagonists can be used as therapeutics to prevent or treat conditions due to altered dopammergic system function.
  • MCH receptor is expressed in regions of the brain involved m control of behavior, memory and learning, mood and sleep.
  • Disorders of behavior include, but are not limited to, autistic disorder, Asperger ' s disorder, aggression, pervasive developmental disorders, Tourette ' s syndrome, attention- deficit hyperactivity disorder and addiction.
  • Disorders of memory and learning include, but are not limited to, Alzheimer's disease; dementia, including dementia due to neurodegenerative diseases, infectious disease, proliferative diseases, endocrine disease, tumors, metabolic disorders, and toxins; and developmental learning disabilities.
  • Disorders of sleep and of the sleep-wake cycle include, but are not limited to, insomnia, bedwettmg, sleepwalking, sleep apnea and narcolepsy.
  • disorders of mood include, but are not limited to, depression; anxiety disorders, such as generalized anxiety disorder, panic attacks, obsessive- compulsive disorder, phobias, acute stress disorder, post-traumatic stress disorder; and psychotic disorders, such as unipolar mania or depression, bipolar disorder and schizophrenia.
  • MCH receptor agonists, antagonists and ligands can be used as therapeutics to prevent or treat disorders of behavior, memory and learning, mood and sleep.
  • MCH receptor is expressed the pituitary, which controls various reproductive functions and developmental growth.
  • an MCH receptor agonist or antagonist can be used as a male or female contraceptive, or m treatment of an MCH receptor-associated reproductive disorder, such as male and female sexual dysfunction, impotence, failure of lactation, infertility and precocious puberty, or an MCH receptor-associated growth disorder, such as dwarfism or acromegaly.
  • an MCH receptor-associated reproductive disorder such as male and female sexual dysfunction, impotence, failure of lactation, infertility and precocious puberty, or an MCH receptor-associated growth disorder, such as dwarfism or acromegaly.
  • the MCH receptor agonist, antagonist or ligand therapeutics of the present invention can be conveniently formulated for administration together with a pharmaceutically acceptable carrier.
  • suitable pharmaceutical carriers for the methods of the invention are well known and include, for example, aqueous solutions such as physiologically buffered saline, and other solvents or vehicles such as glycols, glycerol, oils or m ectable organic esters.
  • a pharmaceutical carrier can contain a physiologically acceptable compound that acts, for example, to stabilize or increase the solubility of a pharmaceutical composition.
  • Such a physiologically acceptable compound can be, for example, a carbohydrate, such as glucose, sucrose or dextrans; an antioxidant, such as ascorbic acid or glutathione; a chelating agent; a low molecular weight protein; or another stabilizer or excipient .
  • a carbohydrate such as glucose, sucrose or dextrans
  • an antioxidant such as ascorbic acid or glutathione
  • a chelating agent such as ascorbic acid or glutathione
  • a chelating agent such ascorbic acid or glutathione
  • a chelating agent such ascorbic acid or glutathione
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • the therapeutic compounds of the invention can be formulated, for example, in liposomes, or chemically derivatized.
  • the choice of the pharmaceutical formulation and the appropriate preparation of the composition will depend on the intended use and mode of administration.
  • Methods of introduction of a therapeutic compound of the invention include, but are not limited to, mtradermal, intramuscular, mtrape ⁇ toneal, intravenous, subcutaneous, oral, mtranasal, mtraspinal and tracerebral routes. Methods of introduction can also be provided by rechargable or biodegradable devices, particularly where gradients of concentrations of drug in a tissue is desired.
  • Various slow release polymeric devices are known the art for the controlled delivery of drugs, and include both biodegradable and non- degraoable polymers and hy ⁇ rogels.
  • An effective dose of a therapeutic composition of the invention can be determined by extrapolation from the concentration required for modulating MCH receptor signaling or binding m m vi tro assays described herein, and from the dose required for efficacy in an animal model of the MCH receptor-associated conditions described herein.
  • an appropriate dose can be in the range of 0.001-100 mg/kg of body weight, but can be determined by those skilled m the art depending on the bioactivity of the particular compound, the desired route of administration, the gender and health of the individual, the number of doses and duration of treatment, and the particular condition being treated.
  • the invention also provides methods of identifying an individual having or susceptible to an MCH receptor-associated condition. Such knowledge allows optimal medical care for the individual, including appropriate genetic counseling and prophylactic and therapeutic intervention.
  • the method consists of detecting MCH receptor nucleic acid molecule m a sample from the individual. Abnormal structure or expression of MCH receptor nucleic acid molecule in the sample indicates that the individual has, or is at greater risk than a normal individual of developing, an MCH receptor-associated condition.
  • MCH receptor nucleic acid molecule refers to a DNA or RNA molecule that corresponds to at least a part of a nucleotide sequence of a gene that encodes an MCH receptor.
  • an MCH receptor nucleic acid molecule can be MCH receptor genomic DNA, mRNA, or a nucleic acid molecule derived therefrom, such as a PCR amplification product or cDNA.
  • An MCH receptor nucleic acid molecule can correspond to the sense or antisense strand, and can include coding or non-coding sequence, or both, of an MCH receptor gene.
  • Normal human MCH receptor cDNA has substantially the nucleotide sequence presented m Figure 1 (SEQ ID NO : 1 ) , and encodes substantially the ammo acid sequence presented in Figure 1 (SEQ ID NO: 2) .
  • altered expression or structure of the nucleic acid molecule By detecting MCH nucleic acid in a sample, either altered expression or structure of the nucleic acid molecule can be determined, and used to diagnose or predict risk of developing an MCH receptor-associated condition.
  • altered expression refers to an increased or decreased amount of MCH receptor nucleic acid in the test sample relative to levels in a normal sample. Altered abundance of a nucleic acid molecule can result, for example, from an altered rate of transcription, from altered transcript stability, or from altered copy number of the corresponding gene.
  • altered structure of a nucleic acid molecule refers to differences, such as point mutations, insertions, deletions, chromosomal translocations, splice variations and other rearrangements, between the structure of a nucleic acid molecule of the invention m a test sample and the structure of the nucleic acid molecule m a normal sample.
  • mutations that alter the structure of a nucleic acid molecule can also alter its expression.
  • Abundance or structure of MCH receptor n a normal sample can, if desired, be determined simultaneously with the test sample, or can be a previously established value.
  • sample refers to any biological fluid, cell, tissue, organ or portion thereof, that is appropriate to detect MCH receptor nucleic acids and polypeptides, and includes samples present in an individual as well as samples obtained or derived from the individual.
  • a sample can be a histologic section of a specimen obtained by biopsy, or cells that are placed in or adapted to tissue culture.
  • a sample further can be a subcellular fraction or extract, or a crude or substantially pure nucleic acid or protein preparation.
  • the appropriate source and method of preparing the sample can be determined by those skilled the art, depending on the application of the detection method.
  • any convenient source of DNA such as blood cells, lymph cells, cheek cells or sk n cells, can be used.
  • a sample should be obtained from a tissue that expresses MCH receptor, such as neural tissue or, more conveniently, tongue or skeletal muscle.
  • RNA abundance Various qualitative and quantitative assays to detect altered expression or structure of a nucleic acid molecule in a sample are well known in the art, and generally involve hybridization of a detectable agent, such as a complementary primer or probe, to the nucleic acid molecule.
  • a detectable agent such as a complementary primer or probe
  • Such assays include, for example, in si tu hybridization, which can be used to detect altered chromosomal location of the nucleic acid molecule, altered gene copy number, or altered RNA abundance, depending on the format used.
  • RNA assays include, for example, Northern blots and RNase protection assays, which can be used to determine the abundance and integrity of RNA; Southern blots, which can be used to determine the copy number and integrity of DNA; SSCP analysis, which can detect single point mutations in DNA, such as in a PCR or RT-PCR product; direct sequencing of nucleic acid fragments, such as PCR amplification fragments; and coupled PCR, transcription and translation assays, such as the Protein Truncation Test, m which a mutation in DNA is determined by an altered protein product on an electrophoresis gel.
  • Northern blots and RNase protection assays which can be used to determine the abundance and integrity of RNA
  • Southern blots which can be used to determine the copy number and integrity of DNA
  • SSCP analysis which can detect single point mutations in DNA, such as in a PCR or RT-PCR product
  • direct sequencing of nucleic acid fragments such as PCR amplification fragments
  • An appropriate assay format and detectable agent to detect an alteration m the expression or structure of an MCH receptor nucleic acid molecule can be determined depending on the alteration it is desired to identify. Methods of performing such assays are well known in the art.
  • the invention also provides a method of identifying an individual having or susceptible to an MCH receptor-associated condition, by detecting MCH receptor polypeptide in a sample from the individual. Abnormal structure or activity of MCH receptor polypeptide m the sample indicates that the individual has or is susceptible to an MCH receptor-associated condition.
  • altered expression of a polypeptide refers to an increased or decreased amount, or altered subcellular localization, of the polypeptide in the test sample relative to known levels or localization m a normal sample.
  • Altered abundance of a polypeptide can result, for example, from an altered rate of translation or altered copy number of the corresponding message, or from altered stability of the protein.
  • Altered subcellular localization can result, for example, from truncation or mactivation of a sorting sequence, from fusion with another polypeptide sequence, or altered interaction with other cellular polypeptides.
  • assays to detect altered expression of polypeptides are known the art, and generally involve hybridization of a detectable agent, such as a labeled ligand, to the polypeptide in a sample, or within the body in diagnostic imaging procedures.
  • Assays to detect altered expression of MCH receptor can be performed m si tu, in which a detectably labeled ligand, such as an antibody or other ligand identified by the methods described herein, contacts MCH receptor in a whole cell.
  • Other assays to detect altered expression of MCH receptor polypeptide include, for example, ELISA assays, lmmunoprecipitation, and immunoblot analysis, which can be performed with cell or tissue extracts.
  • An appropriate assay format and detectable agent to detect an alteration in the expression of MCH receptor polypeptide can be determined depending by those skilled in the art depending on the alteration it is desired to identify. Methods of performing such assays are well known in the art .
  • This example shows an assay system and signaling composition that can be used to identify MCH receptor agonists and antagonists.
  • This assay system was used to identify MCH as an endogenous agonist of MCH receptor .
  • SLC-1 exhibits about 40% amino acid identity to five known human somatostatin recetors (SSTRs ) , which are G ⁇ -linked receptors.
  • SSTRs human somatostatin recetors
  • SLC-1 would also bind a peptidic ligand and couple to G ⁇ i proteins, different tissues were harvested that were known to express SLC-1, and processed for peptide extraction following protocols described in Reinscheid et al . , Science 270:792-794 (1995); Meunier et al . , Nature 377:532-535 (1995); and Hinuma et al . , Nature 393:272-276 (1998).
  • chromatographic fractions prepared as described in Example II, were tested for their ability to induce a decrease in cAMP levels in forskolin- stimulated, SLC-1-transfected CHO cells. None of the fractions showed a response which could be reproducibly followed over several purification steps due to the lability of the cAMP assays.
  • a new assay system was therefore developed to monitor SLC-1 reactivity through recording of calcium influxes, by forcing SLC-1 to couple to a G ⁇ q protein. Because it has been shown that the five C-terminal residues of G ⁇ are sufficient for receptor contact, while the rest of the subunit serves to interact with the effector molecule, a G ⁇ q/i3 chimera designed to drive SLC-1 to G ⁇ q activation was constructed.
  • the G ⁇ q/i3 chimera contained the five C-terminal residues of G ⁇ i3 (ECGLY) while retaining the rest of the G ⁇ q sequence, residues 1-354.
  • the resulting l.lkb PCR products was subcloned into pcDNA 3.1 (+) expression vector and sequenced.
  • the SLC-1 cDNA subcloned into pcDNA 3.1 (+) was transfected with the G ⁇ q/i3 chimera into CHO dhfr (-) cells using LipofectAMINE PLUS transfection reagent and following the manufacture' s instructions (GIBCO-BRL) .
  • Calcium influx assays were performed as described m Coward et al . , Proc. Natl. Acad. Sci. USA 95:352-357 (1998). In brief, transfected or control cells were seeded into 96 wells at 5.5 x 10 4 cells/well.
  • the cells were loaded with Fluo-3 AM (Molecular Probes) m standard bath solution (130mM NaCl, 2mM CaCl 2 , 5mM KC1, lOmM glucose, 0.45mM KH 2 P0 4 , 0.4mM Na 2 HP0 4 , 8mM MgS0 4 , 4.2mM NaHC0 3 , 20mM HEPES, and 10 ⁇ M probenecid) with 0.1% fetal bovine serum for 1 hr at 37°C, then washed with a standard bath solution. Transient changes in [Ca 2+ ]. evoked by fractions were monitored by the FLIPR system (Fluorometric Imaging Plate Reader, Molecular Devices) in 96 well plates at 488 nm for 210 seconds.
  • Fluo-3 AM Molecular Probes
  • MCH receptor agonists induce dose-dependent transient increases in cytoplasmic calcium levels in SLC-l-G ⁇ q/ ⁇ 3 transfected cells using the above-described assay system.
  • This example shows the purification of an endogenous agonist of the orphan G-protem coupled receptor SLC-1 (GPR24) and its identification as the neuropeptide melanin-concentrating hormone (MCH) .
  • the purification of the endogenous SLC-1 ligand was performed as follows. 400g rat frozen brain (Pel- Freez) were extracted 1M acetic acid and centrifuged at 20,000 x g for 15mm at 4 C. The resulting supernatant was precipitated with acetone and extracted with diethylether . The aqueous pnase was concentrated and loaded onto a C18 reverse phase HPLC column (PrepPAK- Delta-Pac 25 x 100mm, Waters) and eluted with a linear gradient of 5-48% CH.CN in 0.1% trifluoroacetic acid (TFA) at a flow rate of 1 ml/mm. Fractions were monitored for anility to induce increases m [CaA_ . m CHO cells transiently cotransfected with the G ⁇ q/ ⁇ 3 chimera and SLC-1, using the calcium influx assay described in Example I, above.
  • Active fractions 56 and 57 from the reverse phase HPLC were pooled and further purified by six more chromatographic steps. Briefly, pooled fractions 56 ana 57 were further purified on a cation-exchange column AP- 1/SP-8HR (Waters) with a linear gradient of 0.15-0.35M NaCl 6mM HC1 and 30% CH.CN . Active fractions were further purified on an analytical CIS Select B column
  • the final active compound was subjected to a structural analysis by MALDI mass spectrometry and Edman degradation.
  • Ammo acid sequences were determined in a pulse liquid automatic sequencer. Am o acid sequence analysis revealed an N-termmal sequence in which three of the first five residues were identical to that of the rat melanin-concentrating hormone (MCH) described by Vaughan et al., Endocrinology 125:1660-1665 (1989).
  • MCH melanin-concentrating hormone
  • Synthetic rat MCH was shown to behave identically to the purified active peptide m retention time (by reverse phase HPLC) and molecular size (by mass data) . Therefore, it was inferred that the isolated peptide was MCH. Final yields of MCH was approximately 25pmol/kg of rat brain (wet weight) .
  • MCH from other species were also tested in SLC- l-G ⁇ q/i3 transfected cells.
  • Salmon MCH described by Kawauchi et al., Nature 305;321-323 (1983), which has a high degree of homology to rat MCH in its central and C- terminal portions, activated SLC-1 with an EC50 of 18.6 ⁇ 2.3 nM.
  • a longer isoform of human SLC-1 described by Kolakowski et al., FEBS Lett. 398:255-258 (1996), was inactive in this assay system.
  • MCH-gene related peptides MCH- precursor-derived peptide NEI, MCH-gene-overprinted- polypeptide, MGOP-14, or -27
  • MCH-gene related peptides MCH- precursor-derived peptide NEI, MCH-gene-overprinted- polypeptide, MGOP-14, or -27
  • MCH and ⁇ -MSH demonstrate opposite actions on skin coloration in teleost fishes (Baker, Ann. NY Acad.
  • MCH receptor couples to G ⁇ i- and G ⁇ q-containing G protein signal transduction pathways.
  • a CHO cell line stably expressing stably SLC-1 was established as follows.
  • the SLC-1 cDNA subcloned into pcDNA 3.1 (+) was transfected into CHO dhfr (-) cells by the calcium-phosphate method described in Saito et al., J. Neurosci. Res. 48:397-406 (1997), and stable cell lines were established.
  • these cell lines were analyzed by Northern blot and one clone was chosen for further experiments.
  • This example shows the expression of the MCH receptor in mammaliam tissues, as determined by Northern Plot analysis and m si tu hybridization.
  • the l.lkb insert of SLC-1 was labeled with ⁇ P-dCTP and used as a probe m Northern blot analysis.
  • Northern blots containing 3 ⁇ g of poly (A) + RNA from various rat tissues were hybridized and washed under high stringency conditions. Blots were exposed to Kodak X- OMAT film at -80°C with two intensifying screens.
  • m si tu hybridization was performed using a cRNA probe on rat brain sections.
  • a 0.6 kb BamHl-Xbal fragment of human SLC-1 cDNA was generated and subcloned into the pBluesc ⁇ pt II SK (+) vector. The homology between human ano rat sequences is 92% in this fragment.
  • Sense and anti-sense riboprobes were generated by T7 and T3 RNA polymerases, respectively, in the presence of 35S-UTP.
  • In situ hybridization to adult rat whole brain sections was performed as described by W zer-Serhan et al., Br. Res. Prots. 3:229-241 (1999) . Control hybridization with a sense strand cRNA produced no specific signal (data not shown) .
  • Moderate expression of SLC-1 mRNA was found m the substantia nigra, ventral tegmental area and m the amygdala ( Figure 5B, Panels b and c) , indicating that MCH may modulate the dopammergic system.
  • Moderate expression of SLC-1 was also detected m the locus coeruleus ( Figure 5B, Panel d) which suggests that MCH may participate in the control of various noradrenergic- modulated responses including vigilance, attention, memory, and sleep.

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Abstract

L'invention concerne un procédé d'identification d'un agoniste ou d'un antagoniste du récepteur de l'hormone concentratrice de la mélanine (MCH), qui consiste à mettre un récepteur de MCH en contact avec un ou plusieurs composés d'intérêt potentiel, dans des conditions dans lesquelles le récepteur de MCH produit un signal prédéterminé en réponse à un agoniste du récepteur MCH, et à identifier un composé qui modifie la production du signal prédéterminé. L'invention porte également sur un procédé d'identification d'un ligand de récepteur MCH, qui consiste à mettre un récepteur de MCH en contact avec un ou plusieurs composés d'intérêt potentiel, dans des conditions permettant une liaison sélective entre le récepteur MCH et un ligand de récepteur MCH, et à identifier un composé qui se fixe sélectivement au récepteur MCH. L'invention se rapporte encore à un procédé d'identification d'un individu souffrant d'un état associé au récepteur MCH ou sujet audit état, qui consiste à détecter l'acide nucléique ou un polypeptide du récepteur MCH dans un échantillon. Des compositions de signalisation contenant un récepteur MCH exprimé par recombinaison et une sous-unité alpha exprimée par recombinaison d'une protéine G, ou contenant un récepteur MCH exprimé par recombinaison, une protéine G et un indicateur de calcium, sont également décrites.
PCT/US2000/015503 1999-06-08 2000-06-06 Recepteur de l'hormone concentratrice de la melanine Ceased WO2000075166A1 (fr)

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Cited By (12)

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WO2002008290A1 (fr) * 2000-07-21 2002-01-31 Merck & Co., Inc. Recepteur de l'hormone concentrant la melanine du chien
EP1288310A1 (fr) * 2001-08-31 2003-03-05 Aventis Pharma Deutschland GmbH Utilisation de polymorphismes du nucléotide simple (SNP's) du récepteur (MCH-R) d'hormone concentrant la mélanine pour l'identification d'affections génétiques associées au maintien du poids du corps
WO2003104489A3 (fr) * 2002-06-05 2004-03-11 Univ Marburg Philipps Variante du mchr1 associee a l'obesite humaine
WO2004035614A1 (fr) * 2001-07-11 2004-04-29 Karo Bio Ab Peptides synthetiques ou partiellement purifies pouvant se lier a des sous-unites specifiques de proteines g, et leurs utilisations
WO2005040824A3 (fr) * 2003-10-24 2005-06-30 Bayer Healthcare Ag Diagnostics et therapeutiques pour maladies liees au recepteur 24 couple a la proteine g (gpr24)
US7078187B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7078484B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7125885B2 (en) 2001-05-04 2006-10-24 Amgen Inc. Fused heterocyclic compounds
US7141391B2 (en) 2001-11-13 2006-11-28 Neurogen Corporation Monkey and canine melanin concentrating hormone receptors
US7208282B2 (en) 2001-05-31 2007-04-24 Merck & Co., Inc. Rhesus monkey, dog and ferret melanin-concentrating hormone type 2 receptor
US7253179B2 (en) 2002-11-06 2007-08-07 Amgen Inc. Fused heterocyclic compounds
US7393655B2 (en) 1998-12-31 2008-07-01 H. Lundbeck A/S Methods of identifying melanin concentrating hormone receptor antagonists

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EP0848060A2 (fr) * 1996-12-11 1998-06-17 Smithkline Beecham Corporation Variante d'épissure de 11CB humaine
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WO2000039279A2 (fr) * 1998-12-31 2000-07-06 Synaptic Pharmaceutical Corporation Adn codant un recepteur d'hormone (mch1) concentrant la melamine humaine et utilisations associees

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WO1996018651A1 (fr) * 1994-12-16 1996-06-20 Smithkline Beecham Corporation Recepteur humain somatostatinoide
US6008012A (en) * 1995-12-15 1999-12-28 Smithkline Beecham Corporation, Human somatostatin-like receptor
EP0848060A2 (fr) * 1996-12-11 1998-06-17 Smithkline Beecham Corporation Variante d'épissure de 11CB humaine
US6033872A (en) * 1996-12-11 2000-03-07 Smithkline Beecham Corporation Polynucleotides encoding a novel human 11cb splice variant
WO2000039279A2 (fr) * 1998-12-31 2000-07-06 Synaptic Pharmaceutical Corporation Adn codant un recepteur d'hormone (mch1) concentrant la melamine humaine et utilisations associees

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7393655B2 (en) 1998-12-31 2008-07-01 H. Lundbeck A/S Methods of identifying melanin concentrating hormone receptor antagonists
US7148026B2 (en) 2000-07-21 2006-12-12 Merck & Co., Inc. Dog melanin-concentrating hormone receptor
WO2002008290A1 (fr) * 2000-07-21 2002-01-31 Merck & Co., Inc. Recepteur de l'hormone concentrant la melanine du chien
US7078187B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7078484B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7125885B2 (en) 2001-05-04 2006-10-24 Amgen Inc. Fused heterocyclic compounds
US7208282B2 (en) 2001-05-31 2007-04-24 Merck & Co., Inc. Rhesus monkey, dog and ferret melanin-concentrating hormone type 2 receptor
WO2004035614A1 (fr) * 2001-07-11 2004-04-29 Karo Bio Ab Peptides synthetiques ou partiellement purifies pouvant se lier a des sous-unites specifiques de proteines g, et leurs utilisations
EP1288310A1 (fr) * 2001-08-31 2003-03-05 Aventis Pharma Deutschland GmbH Utilisation de polymorphismes du nucléotide simple (SNP's) du récepteur (MCH-R) d'hormone concentrant la mélanine pour l'identification d'affections génétiques associées au maintien du poids du corps
WO2003020965A3 (fr) * 2001-08-31 2003-10-23 Aventis Pharma Gmbh Utilisation de polymorphismes de nucleotide simple de recepteur mch dans l'identification de troubles genetiques dans la conservation du poids corporel normal
US7141391B2 (en) 2001-11-13 2006-11-28 Neurogen Corporation Monkey and canine melanin concentrating hormone receptors
WO2003104489A3 (fr) * 2002-06-05 2004-03-11 Univ Marburg Philipps Variante du mchr1 associee a l'obesite humaine
US7253179B2 (en) 2002-11-06 2007-08-07 Amgen Inc. Fused heterocyclic compounds
WO2005040824A3 (fr) * 2003-10-24 2005-06-30 Bayer Healthcare Ag Diagnostics et therapeutiques pour maladies liees au recepteur 24 couple a la proteine g (gpr24)

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