WO1996023809A1 - Neuropeptide y receptor - Google Patents

Neuropeptide y receptor Download PDF

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Publication number
WO1996023809A1
WO1996023809A1 PCT/US1996/001444 US9601444W WO9623809A1 WO 1996023809 A1 WO1996023809 A1 WO 1996023809A1 US 9601444 W US9601444 W US 9601444W WO 9623809 A1 WO9623809 A1 WO 9623809A1
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Prior art keywords
sequence
receptor
npy
dna
neuropeptide
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PCT/US1996/001444
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French (fr)
Inventor
Margaret A. Cascieri
David L. Linemeyer
Douglas J. Macneil
Lin-Lin Shiao
Catherine Strader
David H. Weinberg
Carina P. Tan
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Merck and Co Inc
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Merck and Co Inc
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Priority to US08/894,236 priority Critical patent/US5939263A/en
Priority to EP96904553A priority patent/EP0809648A4/en
Priority to JP8523748A priority patent/JPH11500610A/en
Publication of WO1996023809A1 publication Critical patent/WO1996023809A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • Neuropeptide Y is a 36 residue, amidated polypeptide. It is anatomically co-distributed and co-released with norepinephrine in and from sympathetic postganglionic neurons ([1], [2], [3], [4], [5], [6]). Stimulation of the sympathetic nervous system under physiological circumstances such as exercise ([7], [8]) or exposure to the cold ([9], [10]) promotes an elevation of both norepinephrine and NPY.
  • NPY is believed to act in the regulation of appetite control ([ 1 1], [12]) and vascular smooth muscle tone ([13], [14]) as well as regulation of blood pressure ([6], [15], [16], [17]). NPY also decreases cardiac contractility ([18], [19], [20], [21], [22]). Congestive heart failure and cardiogenic shock are associated with probable releases of NPY into the blood ([23], [24], [25]). Regulation of NPY levels may be beneficial to these disease states [26].
  • neuropeptide Y binds to a G -protein coupled receptor ([27], [28], [29], [30]).
  • Neuropeptide Y is involved in regulating eating behavior and is an extremely potent orixigenic agent ([1 1], [12], [31]).
  • VN hypothalamic paraventricular nucleus
  • NPY levels in the PVN increase upon fasting ([40], [41], [42], [43], [44]), before a scheduled meal ([31], [36], [40]), and in both streptozotocin-induced and spontaneous diabetes ([36], [45], [46], [47], [48], [49]).
  • NPY levels are increased in genetically obese and hyperphagic Zucker rats ([36], [50], [51]).
  • a specific centrally acting antagonist for the appropriate NPY receptor subtype may be therapeutically useful for treating obesity and diabetes.
  • disorders which might be targeted therapeutically include anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases ([26], [52]).
  • the Y 1 receptor is stimulated by NPY or PYY (peptide YY) and appears to be the major vascular receptor ([16], [53], [54], [55]).
  • the Y2 receptor is stimulated by C-terminal fragments of NPY or PYY and is abundantly expressed both centrally and peripherally ([55], [56], [57], [58]).
  • a third receptor (Y3) is exclusively responsive to NPY and is likely present in adrenal medulla, heart, and brain stem ([27], [59]).
  • other subtypes of this receptor family are known to exist, based on pharmacological and physiological characterization ([60], [61].
  • the feeding behavior is stimulated potently by NPY, NPY2- 36 and the Yl agonist [Leu31, Pro34]NPY, but is not stimulated by the Y2 agonist NPY 13-36 ([1 1], [64], [65], [66]).
  • This pharmacology is not characteristic of the defined Yl, Y2 or Y3 receptors and can thus be attributed to a unique receptor, termed "atypical Yl " ([ 11], [65], [66]), that is responsible for evoking the feeding response.
  • Y 1 receptor has been cloned and shown to be a G- protein coupled receptor ([53], [69], [70]). Recently, the Y2 receptor has been cloned ( ⁇ 71], [72]).
  • PP1 [73]
  • Y4 [(74)
  • NPY Neuropeptide Y
  • NPY Neuropeptide Y
  • the present invention relates to an isolated DNA sequence encoding a novel neuropeptide Y receptor, hereinafter referred to as the neuropeptide Y Yx receptor.
  • the DNA sequence encoding the NPY Yx receptor has a sequence selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3, a fragment of the sequence of substantial homology to the sequence shown in Figure 3, a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5.
  • the isolated DNA sequence wherein the sequence is selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3 or a fragment of the sequence of substantial homology to the sequence shown in Figure 3.
  • a class of the invention is the DNA sequence which encodes a murine NPY Yx receptor.
  • a subclass of the invention is the DNA sequence having a sequence selected from the sequence shown in Figure 3, the sequence of substantial homology to the sequence shown in Figure 3 or the fragment of the sequence shown in Figure 3 comprising bases 822 to 1934.
  • a second embodiment of the invention is the isolated DNA sequence wherein the DNA sequence has a sequence selected from a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5.
  • a second class of the invention is the DNA sequence which encodes a human NPY Yx receptor.
  • a second subclass is the DNA sequence having a sequence selected from the sequence shown in Figure 5, the sequence of substantial homology to the sequence shown in Figure 5 or the fragment of the sequence shown in Figure 5 comprising bases 182 to 1291.
  • Illustrative of the invention is an expression vector containing any of the DNA sequences described above.
  • An illustration of the invention is a cell transformed by the expression vector.
  • Exemplifying the invention is a method of producing the neuropeptide Y Yx receptor, comprising culturing the cell under conditions which allow the production of the neuropeptide Y Yx receptor and optionally recovering the neuropeptide Y Yx receptor.
  • An example of the invention is a neuropeptide Y Yx receptor produced by this process.
  • More particularly illustrating the invention is an antibody immunologically reactive with the NPY Yx receptor.
  • Another illustration of the invention is an isolated RNA encoded by any of the DNA sequences described above or their complementary sequences.
  • RNA which encodes the NPY Yx receptor
  • another example of the invention is isolated DNA containing this complementary sequence or a fragment thereof.
  • a neuropeptide Y Yx receptor in substantially pure form comprising an amino acid sequence selected from a sequence as shown in Figure 3 (SEQ. I.D. NO. 6), a sequence as shown in Figure 6 (SEQ. I.D. NO. 12), or a functional derivative thereof.
  • the neuropeptide Y Yx receptor has an amino acid sequence as shown in Figure 3 (SEQ. I.D. NO. 6) or a functional derivative thereof.
  • the neuropeptide Y Yx receptor has an amino acid sequence as shown in Figure 6 (SEQ. I.D. NO. 12) or a functional derivative thereof.
  • FIG. 1 Schematic diagram of FRAMES analysis using the Genetics Computing Group software (Madison, WI) identifying a 371 amino acid open reading frame (aa ORF).
  • Figure 2. Schematic diagram of hydrophobicity analysis using PEPPLOT (Genetics Computing Group software (Madison, WI)) indicating the open reading frame (ORF) has 7 transmembrane spanning domains characteristic of G-protein coupled receptor.
  • Figure 3 shows the genomic DNA sequence and deduced amino acid sequence of the murine NPY Yx receptor.
  • the sequence disclosure of Fig. 3 is represented as SEQ- I.D. NO. 5 and SEQ. I.D. NO. 6.
  • Figure 4 shows a comparative alignment determined by BESTFIT (Genetics Computing Group software (Madison, WI)) of the mouse NPY Yl receptor and the mouse NPY Yx receptor, described herein.
  • the seven transmembrane spanning domains are indicated as are three putative N-linked glycosylation sites (indicated by a *).
  • the sequence disclosures of the mouse NPY Yl receptor and mouse NPY Yx receptor of Fig. 4 are represented as SEQ. I.D. NO. 7 and SEQ. I.D. NO. 6, respectively.
  • Figure 5 shows the DNA sequence of the human NPY Yx receptor.
  • the sequence disclosure of Fig. 5 is represented as SEQ. I.D. NO. 1 1.
  • Figure 6 shows the amino acid sequence of the human NPY Yx receptor.
  • the sequence disclosure of Fig. 6 is represented as SEQ. I.D. NO. 12. DETAILED DESCRIPTION OF THE INVENTION
  • G-protein coupled receptors refers to any receptor protein that mediates its endogenous signal transduction through activation of one or more guanine nucleotide binding regulatory proteins (G-proteins). These receptors share common structural features, including seven hydrophobic transmembrane domains.
  • G-protein coupled receptors include receptors that bind to small biogenic amines, including but not limited to beta- adrenergic receptors ( ⁇ AR), alpha-adrenergic receptors ( ⁇ AR) and muscarinic receptors, as well as receptors whose endogenous ligands are peptides, such as neurokinin, neuropeptide Y and glucagon receptors.
  • ⁇ AR beta- adrenergic receptors
  • ⁇ AR alpha-adrenergic receptors
  • muscarinic receptors as well as receptors whose endogenous ligands are peptides, such as neurokinin, neuropeptide Y and glucagon receptors.
  • ⁇ AR include beta-1, beta-2, and beta-3 adrenergic receptors.
  • G-protein coupled receptors are cell surface proteins that mediate the responses of a cell to a variety of environmental signals. Upon binding an agonist, the receptor interacts with one or more specific G-proteins,
  • G-protein coupled receptors function as a complex information processing network within the plasma membrane of the cell, acting to coordinate a cell's response to multiple environmental signals.
  • G-protein coupled receptors consist of seven hydrophobic domains connecting eight hydrophilic domains.
  • the hydrophobicity or hydrophilicity of the domains may be determined by standard hydropathy profiles, such as Kyte-Doolittle analysis (Kyte, J. and Doolittle, R.J.F. J. Mol. Biol. 157: 105 (1982)).
  • the receptors are thought to be oriented in the plasma membrane of the cell such that the N-terminus of the receptor faces the extracellular space and the C- terminus of the receptor faces the cytoplasm, so that each of the hydrophobic domains crosses the plasma membrane.
  • the receptors have been modeled and the putative boundaries of the extracellular, transmembrane and intracellular domains are generally agreed (for a review, see Baldwin, EMBO J. 12:1693, 1993).
  • the transmembrane domains are comprised of stretches of 20-25 amino acids in which most of the amino acid residues have hydrophobic side chains (including cysteine, methionine, phenylalanine, tyrosine, tryptophan, proline, glycine, alanine, valine, leucine, isoleucine), whereas the intracellular and extracellular loops are defined by contiguous stretches of several amino acids that have hydrophilic or polar side chains (including aspartate, glutamate, asparagine, glutamine, serine, threonine, histidine, lysine, and arginine).
  • NPY Yx mammalian neuropeptide Y receptor subtype
  • a method of making the NPY Yx receptor is also provided.
  • the invention includes DNA encoding the NPY Yx receptor, the NPY Yx receptor, assays employing the NPY Yx receptor, cells expressing the NPY Yx receptor, antibodies which bind specifically to the NPY Yx receptor, RNA encoded by the DNA sequence or its complementary sequence, and single-stranded DNA with a sequence complementary to the RNA which encodes the NPY Yx receptor.
  • the NPY Yx receptor and assays employing the NPY Yx receptor are useful for identifying compounds which bind to the receptor, including specific modulators of the receptor. Modulators, as described herein, include but are not limited to agonists, antagonists, suppressors and inducers.
  • modulators may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.
  • Modulators identified by the processes described herein are useful as therapeutic agents.
  • compounds identified using the mammalian NPY Yx receptor are useful for treating one or more disease conditions, including obesity, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases.
  • the receptor may include genetic variants, both natural and induced. Induced receptors may be derived by a variety of methods, including but not limited to, site-directed mutagenesis. Techniques for nucleic acid and protein manipulation are well-known in the art and are described generally in Methods in Enzymology and in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989).
  • this invention is also directed to those DNA sequences which contain alternative codons which code for the eventual translation of the identical amino acid.
  • a sequence bearing one or more replaced codons will be defined as a degenerate variation.
  • mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate functional properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
  • DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide.
  • Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
  • a fragment of a DNA sequence refers to any polynucleotide subset of the DNA sequence or its complementary sequence. Also included within the scope of the present invention are fragments of DNA sequence capable of producing functional NPY Yx protein.
  • a "functional derivative" of a receptor is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of the receptor.
  • the term “functional derivative” is intended to include the "fragments,” “variants,” “degenerate variants,” “analogs” and “homologues” or to "chemical derivatives” of the receptor.
  • fragment is meant to refer to any polypeptide subset of receptor.
  • variant is meant to refer to a molecule substantially similar in structure and function to either the entire receptor molecule or to a fragment thereof.
  • a molecule is "substantially similar" to a receptor if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical.
  • analog refers to a molecule substantially similar in function to either the entire receptor molecule or to a fragment thereof.
  • ligand refers to a molecule which binds to the receptor; the term “ligand” includes both agonists and antagonists.
  • Substantial homology when referring to nucleic acids means that the segments or their complementary strands, when optimally aligned and compared, are identical with appropriate nucleotide insertions or deletions, in at least about 30% of the nucleotides, usually >40% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize to a strand or its complement under standard conditions. Standard hybridization conditions are described in Sambrook, J., et al., supra.
  • the terms “substantial homology” and “substantial similarity” are intended to cover minor variation in the DNA sequence which, due to degeneracy in the DNA code, do not result in the sequence encoding a different polypeptide; further these terms are intended to cover alterations in the DNA code which lead to changes in the encoded polypeptide but in which such changes do not affect the biological activity of the peptide.
  • nucleic acids claimed herein may be present in whole cells or in cell lysates or in a partially purified or substantially purified form.
  • isolated and
  • nucleic acid is considered to be isolated and/or substantially purified when it is purified away from environmental contaminants.
  • a nucleic acid sequence isolated from cells is considered to be isolated and/or substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
  • the term “substantially pure” is used relative to nucleic acids, proteins or peptides with which the nucleic acids of the instant invention are associated in nature, and are not intended to exclude compositions in which the nucleic acid of the invention is admixed with nonproteinous pharmaceutical carriers or vehicles.
  • Nucleic acid compositions of this invention may be derived from genomic DNA, cDNA, or RNA, prepared by synthesis or by a combination of techniques.
  • the natural or synthetic nucleic acids encoding the G- protein coupled receptor of the present invention may be incorporated into expression vectors.
  • the expression vectors incorporating the receptor will be suitable for replication in a host.
  • acceptable hosts include, but are not limited to, prokaryotic and eukaryotic cells.
  • recombinant expression system means a substantially homogenous culture of suitable host organisms that stably carry a recombinant expression vector.
  • suitable hosts include, but are not limited to, bacteria, yeast, fungi, insect cells, plant cells and mammalian cells.
  • cells of the expression system are the progeny of a single ancestral transformed cell.
  • the cloned receptor DNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant receptor. Techniques for such manipulations are fully described in Sambrook, J., et al., supra, and are well known in the art.
  • Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells, fungal cells and animal cells.
  • Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells or bacteria-fungi or bacteria-invertebrate cells.
  • An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
  • a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
  • a strong promoter is one which causes mRNAs to be initiated at high frequency.
  • Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
  • mammalian expression vectors may be used to express recombinant receptor in mammalian cells.
  • Commercially available mammalian expression vectors which may be suitable for recombinant receptor expression include but are not limited to, pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), 6/23809 PCI7US96/01444
  • pSG5 (Stratagene), pCI-neo (Promega), EBO-pSV2-neo (ATCC 37593) pBPV- 1(8-2) (ATCC 371 10), pdBPV-MMTneo(342- 12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and ⁇ ZD35 (ATCC 37565).
  • bacterial expression vectors may be used to express recombinant receptor in bacterial cells.
  • Commercially available bacterial expression vectors which may be suitable for recombinant receptor expression include, but are not limited to pGEM-3ZF (Promega), pETl la (Novagen), lambda gtl 1
  • fungal cell expression vectors may be used to express recombinant receptor in fungal cells.
  • Commercially available fungal cell expression vectors which may be suitable for recombinant receptor expression include but are not limited to pYES2 (Invitrogen), Pichia expression vector (Invitrogen).
  • insect cell expression vectors may be used to express recombinant receptor in insect cells.
  • Commercially available insect cell expression vectors which may be suitable for recombinant expression of receptor include but are not limited to pBlue Bac ELI (Invitrogen).
  • An expression vector containing DNA encoding receptor may be used for expression of receptor in a recombinant host cell.
  • Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli.
  • fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines.
  • L cells L-M(TK") ATCC CCL 1.3
  • L cells L-M ATCC CCL 1.2
  • 293 ATCC CRL 1573
  • Raji ATCC CCL 86
  • CV-1 ATCC CCL 70
  • COS-1 ATCC CRL 1650
  • COS-7 ATCC CRL 1651
  • CHO-K1 ATCC CCL 61
  • 3T3 ATCC CCL 92
  • NIH/3T3 ATCC CRL 1658
  • HeLa ATCC CCL 2
  • C127I ATCC CRL 1616
  • BS-C-1 ATCC CCL 26
  • MRC-5 ATCC CCL 171 .
  • the expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, lipofection, protoplast fusion, and electroporation.
  • the expression vector-containing cells are clonally propagated and analyzed to determine whether they produce receptor protein. Identification of receptor expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti -receptor antibodies or specific ligand binding. Expression of receptor DNA may also be performed using in vitro produced synthetic mRNA or native mRNA.
  • Synthetic mRNA or mRNA isolated from receptor producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with micro injection into frog oocytes being preferred.
  • substantially homology when referring to polypeptides, indicates that the polypeptide or protein in question exhibits at least about 30% homology with the naturally occurring protein in question, usually at least about 40% homology.
  • the receptor may be expressed in an appropriate host cell and used to discover compounds that affect the receptor.
  • the receptor is expressed in a mammalian cell line, including but not limited to, COS-7, CHO or L cells, or an insect cell line, including but not limited to, Sf9 or Sf21 , and may be used to discover ligands that bind to the receptor and alter or stimulate its function.
  • the receptor may also be produced in bacterial, fungal or yeast expression systems. The expression of the receptor may be detected by use of a radiolabeled ligand specific for the receptor.
  • such a ligand may be 125j_j 0 d OC y an ⁇ p m( j 0 ⁇ 0 ] (125i_CYP).
  • a ligand may be 12 5 ⁇ _NPY 0 r 125 ⁇ _Pe P tide YY (PYY).
  • the specificity of binding of compounds showing affinity for the receptor is shown by measuring the affinity of the compounds for cells transfected with the cloned receptor or for membranes from these cells. Expression of the cloned receptor and screening for compounds that inhibit the binding of radiolabeled ligand to these cells provides a rational way for rapid selection of compounds with high affinity for the receptor.
  • These compounds identified by the above assays may be agonists or antagonists of the receptor and may be peptides, proteins, or non-proteinaceous organic molecules.
  • functional assays of the receptor may be used to screen for compounds which affect the activity of the receptor. Such functional assays range from ex vivo muscle contraction assays to assays which determine second messenger levels in cells expressing the receptor.
  • the second messenger assays include but are not limited to assays to measure cyclic AMP or calcium levels or assays to measure adenyl cyclase activity.
  • These compounds identified by the above assays may be agonists, antagonists, suppressors, or inducers of the receptor. The functional activity of these compounds is best assessed by using the receptor either natively expressed in tissues or cloned and exogenously expressed.
  • the recombinant receptor is in a well-characterized environment.
  • the membranes from the recombinant cells expressing the receptor are then isolated according to methods known in the art.
  • the isolated membranes or whole cells may be used in a variety of membrane-based or whole cell-based receptor binding assays.
  • Ligands either agonists or antagonists
  • Ligands may be identified by standard radioligand binding assays. These assays will measure the intrinsic affinity of the ligand for the receptor.
  • the activity of receptor ligands or modulators of the receptor may be measured in functional assays as described above.
  • the present invention provides methods of identifying compounds that bind to a novel mammalian NPY Yx receptor.
  • Methods of identifying compounds are exemplified by an assay, comprising: a) cloning DNA which encodes a mammalian neuropeptide Y Yx receptor; b) splicing the DNA into an expression vector to produce a construct such that the NPY Yx receptor is operably linked to transcription and translation signals sufficient to induce expression of the NPY Yx receptor upon introduction of the construct into a prokaryotic or eukaryotic cell; c) introducing the construct into a prokaryotic or eukaryotic cell which does not express the NPY Yx receptor in the absence of the introduced construct; and d) incubating cells or membranes isolated from cells produced in step c with a quantifiable compound known to bind to the NPY Yx receptor, and subsequently adding test compounds at a range of concentrations so that the test compounds compete with the quantifiable
  • the present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding NPY Yx receptor or which modulate the function of NPY Yx receptor protein.
  • Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non- proteinaceous organic molecules.
  • Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding receptor, or the function of receptor protein.
  • Compounds that modulate the expression of DNA or RNA encoding receptor or the function of receptor protein may be detected by a variety of assays.
  • the assay may be a simple "yes/no" assay to determine whether there is a change in expression or function.
  • the assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.
  • the present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RN A encoding the NPY Yx receptor as well as the function of the NPY Yx receptor protein in vivo.
  • Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non- proteinaceous organic molecules.
  • Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding the receptor, or the function of the receptor protein.
  • Compounds that modulate the expression of DNA or RNA encoding the receptor or the function of the receptor protein may be detected by a variety of assays.
  • the assay may be a simple "yes/no" assay to determine whether there is a change in expression or function.
  • the assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.
  • Kits containing receptor DNA, antibodies to receptor, or receptor protein may be prepared. Such kits are used to detect DNA which hybridizes to receptor DNA or to detect the presence of receptor protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic, taxonomic or epidemiological studies.
  • the DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of receptor DNA, receptor RNA or receptor protein.
  • the recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of receptor.
  • Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container.
  • the carrier would further comprise reagents such as recombinant receptor protein or anti-receptor antibodies suitable for detecting receptor.
  • the carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.
  • Compounds identified by the screening methods identified herein are formulated into pharmaceutical compositions according to standard methods.
  • the compounds or pharmaceutical compositions are used either alone or in combination with other compounds or compositions for the treatment of animals (including humans) in need of treatment.
  • Conditions which can be treated with compounds identified by the methods of the present invention include but are not limited to obesity, regulation of appetite, congestive heart failure, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases.
  • animals having a condition, the condition being characterized by factors selected from altered levels of neuropeptide Y, altered activities of neuropeptide Y, altered levels of neuropeptide Y receptor activity, altered neuropeptide Y receptor activity, and combinations thereof, can be treated with compounds or derivatives of compounds (or pharmaceutical compositions comprising the compounds or derivatives of compounds) identified by the screening methods described herein.
  • compositions comprising modulators of receptor activity, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences.
  • a pharmaceutically acceptable composition suitable for effective administration such compositions will contain a pharmacologically effective amount of the protein, DNA, RNA, or modulator.
  • pharmacologically effective amount means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response that is being sought by a researcher or clinician.
  • compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders.
  • the effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
  • the pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
  • derivative or “chemical derivative” describe a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
  • the present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention.
  • the compositions containing compounds identified according to this invention as the active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
  • the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
  • they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • compounds identified by the methods of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • these compounds can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • the individual components of the combination can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms.
  • the instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly.
  • the dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, 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 thereof employed.
  • a physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • 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.
  • a mouse genomic DNA library was constructed using published procedures (Mudgett, J.S. and Machines, M.A. Genomics 8, 623-633(1990)) in the cosmid sCOS vector using genomic DNA isolated from the embryonic stem cell line J l (ES-J1) (Dr. R. Jaenisch, The Whitehead Institute) which was derived from J129 SV mice (J. Mudgett, MRL).
  • the recombinant DNA library constructed from genomic DNA of ES-J 1 cells was plated on Colony/Plaque screen hybridization transfer membrane (Dupont/NEN) at a density of approximately 30,000 colonies per plate.
  • ES-14TG2A mouse pluripotent stem cells can be used in place of the ES-J 1 stem cell line.
  • Replicas of master plates were lysed and processed for hybridization using standard protocols (Sambrook, J., Fritsch, E.F., Maniatis, T. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).
  • the DNA was UV crosslinked to the membrane with a Stratalinker (Stratagene).
  • the filters were incubated overnight at 42°C with radiolabeled probe in 35% formamide hybridization solution, [5 x SSC, 0.02% SDS, 0.1 % n- lauroyl sarcosine, 0.02% (w/v) blocking buffer (Boehringer Mannheim Biochemicals)].
  • the probe a DNA fragment containing coding sequences of the human Y 1 neuropeptide Y receptor from transmembrane domains 3 to 7, was generated by PCR (Perkin Elmer Cetus) using primers 554 (5'
  • Filters containing Pstl fragments cloned from the mouse cosmid library were hybridized to a human NPY Yl receptor cDNA probe.
  • the probe was prepared using a PCR labeling kit (Bethesda Research Labs, Bethesda, MD).
  • the forward primer was (5'
  • the reverse primer was (5' GCATCAAGTG TTACATTTTGGAAC 3') (SEQ. I.D. NO. 4)
  • the template was a 928 bp EcoRI-EcoRV restriction fragment prepared with Qiaex resin (Qiagen Inc., Chatsworth, CA) from an agarose gel slice of restricted and electrophoresed NPY Yl cDNA.
  • the PCR product was a 463 bp fragment spanning transmembrane domains 1 to 4 of the NPY Y 1 receptor.
  • Hybridization was performed using prehybridization and hybridization solutions (5 Prime -> 3 Prime, Inc., Boulder, CO) with an Autoblot oven and bottles (Bellco Glass Co., Vineland, NJ). After a prehybridization at 60°C for 3 hrs, 2x 10? CPM of the heat denatured probe was added to each hybridization bottle and the filters were hybridized at 60°C for 1 hr, then 55°C for 1 1 hrs, and 50°C for 3 hrs.
  • the filters were washed in 2 X SSC, 0.1 % SDS, first at room temperature for 60 min, then at 30°C for 20 min, then at 45 °C for 30 min and the filters were then exposed to Kodak X-OMAT AR film for 4 hrs.
  • Several putative hybridizing colonies were identified and DNA was purified from these clones by Qiaprep system (Qiagen, Inc., Chatsworth, CA), subjected to digestion with EcoRI and EcoRV, separated by electrophoresis in an agarose gel, and transferred to a nylon filter (Zeta-Probe, Bio-Rad Laboratories, Hercules, CA) using the Stratagene Posiblot system.
  • the resulting filters were hybridized to the PCR labeled 463 bp NPY Yl receptor cDNA fragment as described above. After a final stringent wash at 55°C for 45 min in 2 X SSC, 0.1 SDS the filters were then exposed to Kodak X- OMAT AR film for 1 hr.
  • One hybridizing clone, designated ET 15952 and containing plasmid pVE2841 was selected for DNA sequence analysis.
  • DNA was prepared from overnight cultures of ET15952 using the Wizard DNA Purification System (Promega Corp., Madison, WI) and subjected to automated sequence analysis using the PRISM Dye Deoxy terminator cycle sequencing kit (Applied Biosy stems, Foster City, CA). Initial sequencing primers were complementary to the T7 and SP6 promoter sites in pGEM 3ZF, additional primers were made complementary to the insert DNA in pVE2841. Sequencing indicated that pVE2841 contains a 2280 bp PstI insert. FRAMES analysis using the Genetics Computing Group software (Madison, WI) identified a 371 amino acid open reading frame (Fig. 1).
  • Hydrophobicity analysis using PEPPLOT indicates the open reading frame has 7 hydrophobic domains characteristic of the transmembrane spanning domains of G-protein coupled receptors (Fig. 2).
  • the DNA sequence of the PstI fragment and the region encoding the open reading frame are shown in Fig. 3.
  • the DNA encoding the open reading frame appears to lack introns and is 59% identical to mouse NPY Yl receptor cDNA, while the codon sequence of the open reading frame is 52% identical to the mouse NPY Yl receptor.
  • Fig. 4 A comparative alignment determined by BESTFIT (Genetics Computing Group software (Madison, WI)) of the mouse NPY Y 1 receptor and the novel homolog described herein is presented in Fig. 4. In Fig. 4 the seven potential transmembrane spanning domains are indicated as are three putative N-linked glycosylation sites (indicated by a *). EXAMPLE 4
  • CA CA
  • EcoRV restriction enzyme
  • EcoRV EcoRV
  • the 5' ends of the vector were dephosphorylated by adding 2 ⁇ l of an alkaline phosphatase solution (Boehringer Mannheim, Indianapolis, IN) (0.1 U in 10 mM Tris pH7, 10 mM MgCl2, 0.1 mM ZnCl2, 50% glycerol) and incubating at 37°C. After an hour, the enzymes were heat inactivated at 65°C for 10 min.
  • 5 ⁇ g of pVE2841 were digested with restriction enzyme PstI, according to the manufacture's directions (Bethesda Research Labs, Bethesda, MD), in a 40 ⁇ L solution.
  • the 3' overhanging ends of the 2.3 kb PstI fragment were converted to blunt ends by adding 8.8 ⁇ L of 5x T4 DNA Polymerase buffer and 5 U of T4 DNA polymerase (Bethesda Research Labs, Bethesda, MD) and continuing the incubation at 37°C. After an hour, the enzymes were heat inactivated at 65°C for 10 min.
  • the reaction products were separated on a 1 % agarose gel (Sigma Chemical Co., St.
  • the 2.3 kb fragment was ligated to the pcDNA3 vector DNA in a 20 ⁇ L reaction containing 1 ⁇ L of purified vector, 3 ⁇ L of purified 2.3 kb fragment, 4 ⁇ L of 5x T4 DNA ligation buffer (Bethesda Research Labs, Bethesda, MD), 1 1 ⁇ L of water, and 1 ⁇ l of T4 DNA ligase (1U, Bethesda Research Labs, Bethesda. MD). After ligation for 2 hr at 20°C, 1 ⁇ L of the ligation mixture was transformed into 20 ⁇ L of XL 1 Blue competent E. coli cells (Stratagene, La Jolla, CA), according to the manufacturer's directions.
  • Transformants were isolated as ampicillin-resistant colonies and plasmid DNA was isolated from transformants by Qiaprep system (Qiagen Corp. Chatsworth, CA). Among the transformants containing the 2.3 kb insert, one isolate was identified and designated pVE2863 which contained the 2.3 kb in the correct orientation for expression from the CMV promoter.
  • MNPY5FRI (5'GATCGAATTCG CCATGGAAGTGCTCACAAAC 3 ) (SEQ. I.D. NO. 13) and MNPY5RXBA (5'GATCTCTAGACTA TATGCCTGTTGGTATGTG 3 ) (SEQ. I.D. NO. 14) in buffer supplied by the manufacturer.
  • the reactions were carried out for 25 cycles of 95 degrees for 1 minute, 50 degrees for 30 seconds, and 72 degrees for 1 minute 30 seconds.
  • the products of the reaction were resolved on a 1 % agarose gel and the single predominant product was isolated and purified using a Wizard PCR Preps kit (Promega Corp., Madison, WI).
  • the purified PCR fragment was digested in a 50 ⁇ L reaction containing 10 units of EcoRI and 10 units of Xbal according to the manufacturer's directions (Bethesda Research Labs, Bethesda, MD) for 16 hours.
  • the PCR fragment was repurified as before using the Wizard PCR Preps kit and resuspended in 50 ⁇ L of water.
  • the PCR fragment was ligated to the pCI-neo vector DNA in a 10 ⁇ L reaction containing 1 ⁇ L of purified vector, 3 ⁇ L of purified PCR fragment, 2 ⁇ L of 5x T4 DNA ligation buffer (Bethesda Research Labs, Bethesda, MD), 3 ⁇ L of water, and 1 ⁇ L of T4 DNA ligase (1 U, Bethesda Research Labs, Bethesda, MD). After ligation for 16 hr at 12°C, 1 ⁇ L of the ligation mixture was transformed into 50 ⁇ L of Max -Efficiency DH5 alpha competent E.
  • Transformants were isolated as ampicillin-resistant colonies and plasmid DNA was isolated from transformants by Qiaprep system (Qiagen Corp. Chatsworth, CA). Transformants containing the appropriate fragment insert were identified by restriction with EcoRI and Xba I. Fidelity of the Yx gene was verified by automated sequence analysis using the PRISM Dye Deoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). A single clone was selected and designated pCI-mY5.
  • COS-7 cells were transfected with the pcDNA3 or the pCI-neo expression vector containing the genomic DNA for the murine NPY Yx receptor (pVE2863 or pCI-mY5, respectively).
  • pVE2863 or pCI-mY5 Two days post transfection, cells were dissociated with enzyme-free dissociation buffer, centrifuged at 1000 rpm for 10 minutes at 4°C, and resuspended in 10 mM Tris, pH 7.4 containing 0.1 M PMSF (phenylmethylsulfonyl fluoride), 10 ⁇ M phosphoramidon, and 40 ⁇ g/ml bacitracin.
  • PMSF phenylmethylsulfonyl fluoride
  • the cell suspension was homogenized with a glass homogenizer, and centrifuged at 2200 rpm for 10 minutes at 4°C to remove cell debris.
  • the plasma membrane fraction was recovered by centrifuging the supernatant at 18,000 rpm in a Sorvall SS-34 rotor for 15 minutes at 4°C.
  • the membrane pellet was resuspended in the Tris/PMSF/phosphoramidon/bacitracin buffer by trituration 5 times using a 25 g needle and syringe.
  • the membranes were stored frozen at -80°C until use.
  • Membranes (50 ⁇ g protein) were incubated with 1 5 ⁇ _ PYY (90 pM) in the presence or absence of 1 ⁇ M unlabeled PYY (non-specific binding) or serial dilutions of other competing peptides in 0.25 ml of 50 mM Tris, pH 7.4 containing 2 mM CaCl2, 1 mM MgCl2, 5 mM KC1, 0.2% bovine serum albumin, 10 ⁇ M phosphoramidon, 4 ⁇ g/ml leupeptin and 40 ⁇ g/ml bacitracin.
  • a hybridization probe with identity to the human NPY Yx receptor was generated by PCR using human genomic DNA as the target. Oligonucleotide primer pairs based upon the DNA sequence of the mouse NPY Yx receptor (primer 1 ; 5 ACCAGTGGCAAGAGCAACAAC (SEQ. I.D. NO. 8), primer 2; 5'CTCATTGGTGAGGTGGTAGGAC (SEQ. I.D. NO. 9)) were used in a primary PCR reaction. The products of this reaction were used as the template for a second PCR reaction using primer 2 and primer 3
  • Primer 3 a 'nested' primer, was designed to hybridize to mouse NPY Yx receptor sequences which are internal to those with homology to primers 1 and 2.
  • a product obtained from the second PCR reaction was cloned and found to share homology to the mouse NPY Yx receptor by DNA sequence analysis in both directions using an ABI 373A automated sequencing unit (Perkin Elmer).
  • the filters were prewashed with 500 mis of 0.1 X SSC, 0.1 % SDS at 65°C for 1 hour and then prehybridized in a solution of 0.25M NaP ⁇ 4, 7% SDS pH7.5 at 60°C for 1 hour.
  • the PCR fragment having identity to the human NPY Yx receptor described above was labeled by random priming (Rediprime, Amersham) in the presence of [cx-32p]dCTP (3000 Ci/mmole).
  • the membranes were allowed to hybridize to the [r ⁇ -32p]-iabeled PCR fragment in a solution of 0.25M NaP04, 7% SDS, 10% dextran sulfate pH7.5 for 16 hours at 60°C.
  • the membranes were washed in 1 liter of IX SSC, 0.1 % SDS at 45 °C and then exposed to Kodak XAR-5 Xray film. Primary positives identified by this procedure were selected, re-plated, and subjected to a second round of hybridization screening to allow identification and selection of individual hybridization positive plaques. Plasmids containing the cDNA inserts were rescued from the phage using methodology supplied by the manufacturer (Stratagene) which results in the generation of bacterial colonies.
  • Bacterial colonies with plasmids containing the human NPY Yx receptor cDNA sequences were identified by filter hybridization with the [ ⁇ -32p] -labeled human NPY Yx receptor probe using standard methodologies (Sambrook, J., Fritsch, E.F., Maniatis, T. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989) .
  • the cloned cDNA, pCD13 was analyzed by sequencing in both directions using an ABI 373A automated sequencing unit (Perkin Elmer).
  • the DNA sequence of the approximately 1499 bp clone of human NPY Yx receptor cDNA, pCD13 contains an open reading frame of about 11 10 bp.
  • the proposed initiator methionine at nucleotide 182 conforms to the Kozak sequence (M. Kozak, Journal of Cell Biology 108, 229-241 (1989)) at the +4 position but not at the -3 position.
  • the proposed 370 amino acid sequence (SEQ. I.D. NO. 12) encoded by the open reading frame is 82% identical to the amino acid sequence encoded by the open reading frame of the mouse NPY Yx receptor clone.
  • the human NPY Yx receptor cDNA is 78% identical to the DNA of the mouse NPY Yx receptor.
  • the human NPY Yx receptor cDNA described herein contains 181 untranslated nucleotides at the 5 prime end and 208 nucleotides following the stop codon.
  • CA CA
  • EcoRI restriction enzyme
  • the DNA was resolved by electrophoresis on a 1% agarose gel (Sigma Chemical Co., St. Louis MO) and the linearized vector was identified and excised from the gel.
  • the DNA was purified by Gene Clean using the procedure supplied by the manufacturer (BIO 101 Inc., Vista, CA).
  • the 5' ends of the vector were dephosphorylated by adding 1 ⁇ l (0.1 unit) of an alkaline phosphatase solution (Boehringer Mannheim, Indianapolis, IN) in 50 ⁇ l of 10 mM Tris pH7, 10 mM MgCl2, 0.1 mM ZnCl2, 50% glycerol and incubating at 37°C for 1 hour.
  • the DNA was subjected to a second round of Gene Clean purification and resuspended in 50 ⁇ l of TE (10 mM Tris pH 7.4, 1 mM EDTA).
  • Eco RI Eco RI
  • the 1.6 kb fragment was ligated to the linearized pcDNA3 vector DNA in a 10 ⁇ l reaction containing 1 ⁇ l of purified vector, 3 ⁇ l of purified 1.6 kb fragment, 1 ⁇ l of lOx T4 DNA ligation buffer (Boehringer Mannheim, Indianapolis, IN), 5 ⁇ l of water, and 1 ⁇ l of T4 DNA ligase (1 U, Boehringer Mannheim, Indianapolis, IN). After ligation for 2 hr at 20°C, 1 ⁇ l of the ligation mixture was transformed into 50 ⁇ l of Max Efficiency DH5alpha competent E. coli cells (Bethesda Research Labs, Bethesda, MD), according to the manufacturer's directions.
  • Transformants were isolated as ampicillin-resistant colonies and plasmid DNA was isolated from transformants using the WIZARD miniprep system (Promega Corporation, Madison WI). Among the transformants containing the 1.6kb insert, one isolate was identified and designated pcDNA3HYx-RI, which contained the 1.6 kb in the correct orientation for expression from the CMV promoter.
  • the approximately 1.6kb fragment containing the receptor coding sequences was purified by Gene Clean and resuspended in 50 ⁇ l of TE.
  • the 1.6 kb fragment was ligated to the pcDNA3 vector DNA in a 10 ⁇ l reaction containing 1 ⁇ l of purified vector, 3 ⁇ l of purified 1.6 kb fragment, 1 ⁇ l of lOx T4 DNA ligation buffer (Boehringer Mannheim, Indianapolis, IN), 5 ⁇ l of water, and 1 ⁇ l of T4 DNA ligase (1U, Boehringer Mannheim, Indianapolis, IN).
  • Recombinant receptor is produced in a bacterial expression system such as coli.
  • the receptor expression cassette is transferred into an coli expression vector; expression vectors include but are not limited to, the pET series (Novagen).
  • the pET vectors place receptor expression under control of the tightly regulated bacteriophage T7 promoter.
  • expression of receptor is induced by addition of an appropriate lac substrate (IPTG) added to the culture.
  • IPTG lac substrate
  • Baculovirus vectors derived from the genome of the AcNPV virus are designed to provide high level expression of DNA in the Sf9 line of insect cells (ATCC CRL# 171 1).
  • Recombinant baculovirus expressing receptor DNA is produced by the following standard methods (I Vitrogen Maxbac Manual): the receptor DNA constructs are ligated into the polyhedrin gene in a variety of baculovirus transfer vectors, including the pAC360 and the BlueBac vector (InVitrogen).
  • Recombinant baculoviruses are generated by homologous recombination following co-transfection of the baculovirus transfer vector and linearized AcNPV genomic DNA [Kitts, P.A., Nuc. Acid. Res.
  • Recombinant pAC360 viruses are identified by the absence of inclusion bodies in infected cells and recombinant pBlueBac viruses are identified on the basis of B-galactosidase expression (Summers, M. D. and Smith, G. E., Texas Agriculture Exp. Station Bulletin No. 1555).
  • receptor expression is measured by assays described herein. Authentic receptor is found in association with the infected cells. Active receptor is extracted from infected cells by hypotonic or detergent lysis.
  • the receptor is expressed in the Drosophila Schneider 2 cell line by cotransfection of the Schneider 2 cells with a vector containing the receptor DNA downstream and under control of an inducible metallothionin promoter, and a vector encoding the G418 resistant neomycin gene. Following growth in the presence of G418, resistant cells are obtained and induced to express receptor by the addition of Q1SO4. Identification of modulators of the receptor is accomplished by assays using either whole cells or membrane preparations.
  • EXAMPLE 1 Cloning of NPY Yx Receptor DNA into a Yeast Expression Vector Recombinant receptor is produced in the yeast cerevisiae following the insertion of the receptor DNA cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins.
  • vectors such as EmBLyex4 or the like are ligated to the receptor cistron [Rinas, U. et al., Biotechnology 8, 543-545 (1990); Horowitz B. et al., J. Biol. Chem. 265, 4189-4192 (1989)].
  • the receptor cistron is ligated into yeast expression vectors which fuse a secretion signal. The levels of expressed receptor are determined by the assays described herein.
  • EXAMPLE 12 Purification of Recombinant NPY Yx Receptor
  • Recombinantly produced receptor may be purified by a variety of procedures, including but not limited to antibody affinity chromatography.
  • Receptor antibody affinity columns are made by adding the anti-receptor antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1 M ethanolamine HC1 (pH 8). The column is washed with water followed by 0.23 M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
  • the column is then equilibrated in phosphate buffered saline (pH 7.3) together with appropriate membrane solubilizing agents such as detergents, and the cell culture supernatants or cell extracts containing solubilized receptor or receptor subunits are slowly passed through the column.
  • the column is then washed with phosphate-buffered saline (PBS) supplemented with detergents until the optical density (A280) falls to background; then the protein is eluted with 0.23 M glycine-HCl (pH 2.6) supplemented with detergents.
  • PBS phosphate-buffered saline
  • the purified receptor protein is then dialyzed against PBS.
  • Recombinant cells containing DNA encoding the novel murine or human NPY Yx receptor, membranes derived from the recombinant cells, or recombinant receptor preparations derived from the cells or membranes may be used to identify compounds that modulate the murine or human NPY Yx receptor activity. Modulation of such activity may occur at the level of DNA, RNA, protein or combinations thereof.
  • One method of identifying compounds that modulate NPY Yx receptor activity or modulate binding of ligands to the protein comprises:
  • This screening assay will detect a compound that has affinity for the NPY Yx receptor.
  • Such compounds may be either agonists or antagonists and may be peptides, proteins, or non- proteinaceous organic molecules.
  • Another method of identifying compounds that modulate the NPY Yx receptor comprises:
  • the cells are transformed by an expression vector containing a sequence selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3, a fragment of the sequence of substantial homology to the sequence shown in Figure 3, a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5.
  • the expression vector contains a DNA sequence selected from a DNA sequence having a sequence selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3 or a fragment of the sequence of substantial homology to the sequence shown in Figure 3.
  • the expression vector contains a DNA sequence selected from the sequence shown in Figure 3, the sequence of substantial homology to the sequence shown in Figure 3 or the fragment of the sequence shown in Figure 3 comprising bases 822 to 1934.
  • the expression vector contains a DNA sequence selected from a DNA sequence having a sequence selected from a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5.
  • the expression vector contains a DNA sequence selected from the sequence shown in Figure 5, the sequence of substantial homology to the sequence shown in Figure 5 or the fragment of the sequence shown in Figure 5 comprising bases 182 to 1291.
  • This screening assay will detect a compound which modulates NPY Yx receptor activity.
  • Such compounds may be either agonists, antagonists, suppressors or inducers.
  • Such compounds may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • CTGCAGTCTA TTGGATGAAG AGTGTACATA TTCATATAAT TCTTAAAGTA GGCAGAAATT 60
  • AACAAAACAA AACCTTCTAC TAATATTCTG GATTCTGTTT GATTTTTAGG ATCTCAAGAG 660
  • CTTTCTTTTTTT TTCCTTTGGA AGAATAATAA CTTAAACAAC CTAGACATCA TTACTGAGGA 2177 AGAGAACAAA AATGAGAGAG CATACAAGGA CAGCAGAGAT GTCTGGGGTA CAAAATTCAC 2237 GTTATTCGCT GGAATAGCTA GAAAGTTATT AGTTGTGCTG CAG 2280
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • CTCTGTGTCC ATATTCTCAC TTGTATTCAC TGCTGTCGAA AGATATCAGC TAATTGTGAA 600

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Abstract

A novel mammalian neuropeptide Y receptor and method of making the receptor are provided. The invention includes DNA encoding the receptor, assays employing the receptor, cells expressing the receptor, antibodies which bind specifically to the receptor, RNA encoded by the DNA sequence or its complementary sequence, and single-stranded DNA with a sequence complementary to the RNA which encodes the receptor. The receptor and assays employing the receptor are useful for identifying compounds which bind to the receptor, including specific modulators of the receptor. Such compounds are useful for treating a variety of disease conditions, including obesity, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases.

Description

TITLE OF THE INVENTION NEUROPEPTIDE Y RECEPTOR
BACKGROUND OF THE INVENTION This application is a continuation-in-part of copending application U.S. Serial No. 08/415,818, filed April 3, 1995, which is a continuation-in-part of copending application U.S. Serial No. 08/383,746, filed February 3, 1995, the contents of both of which are hereby incorporated by reference. Neuropeptide Y (NPY) is a 36 residue, amidated polypeptide. It is anatomically co-distributed and co-released with norepinephrine in and from sympathetic postganglionic neurons ([1], [2], [3], [4], [5], [6]). Stimulation of the sympathetic nervous system under physiological circumstances such as exercise ([7], [8]) or exposure to the cold ([9], [10]) promotes an elevation of both norepinephrine and NPY.
NPY is believed to act in the regulation of appetite control ([ 1 1], [12]) and vascular smooth muscle tone ([13], [14]) as well as regulation of blood pressure ([6], [15], [16], [17]). NPY also decreases cardiac contractility ([18], [19], [20], [21], [22]). Congestive heart failure and cardiogenic shock are associated with probable releases of NPY into the blood ([23], [24], [25]). Regulation of NPY levels may be beneficial to these disease states [26].
At the cellular level, neuropeptide Y binds to a G -protein coupled receptor ([27], [28], [29], [30]). Neuropeptide Y is involved in regulating eating behavior and is an extremely potent orixigenic agent ([1 1], [12], [31]). When administered intracerebroventricularly or injected into the hypothalamic paraventricular nucleus (PVN) it elicits eating in satiated rats ([32], [33], [34]) and intraventricular injection of antisera to NPY decreases eating ([11 ], [31]). It has been shown to stimulate appetite in a variety of species and at different stages of development ([12]). Other effects on energy metabolism include decreased thermogenesis, body temperature and uncoupling protein, and increased white fat storage and lipoprotein lipase activity ([9], [35], [36], [37], [38], [39]). NPY levels in the PVN increase upon fasting ([40], [41], [42], [43], [44]), before a scheduled meal ([31], [36], [40]), and in both streptozotocin-induced and spontaneous diabetes ([36], [45], [46], [47], [48], [49]). Also, NPY levels are increased in genetically obese and hyperphagic Zucker rats ([36], [50], [51]). Thus, a specific centrally acting antagonist for the appropriate NPY receptor subtype may be therapeutically useful for treating obesity and diabetes. Other disorders which might be targeted therapeutically include anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases ([26], [52]).
At least four receptor subtypes of the NPY family have been proposed based on pharmacological and physiological properties. The Y 1 receptor is stimulated by NPY or PYY (peptide YY) and appears to be the major vascular receptor ([16], [53], [54], [55]). The Y2 receptor is stimulated by C-terminal fragments of NPY or PYY and is abundantly expressed both centrally and peripherally ([55], [56], [57], [58]). A third receptor (Y3) is exclusively responsive to NPY and is likely present in adrenal medulla, heart, and brain stem ([27], [59]). In addition, other subtypes of this receptor family are known to exist, based on pharmacological and physiological characterization ([60], [61]. [62], [63]). The feeding behavior is stimulated potently by NPY, NPY2- 36 and the Yl agonist [Leu31, Pro34]NPY, but is not stimulated by the Y2 agonist NPY 13-36 ([1 1], [64], [65], [66]). This pharmacology is not characteristic of the defined Yl, Y2 or Y3 receptors and can thus be attributed to a unique receptor, termed "atypical Yl " ([ 11], [65], [66]), that is responsible for evoking the feeding response. In addition, data indicate the existence of additional members of this receptor family including one subtype specific for peptide PP ([62], [63]), one with affinity for short C-terminal fragments of NPY which induce hypotension when administered systemically ([15], [17], [30], [67], [68]), and one associated with binding of NPY and PYY to brain sigma and phencyclidine binding sites ([61]). The Y 1 receptor has been cloned and shown to be a G- protein coupled receptor ([53], [69], [70]). Recently, the Y2 receptor has been cloned ({71], [72]). In addition a peptide PP preferring receptor, termed PP1 ([73]) or Y4 [(74)], has been cloned. Until the invention described herein of a novel NPY receptor, other NPY receptors had not been cloned.
References
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SUMMARY OF THE INVENTION
The present invention relates to an isolated DNA sequence encoding a novel neuropeptide Y receptor, hereinafter referred to as the neuropeptide Y Yx receptor. The DNA sequence encoding the NPY Yx receptor has a sequence selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3, a fragment of the sequence of substantial homology to the sequence shown in Figure 3, a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5.
In one embodiment of the invention is the isolated DNA sequence wherein the sequence is selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3 or a fragment of the sequence of substantial homology to the sequence shown in Figure 3.
In a class of the invention is the DNA sequence which encodes a murine NPY Yx receptor. In a subclass of the invention is the DNA sequence having a sequence selected from the sequence shown in Figure 3, the sequence of substantial homology to the sequence shown in Figure 3 or the fragment of the sequence shown in Figure 3 comprising bases 822 to 1934.
In a second embodiment of the invention is the isolated DNA sequence wherein the DNA sequence has a sequence selected from a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5. In a second class of the invention is the DNA sequence which encodes a human NPY Yx receptor.
In a second subclass is the DNA sequence having a sequence selected from the sequence shown in Figure 5, the sequence of substantial homology to the sequence shown in Figure 5 or the fragment of the sequence shown in Figure 5 comprising bases 182 to 1291.
Illustrative of the invention is an expression vector containing any of the DNA sequences described above.
An illustration of the invention is a cell transformed by the expression vector. Exemplifying the invention is a method of producing the neuropeptide Y Yx receptor, comprising culturing the cell under conditions which allow the production of the neuropeptide Y Yx receptor and optionally recovering the neuropeptide Y Yx receptor. An example of the invention is a neuropeptide Y Yx receptor produced by this process. Preferably, the neuropeptide Y Yx receptor produced by this process is characterized by a pharmacological binding profile with affinities of PYY = NPY = [Leu31pr034]NPY = NPY (2-36) > NPY(13- 36). More specifically exemplifying the invention is the method wherein the cell is a mammalian cell; preferably, a COS-7 cell.
Further illustrating the invention is a neuropeptide Y Yx receptor, or a functional derivative thereof, which is characterized by a pharmacological binding profile with affinities of PYY ≡ NPY ≡ [Leu31Pro34]NPY = NPY(2-36) > NPY( 13-36), in substantially pure form.
More particularly illustrating the invention is an antibody immunologically reactive with the NPY Yx receptor.
Another illustration of the invention is an isolated RNA encoded by any of the DNA sequences described above or their complementary sequences.
Since a single-stranded DNA with a sequence complementary to the sequence of RNA which encodes the NPY Yx receptor could be used to modulate the expression of the receptor, another example of the invention is isolated DNA containing this complementary sequence or a fragment thereof.
Further exemplifying the invention is a neuropeptide Y Yx receptor in substantially pure form comprising an amino acid sequence selected from a sequence as shown in Figure 3 (SEQ. I.D. NO. 6), a sequence as shown in Figure 6 (SEQ. I.D. NO. 12), or a functional derivative thereof. Preferably, the neuropeptide Y Yx receptor has an amino acid sequence as shown in Figure 3 (SEQ. I.D. NO. 6) or a functional derivative thereof. More preferably, the neuropeptide Y Yx receptor has an amino acid sequence as shown in Figure 6 (SEQ. I.D. NO. 12) or a functional derivative thereof. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Schematic diagram of FRAMES analysis using the Genetics Computing Group software (Madison, WI) identifying a 371 amino acid open reading frame (aa ORF). Figure 2. Schematic diagram of hydrophobicity analysis using PEPPLOT (Genetics Computing Group software (Madison, WI)) indicating the open reading frame (ORF) has 7 transmembrane spanning domains characteristic of G-protein coupled receptor.
Figure 3 shows the genomic DNA sequence and deduced amino acid sequence of the murine NPY Yx receptor. The sequence disclosure of Fig. 3 is represented as SEQ- I.D. NO. 5 and SEQ. I.D. NO. 6.
Figure 4 shows a comparative alignment determined by BESTFIT (Genetics Computing Group software (Madison, WI)) of the mouse NPY Yl receptor and the mouse NPY Yx receptor, described herein. The seven transmembrane spanning domains are indicated as are three putative N-linked glycosylation sites (indicated by a *). The sequence disclosures of the mouse NPY Yl receptor and mouse NPY Yx receptor of Fig. 4 are represented as SEQ. I.D. NO. 7 and SEQ. I.D. NO. 6, respectively.
Figure 5 shows the DNA sequence of the human NPY Yx receptor. The sequence disclosure of Fig. 5 is represented as SEQ. I.D. NO. 1 1.
Figure 6 shows the amino acid sequence of the human NPY Yx receptor. The sequence disclosure of Fig. 6 is represented as SEQ. I.D. NO. 12. DETAILED DESCRIPTION OF THE INVENTION
Neuropeptide Y receptors belong to a class of receptors known as "G-protein coupled receptors." The term "G-protein coupled receptor" refers to any receptor protein that mediates its endogenous signal transduction through activation of one or more guanine nucleotide binding regulatory proteins (G-proteins). These receptors share common structural features, including seven hydrophobic transmembrane domains. G-protein coupled receptors include receptors that bind to small biogenic amines, including but not limited to beta- adrenergic receptors (βAR), alpha-adrenergic receptors (αAR) and muscarinic receptors, as well as receptors whose endogenous ligands are peptides, such as neurokinin, neuropeptide Y and glucagon receptors. Examples of βAR include beta-1, beta-2, and beta-3 adrenergic receptors. G-protein coupled receptors are cell surface proteins that mediate the responses of a cell to a variety of environmental signals. Upon binding an agonist, the receptor interacts with one or more specific G-proteins, which in turn regulate the activities of specific effector proteins. By this means, activation of G-protein coupled receptors amplifies the effects of the environmental signal and initiates a cascade of intracellular events that ultimately leads to defined cellular responses. G-protein coupled receptors function as a complex information processing network within the plasma membrane of the cell, acting to coordinate a cell's response to multiple environmental signals.
G-protein coupled receptors consist of seven hydrophobic domains connecting eight hydrophilic domains. The hydrophobicity or hydrophilicity of the domains may be determined by standard hydropathy profiles, such as Kyte-Doolittle analysis (Kyte, J. and Doolittle, R.J.F. J. Mol. Biol. 157: 105 (1982)). The receptors are thought to be oriented in the plasma membrane of the cell such that the N-terminus of the receptor faces the extracellular space and the C- terminus of the receptor faces the cytoplasm, so that each of the hydrophobic domains crosses the plasma membrane. The receptors have been modeled and the putative boundaries of the extracellular, transmembrane and intracellular domains are generally agreed (for a review, see Baldwin, EMBO J. 12:1693, 1993). In general, the transmembrane domains are comprised of stretches of 20-25 amino acids in which most of the amino acid residues have hydrophobic side chains (including cysteine, methionine, phenylalanine, tyrosine, tryptophan, proline, glycine, alanine, valine, leucine, isoleucine), whereas the intracellular and extracellular loops are defined by contiguous stretches of several amino acids that have hydrophilic or polar side chains (including aspartate, glutamate, asparagine, glutamine, serine, threonine, histidine, lysine, and arginine). Polar amino acids, especially uncharged ones (such as serine, threonine, asparagine, and glutamine) are found in both transmembrane and extramembrane regions. The present invention pertains to a novel mammalian neuropeptide Y receptor subtype (i.e., NPY Yx), particularly exemplified by the murine and human neuropeptide Y Yx receptors described in detail herein. A method of making the NPY Yx receptor is also provided. The invention includes DNA encoding the NPY Yx receptor, the NPY Yx receptor, assays employing the NPY Yx receptor, cells expressing the NPY Yx receptor, antibodies which bind specifically to the NPY Yx receptor, RNA encoded by the DNA sequence or its complementary sequence, and single-stranded DNA with a sequence complementary to the RNA which encodes the NPY Yx receptor. The NPY Yx receptor and assays employing the NPY Yx receptor are useful for identifying compounds which bind to the receptor, including specific modulators of the receptor. Modulators, as described herein, include but are not limited to agonists, antagonists, suppressors and inducers. Such modulators may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules. Modulators identified by the processes described herein are useful as therapeutic agents. Thus, compounds identified using the mammalian NPY Yx receptor are useful for treating one or more disease conditions, including obesity, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases.
The receptor may include genetic variants, both natural and induced. Induced receptors may be derived by a variety of methods, including but not limited to, site-directed mutagenesis. Techniques for nucleic acid and protein manipulation are well-known in the art and are described generally in Methods in Enzymology and in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989).
It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA sequences which contain alternative codons which code for the eventual translation of the identical amino acid. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate functional properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide. Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand. A fragment of a DNA sequence, as used herein, refers to any polynucleotide subset of the DNA sequence or its complementary sequence. Also included within the scope of the present invention are fragments of DNA sequence capable of producing functional NPY Yx protein. An example of one such fragment capable of expressing the NPY Yx receptor protein comprises bases 822 to 1934 of the DNA sequence shown in Figure 3. Another example of a fragment capable of expressing the NPY Yx receptor protein comprises bases 182 to 1291 of the DNA sequence shown in Figure 5. As used herein, a "functional derivative" of a receptor is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of the receptor. The term "functional derivative" is intended to include the "fragments," "variants," "degenerate variants," "analogs" and "homologues" or to "chemical derivatives" of the receptor. The term "fragment" is meant to refer to any polypeptide subset of receptor. The term "variant" is meant to refer to a molecule substantially similar in structure and function to either the entire receptor molecule or to a fragment thereof. A molecule is "substantially similar" to a receptor if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical.
The term "analog" refers to a molecule substantially similar in function to either the entire receptor molecule or to a fragment thereof.
The term "ligand," as used herein, refers to a molecule which binds to the receptor; the term "ligand" includes both agonists and antagonists.
"Substantial homology" or "substantial similarity," when referring to nucleic acids means that the segments or their complementary strands, when optimally aligned and compared, are identical with appropriate nucleotide insertions or deletions, in at least about 30% of the nucleotides, usually >40% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize to a strand or its complement under standard conditions. Standard hybridization conditions are described in Sambrook, J., et al., supra. Thus, the terms "substantial homology" and "substantial similarity" are intended to cover minor variation in the DNA sequence which, due to degeneracy in the DNA code, do not result in the sequence encoding a different polypeptide; further these terms are intended to cover alterations in the DNA code which lead to changes in the encoded polypeptide but in which such changes do not affect the biological activity of the peptide.
The nucleic acids claimed herein may be present in whole cells or in cell lysates or in a partially purified or substantially purified form. When referring to nucleic acids, the terms "isolated" and
"substantially pure" are synonymous. A nucleic acid is considered to be isolated and/or substantially purified when it is purified away from environmental contaminants. Thus, a nucleic acid sequence isolated from cells is considered to be isolated and/or substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors. The term "substantially pure" is used relative to nucleic acids, proteins or peptides with which the nucleic acids of the instant invention are associated in nature, and are not intended to exclude compositions in which the nucleic acid of the invention is admixed with nonproteinous pharmaceutical carriers or vehicles.
Nucleic acid compositions of this invention may be derived from genomic DNA, cDNA, or RNA, prepared by synthesis or by a combination of techniques.
The natural or synthetic nucleic acids encoding the G- protein coupled receptor of the present invention may be incorporated into expression vectors. Usually the expression vectors incorporating the receptor will be suitable for replication in a host. Examples of acceptable hosts include, but are not limited to, prokaryotic and eukaryotic cells.
The phrase "recombinant expression system" as used herein means a substantially homogenous culture of suitable host organisms that stably carry a recombinant expression vector. Examples of suitable hosts include, but are not limited to, bacteria, yeast, fungi, insect cells, plant cells and mammalian cells. Generally, cells of the expression system are the progeny of a single ancestral transformed cell. The cloned receptor DNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant receptor. Techniques for such manipulations are fully described in Sambrook, J., et al., supra, and are well known in the art.
Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells, fungal cells and animal cells.
Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells or bacteria-fungi or bacteria-invertebrate cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. A variety of mammalian expression vectors may be used to express recombinant receptor in mammalian cells. Commercially available mammalian expression vectors which may be suitable for recombinant receptor expression, include but are not limited to, pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), 6/23809 PCI7US96/01444
- 20 -
pSG5 (Stratagene), pCI-neo (Promega), EBO-pSV2-neo (ATCC 37593) pBPV- 1(8-2) (ATCC 371 10), pdBPV-MMTneo(342- 12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and λZD35 (ATCC 37565).
A variety of bacterial expression vectors may be used to express recombinant receptor in bacterial cells. Commercially available bacterial expression vectors which may be suitable for recombinant receptor expression include, but are not limited to pGEM-3ZF (Promega), pETl la (Novagen), lambda gtl 1
(Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia).
A variety of fungal cell expression vectors may be used to express recombinant receptor in fungal cells. Commercially available fungal cell expression vectors which may be suitable for recombinant receptor expression include but are not limited to pYES2 (Invitrogen), Pichia expression vector (Invitrogen).
A variety of insect cell expression vectors may be used to express recombinant receptor in insect cells. Commercially available insect cell expression vectors which may be suitable for recombinant expression of receptor include but are not limited to pBlue Bac ELI (Invitrogen).
An expression vector containing DNA encoding receptor may be used for expression of receptor in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli. fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK") (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171 ).
The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, lipofection, protoplast fusion, and electroporation. The expression vector-containing cells are clonally propagated and analyzed to determine whether they produce receptor protein. Identification of receptor expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti -receptor antibodies or specific ligand binding. Expression of receptor DNA may also be performed using in vitro produced synthetic mRNA or native mRNA. Synthetic mRNA or mRNA isolated from receptor producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with micro injection into frog oocytes being preferred.
The term "substantial homology", when referring to polypeptides, indicates that the polypeptide or protein in question exhibits at least about 30% homology with the naturally occurring protein in question, usually at least about 40% homology.
The receptor may be expressed in an appropriate host cell and used to discover compounds that affect the receptor. Preferably, the receptor is expressed in a mammalian cell line, including but not limited to, COS-7, CHO or L cells, or an insect cell line, including but not limited to, Sf9 or Sf21 , and may be used to discover ligands that bind to the receptor and alter or stimulate its function. The receptor may also be produced in bacterial, fungal or yeast expression systems. The expression of the receptor may be detected by use of a radiolabeled ligand specific for the receptor. For example, for the β2 adrenergic receptor, such a ligand may be 125j_j0dOCyanθpm(j0ι0] (125i_CYP). For the NPY receptor, such a ligand may be 125ι_NPY 0r 125ι_PePtide YY (PYY). The specificity of binding of compounds showing affinity for the receptor is shown by measuring the affinity of the compounds for cells transfected with the cloned receptor or for membranes from these cells. Expression of the cloned receptor and screening for compounds that inhibit the binding of radiolabeled ligand to these cells provides a rational way for rapid selection of compounds with high affinity for the receptor. These compounds identified by the above assays may be agonists or antagonists of the receptor and may be peptides, proteins, or non-proteinaceous organic molecules. Alternatively, functional assays of the receptor may be used to screen for compounds which affect the activity of the receptor. Such functional assays range from ex vivo muscle contraction assays to assays which determine second messenger levels in cells expressing the receptor. The second messenger assays include but are not limited to assays to measure cyclic AMP or calcium levels or assays to measure adenyl cyclase activity. These compounds identified by the above assays may be agonists, antagonists, suppressors, or inducers of the receptor. The functional activity of these compounds is best assessed by using the receptor either natively expressed in tissues or cloned and exogenously expressed.
Once the receptor is cloned and expressed in a mammalian cell line, such as COS-7 cells or CHO cells, the recombinant receptor is in a well-characterized environment. The membranes from the recombinant cells expressing the receptor are then isolated according to methods known in the art. The isolated membranes or whole cells may be used in a variety of membrane-based or whole cell-based receptor binding assays. Ligands (either agonists or antagonists) may be identified by standard radioligand binding assays. These assays will measure the intrinsic affinity of the ligand for the receptor. In addition, the activity of receptor ligands or modulators of the receptor may be measured in functional assays as described above.
The present invention provides methods of identifying compounds that bind to a novel mammalian NPY Yx receptor. Methods of identifying compounds are exemplified by an assay, comprising: a) cloning DNA which encodes a mammalian neuropeptide Y Yx receptor; b) splicing the DNA into an expression vector to produce a construct such that the NPY Yx receptor is operably linked to transcription and translation signals sufficient to induce expression of the NPY Yx receptor upon introduction of the construct into a prokaryotic or eukaryotic cell; c) introducing the construct into a prokaryotic or eukaryotic cell which does not express the NPY Yx receptor in the absence of the introduced construct; and d) incubating cells or membranes isolated from cells produced in step c with a quantifiable compound known to bind to the NPY Yx receptor, and subsequently adding test compounds at a range of concentrations so that the test compounds compete with the quantifiable compound for the NPY Yx receptor, such that an IC50 for the test compound is obtained as the concentration of test compound at which 50% of the quantifiable compound becomes displaced from the NPY Yx receptor.
The present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding NPY Yx receptor or which modulate the function of NPY Yx receptor protein. Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non- proteinaceous organic molecules. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding receptor, or the function of receptor protein. Compounds that modulate the expression of DNA or RNA encoding receptor or the function of receptor protein may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.
The present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RN A encoding the NPY Yx receptor as well as the function of the NPY Yx receptor protein in vivo. Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non- proteinaceous organic molecules. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding the receptor, or the function of the receptor protein. Compounds that modulate the expression of DNA or RNA encoding the receptor or the function of the receptor protein may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.
Kits containing receptor DNA, antibodies to receptor, or receptor protein may be prepared. Such kits are used to detect DNA which hybridizes to receptor DNA or to detect the presence of receptor protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic, taxonomic or epidemiological studies. The DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of receptor DNA, receptor RNA or receptor protein. The recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of receptor. Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant receptor protein or anti-receptor antibodies suitable for detecting receptor. The carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.
Compounds identified by the screening methods identified herein are formulated into pharmaceutical compositions according to standard methods. The compounds or pharmaceutical compositions are used either alone or in combination with other compounds or compositions for the treatment of animals (including humans) in need of treatment. Conditions which can be treated with compounds identified by the methods of the present invention include but are not limited to obesity, regulation of appetite, congestive heart failure, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases. Thus, animals (including humans) having a condition, the condition being characterized by factors selected from altered levels of neuropeptide Y, altered activities of neuropeptide Y, altered levels of neuropeptide Y receptor activity, altered neuropeptide Y receptor activity, and combinations thereof, can be treated with compounds or derivatives of compounds (or pharmaceutical compositions comprising the compounds or derivatives of compounds) identified by the screening methods described herein.
Pharmaceutically useful compositions comprising modulators of receptor activity, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain a pharmacologically effective amount of the protein, DNA, RNA, or modulator. The term "pharmacologically effective amount," as used herein, means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response that is being sought by a researcher or clinician.
Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
The terms "derivative" or "chemical derivative" describe a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages. Alternatively, co- administration or sequential administration of other agents may be desirable.
The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds identified according to this invention as the active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
Advantageously, compounds identified by the methods of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, these compounds can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
For combination treatment with more than one active agent, the individual components of the combination can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly.
The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, 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 thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. 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 following examples are provided to further define the invention without, however, limiting the invention to the particulars of the examples. EXAMPLE 1
Genomic DNA Cloning of a NPY Yx Receptor
A mouse genomic DNA library was constructed using published procedures (Mudgett, J.S. and Machines, M.A. Genomics 8, 623-633(1990)) in the cosmid sCOS vector using genomic DNA isolated from the embryonic stem cell line J l (ES-J1) (Dr. R. Jaenisch, The Whitehead Institute) which was derived from J129 SV mice (J. Mudgett, MRL). The recombinant DNA library constructed from genomic DNA of ES-J 1 cells was plated on Colony/Plaque screen hybridization transfer membrane (Dupont/NEN) at a density of approximately 30,000 colonies per plate. Alternatively, ES-14TG2A mouse pluripotent stem cells (ATCC CRL1821) can be used in place of the ES-J 1 stem cell line. Replicas of master plates were lysed and processed for hybridization using standard protocols (Sambrook, J., Fritsch, E.F., Maniatis, T. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). The DNA was UV crosslinked to the membrane with a Stratalinker (Stratagene). The filters were incubated overnight at 42°C with radiolabeled probe in 35% formamide hybridization solution, [5 x SSC, 0.02% SDS, 0.1 % n- lauroyl sarcosine, 0.02% (w/v) blocking buffer (Boehringer Mannheim Biochemicals)]. The probe, a DNA fragment containing coding sequences of the human Y 1 neuropeptide Y receptor from transmembrane domains 3 to 7, was generated by PCR (Perkin Elmer Cetus) using primers 554 (5'
CACTGGGTCTTTGGTGAGGCGATGTG 3 ) (SEQ. I.D. NO. 1) and 1300 (5* CCCATAAAATATGGGGTTGACACAAGTGG 3 ) (SEQ. I.D. NO. 2) with human Yl cDNA as template and in the presence of [α-32p] dCTP (deoxycytidinetriphosphate) (3000 Ci/mmole). Filters were washed at a final stringency of 0.5 x SSC, 0.1 % SDS at 42°C.
Positives were rescreened to isolate single colonies. DNA was prepared from positive colonies, digested with restriction enzymes, and Southern blot analysis was done to identify restriction fragments for subcloning. A fragment identified by this hybridization was subcloned into pGEM- 3ZF (Promega).
EXAMPLE 2
Isolation of ET15952 (pVE2841)
Filters containing Pstl fragments cloned from the mouse cosmid library were hybridized to a human NPY Yl receptor cDNA probe. The probe was prepared using a PCR labeling kit (Bethesda Research Labs, Bethesda, MD). The forward primer was (5'
TTGGCCATGATATTTACCTTAGCT 3') (SEQ. I.D. NO. 3), the reverse primer was (5' GCATCAAGTG TTACATTTTGGAAC 3') (SEQ. I.D. NO. 4), and the template was a 928 bp EcoRI-EcoRV restriction fragment prepared with Qiaex resin (Qiagen Inc., Chatsworth, CA) from an agarose gel slice of restricted and electrophoresed NPY Yl cDNA. The PCR product was a 463 bp fragment spanning transmembrane domains 1 to 4 of the NPY Y 1 receptor. Hybridization was performed using prehybridization and hybridization solutions (5 Prime -> 3 Prime, Inc., Boulder, CO) with an Autoblot oven and bottles (Bellco Glass Co., Vineland, NJ). After a prehybridization at 60°C for 3 hrs, 2x 10? CPM of the heat denatured probe was added to each hybridization bottle and the filters were hybridized at 60°C for 1 hr, then 55°C for 1 1 hrs, and 50°C for 3 hrs. After hybridization, the filters were washed in 2 X SSC, 0.1 % SDS, first at room temperature for 60 min, then at 30°C for 20 min, then at 45 °C for 30 min and the filters were then exposed to Kodak X-OMAT AR film for 4 hrs. Several putative hybridizing colonies were identified and DNA was purified from these clones by Qiaprep system (Qiagen, Inc., Chatsworth, CA), subjected to digestion with EcoRI and EcoRV, separated by electrophoresis in an agarose gel, and transferred to a nylon filter (Zeta-Probe, Bio-Rad Laboratories, Hercules, CA) using the Stratagene Posiblot system. The resulting filters were hybridized to the PCR labeled 463 bp NPY Yl receptor cDNA fragment as described above. After a final stringent wash at 55°C for 45 min in 2 X SSC, 0.1 SDS the filters were then exposed to Kodak X- OMAT AR film for 1 hr. One hybridizing clone, designated ET 15952 and containing plasmid pVE2841 , was selected for DNA sequence analysis.
EXAMPLE 3
Sequence Analysis of pVE2841
DNA was prepared from overnight cultures of ET15952 using the Wizard DNA Purification System (Promega Corp., Madison, WI) and subjected to automated sequence analysis using the PRISM Dye Deoxy terminator cycle sequencing kit (Applied Biosy stems, Foster City, CA). Initial sequencing primers were complementary to the T7 and SP6 promoter sites in pGEM 3ZF, additional primers were made complementary to the insert DNA in pVE2841. Sequencing indicated that pVE2841 contains a 2280 bp PstI insert. FRAMES analysis using the Genetics Computing Group software (Madison, WI) identified a 371 amino acid open reading frame (Fig. 1). Hydrophobicity analysis using PEPPLOT (Genetics Computing Group software (Madison, WI)) indicates the open reading frame has 7 hydrophobic domains characteristic of the transmembrane spanning domains of G-protein coupled receptors (Fig. 2). The DNA sequence of the PstI fragment and the region encoding the open reading frame are shown in Fig. 3. The DNA encoding the open reading frame appears to lack introns and is 59% identical to mouse NPY Yl receptor cDNA, while the codon sequence of the open reading frame is 52% identical to the mouse NPY Yl receptor. A comparative alignment determined by BESTFIT (Genetics Computing Group software (Madison, WI)) of the mouse NPY Y 1 receptor and the novel homolog described herein is presented in Fig. 4. In Fig. 4 the seven potential transmembrane spanning domains are indicated as are three putative N-linked glycosylation sites (indicated by a *). EXAMPLE 4
Construction of a Vector for Expression of the murine NPY Yx Receptor DNA in Mammalian Cells 3 μg of pcDNA3 DNA (Invitrogen Corp., San Diego,
CA) was digested with restriction enzyme EcoRV according to the manufacturer's directions (Bethesda Research Labs, Bethesda, MD) in a reaction mixture of 40μl. After 2 hours of digestion at 37°C, the 5' ends of the vector were dephosphorylated by adding 2 μl of an alkaline phosphatase solution (Boehringer Mannheim, Indianapolis, IN) (0.1 U in 10 mM Tris pH7, 10 mM MgCl2, 0.1 mM ZnCl2, 50% glycerol) and incubating at 37°C. After an hour, the enzymes were heat inactivated at 65°C for 10 min. 5 μg of pVE2841 were digested with restriction enzyme PstI, according to the manufacture's directions (Bethesda Research Labs, Bethesda, MD), in a 40 μL solution. The 3' overhanging ends of the 2.3 kb PstI fragment were converted to blunt ends by adding 8.8 μL of 5x T4 DNA Polymerase buffer and 5 U of T4 DNA polymerase (Bethesda Research Labs, Bethesda, MD) and continuing the incubation at 37°C. After an hour, the enzymes were heat inactivated at 65°C for 10 min. The reaction products were separated on a 1 % agarose gel (Sigma Chemical Co., St. Louis MO) and the cleaved pcDNA3 vector and 2.3 kb fragment were purified from the agarose gel using Qiaex resin (Qiagen Corp., Chatsworth, CA). The purified DNA fragments were resuspended in 25 μL of TE (10 mM Tris pH 7.4, 1 mM EDTA). The 2.3 kb fragment was ligated to the pcDNA3 vector DNA in a 20 μL reaction containing 1 μL of purified vector, 3 μL of purified 2.3 kb fragment, 4 μL of 5x T4 DNA ligation buffer (Bethesda Research Labs, Bethesda, MD), 1 1 μL of water, and 1 μl of T4 DNA ligase (1U, Bethesda Research Labs, Bethesda. MD). After ligation for 2 hr at 20°C, 1 μL of the ligation mixture was transformed into 20 μL of XL 1 Blue competent E. coli cells (Stratagene, La Jolla, CA), according to the manufacturer's directions. Transformants were isolated as ampicillin-resistant colonies and plasmid DNA was isolated from transformants by Qiaprep system (Qiagen Corp. Chatsworth, CA). Among the transformants containing the 2.3 kb insert, one isolate was identified and designated pVE2863 which contained the 2.3 kb in the correct orientation for expression from the CMV promoter.
EXAMPLE 5
Construction of a second Vector for Expression of the murine NPY Yx Receptor DNA in Mammalian Cells
3 μg of pCI-neo (Promega corp., Madison, WI) was digested with restriction enzymes EcoRI and Xbal according to the manufacturer's directions (Bethesda Research Labs, Bethesda, MD) in a reaction mixture of 50 μL. After 16 hours of digestion at 37°C, the 5' ends of the vector were dephosphorylated by adding 2 μL of an alkaline phosphatase solution (Boehringer Mannheim, Indianapolis, IN) (0.1 U in 10 mM Tris pH7, 10 mM MgCl2, 0.1 mM ZnCl2, 50% glycerol) and incubating at 37°C. After an hour of incubation, the DNA was purified using a Wizard PCR Preps kit (Promega Corp., Madison, WI) and resuspended in 50 μL water. A fragment of the Yx gene containing an EcoRI restriction site 5' to the ATG, and an Xbal restriction site 3' to the stop codon was generated by polymerase chain reaction (PCR). Ten ng of PVE2841 was used in a 50 μL reaction containing 5 units of Pfu polymerase (Promega corp., Madison, WI) and 20 pmoles each of primers
MNPY5FRI (5'GATCGAATTCG CCATGGAAGTGCTCACAAAC 3 ) (SEQ. I.D. NO. 13) and MNPY5RXBA (5'GATCTCTAGACTA TATGCCTGTTGGTATGTG 3 ) (SEQ. I.D. NO. 14) in buffer supplied by the manufacturer. The reactions were carried out for 25 cycles of 95 degrees for 1 minute, 50 degrees for 30 seconds, and 72 degrees for 1 minute 30 seconds. The products of the reaction were resolved on a 1 % agarose gel and the single predominant product was isolated and purified using a Wizard PCR Preps kit (Promega Corp., Madison, WI). The purified PCR fragment was digested in a 50 μL reaction containing 10 units of EcoRI and 10 units of Xbal according to the manufacturer's directions (Bethesda Research Labs, Bethesda, MD) for 16 hours. The PCR fragment was repurified as before using the Wizard PCR Preps kit and resuspended in 50 μL of water. The PCR fragment was ligated to the pCI-neo vector DNA in a 10 μL reaction containing 1 μL of purified vector, 3 μL of purified PCR fragment, 2 μL of 5x T4 DNA ligation buffer (Bethesda Research Labs, Bethesda, MD), 3 μL of water, and 1 μL of T4 DNA ligase (1 U, Bethesda Research Labs, Bethesda, MD). After ligation for 16 hr at 12°C, 1 μL of the ligation mixture was transformed into 50 μL of Max -Efficiency DH5 alpha competent E. coli cells (Bethesda Research Labs, Bethesda, MD), according to the manufacturer's directions. Transformants were isolated as ampicillin-resistant colonies and plasmid DNA was isolated from transformants by Qiaprep system (Qiagen Corp. Chatsworth, CA). Transformants containing the appropriate fragment insert were identified by restriction with EcoRI and Xba I. Fidelity of the Yx gene was verified by automated sequence analysis using the PRISM Dye Deoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). A single clone was selected and designated pCI-mY5.
EXAMPLE 6
Pharmacology of the Recombinant NPY Yx Receptor
Using electroporation, COS-7 cells were transfected with the pcDNA3 or the pCI-neo expression vector containing the genomic DNA for the murine NPY Yx receptor (pVE2863 or pCI-mY5, respectively). Two days post transfection, cells were dissociated with enzyme-free dissociation buffer, centrifuged at 1000 rpm for 10 minutes at 4°C, and resuspended in 10 mM Tris, pH 7.4 containing 0.1 M PMSF (phenylmethylsulfonyl fluoride), 10 μM phosphoramidon, and 40 μg/ml bacitracin. The cell suspension was homogenized with a glass homogenizer, and centrifuged at 2200 rpm for 10 minutes at 4°C to remove cell debris. The plasma membrane fraction was recovered by centrifuging the supernatant at 18,000 rpm in a Sorvall SS-34 rotor for 15 minutes at 4°C. The membrane pellet was resuspended in the Tris/PMSF/phosphoramidon/bacitracin buffer by trituration 5 times using a 25 g needle and syringe. The membranes were stored frozen at -80°C until use.
Membranes (50 μg protein) were incubated with 1 5ι_ PYY (90 pM) in the presence or absence of 1 μM unlabeled PYY (non-specific binding) or serial dilutions of other competing peptides in 0.25 ml of 50 mM Tris, pH 7.4 containing 2 mM CaCl2, 1 mM MgCl2, 5 mM KC1, 0.2% bovine serum albumin, 10 μM phosphoramidon, 4 μg/ml leupeptin and 40 μg/ml bacitracin. After 2 hours at room temperature, the incubation was filtered over GF/C filters, and the radioactivity bound to the filters was quantified with a Packard gamma counter. The percentage of inhibition of 1 5j_ PYY binding at a given concentration of competing peptide was plotted versus the concentration of peptide added, and the concentration of peptide causing 50% inhibition of binding (IC50) was determined using non-linear regression performed by PRISM (GraphPad).
Competitor IC50 (nM) PYY 14
NPY 6
[Leu31 , Pn>34]NPY 9 NPY(2-36) 16 NPY( 13-36) 134
EXAMPLE 7
Human NPY Yx Receptor
Isolation of the hybridization probe. A hybridization probe with identity to the human NPY Yx receptor was generated by PCR using human genomic DNA as the target. Oligonucleotide primer pairs based upon the DNA sequence of the mouse NPY Yx receptor (primer 1 ; 5 ACCAGTGGCAAGAGCAACAAC (SEQ. I.D. NO. 8), primer 2; 5'CTCATTGGTGAGGTGGTAGGAC (SEQ. I.D. NO. 9)) were used in a primary PCR reaction. The products of this reaction were used as the template for a second PCR reaction using primer 2 and primer 3
(5'.GGGCATTTTTGGAAACCTCTC (SEQ. I.D. NO. 10)). Primer 3, a 'nested' primer, was designed to hybridize to mouse NPY Yx receptor sequences which are internal to those with homology to primers 1 and 2. A product obtained from the second PCR reaction was cloned and found to share homology to the mouse NPY Yx receptor by DNA sequence analysis in both directions using an ABI 373A automated sequencing unit (Perkin Elmer).
Isolation of a human NPY Yx receptor cDNA clone. A commercial lambda phage library (Lambda Zap2, Stratagene) containing cDNAs derived from human heart mRNA was screened. Approximately 1 x lθ6 plaques were plated at a density of 50,000 plaques per 150 mm plate and transferred to Colony/Plaque screen hybridization membranes (Dupont/NEN) which were then processed by standard protocols (as supplied by Stratagene). DNA was UV crosslinked to the membrane with a Stratalinker (Stratagene). The filters were prewashed with 500 mis of 0.1 X SSC, 0.1 % SDS at 65°C for 1 hour and then prehybridized in a solution of 0.25M NaPθ4, 7% SDS pH7.5 at 60°C for 1 hour. The PCR fragment having identity to the human NPY Yx receptor described above was labeled by random priming (Rediprime, Amersham) in the presence of [cx-32p]dCTP (3000 Ci/mmole). The membranes were allowed to hybridize to the [rα-32p]-iabeled PCR fragment in a solution of 0.25M NaP04, 7% SDS, 10% dextran sulfate pH7.5 for 16 hours at 60°C. The membranes were washed in 1 liter of IX SSC, 0.1 % SDS at 45 °C and then exposed to Kodak XAR-5 Xray film. Primary positives identified by this procedure were selected, re-plated, and subjected to a second round of hybridization screening to allow identification and selection of individual hybridization positive plaques. Plasmids containing the cDNA inserts were rescued from the phage using methodology supplied by the manufacturer (Stratagene) which results in the generation of bacterial colonies. Bacterial colonies with plasmids containing the human NPY Yx receptor cDNA sequences were identified by filter hybridization with the [α-32p] -labeled human NPY Yx receptor probe using standard methodologies (Sambrook, J., Fritsch, E.F., Maniatis, T. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989) . The cloned cDNA, pCD13, was analyzed by sequencing in both directions using an ABI 373A automated sequencing unit (Perkin Elmer).
Structure of the human NPY Yx receptor cDNA.
The DNA sequence of the approximately 1499 bp clone of human NPY Yx receptor cDNA, pCD13 (SEQ. I.D. NO. 11) contains an open reading frame of about 11 10 bp. The proposed initiator methionine at nucleotide 182 conforms to the Kozak sequence (M. Kozak, Journal of Cell Biology 108, 229-241 (1989)) at the +4 position but not at the -3 position. The proposed 370 amino acid sequence (SEQ. I.D. NO. 12) encoded by the open reading frame is 82% identical to the amino acid sequence encoded by the open reading frame of the mouse NPY Yx receptor clone. The human NPY Yx receptor cDNA is 78% identical to the DNA of the mouse NPY Yx receptor. The human NPY Yx receptor cDNA described herein contains 181 untranslated nucleotides at the 5 prime end and 208 nucleotides following the stop codon.
EXAMPLE 8
Construction of a Vector for Expression of the Human NPY Yx Receptor DNA in Mammalian Cells 2 μg of pcDNA3 DNA (Invitrogen Corp., San Diego,
CA) was digested with restriction enzyme EcoRI according to the manufacturer's directions (Bethesda Research Labs, Bethesda, MD) in a reaction mixture of 20 μl. The DNA was resolved by electrophoresis on a 1% agarose gel (Sigma Chemical Co., St. Louis MO) and the linearized vector was identified and excised from the gel. The DNA was purified by Gene Clean using the procedure supplied by the manufacturer (BIO 101 Inc., Vista, CA). The 5' ends of the vector were dephosphorylated by adding 1 μl (0.1 unit) of an alkaline phosphatase solution (Boehringer Mannheim, Indianapolis, IN) in 50 μl of 10 mM Tris pH7, 10 mM MgCl2, 0.1 mM ZnCl2, 50% glycerol and incubating at 37°C for 1 hour. The DNA was subjected to a second round of Gene Clean purification and resuspended in 50 μl of TE (10 mM Tris pH 7.4, 1 mM EDTA). Approximately 2 μg of an isolated human NPYx receptor clone, pCD13, was digested with restriction enzyme Eco RI (Bethesda Research Labs, Bethesda, MD) in a 20 μl solution. The reaction products were resolved by electrophoresis on a 1 % agarose gel and the approximately 1.6kb fragment containing the receptor coding sequences was purified by Gene Clean as above and resuspended in 50 μl of TE. The 1.6 kb fragment was ligated to the linearized pcDNA3 vector DNA in a 10 μl reaction containing 1 μl of purified vector, 3 μl of purified 1.6 kb fragment, 1 μl of lOx T4 DNA ligation buffer (Boehringer Mannheim, Indianapolis, IN), 5 μl of water, and 1 μl of T4 DNA ligase (1 U, Boehringer Mannheim, Indianapolis, IN). After ligation for 2 hr at 20°C, 1 μl of the ligation mixture was transformed into 50 μl of Max Efficiency DH5alpha competent E. coli cells (Bethesda Research Labs, Bethesda, MD), according to the manufacturer's directions. Transformants were isolated as ampicillin-resistant colonies and plasmid DNA was isolated from transformants using the WIZARD miniprep system (Promega Corporation, Madison WI). Among the transformants containing the 1.6kb insert, one isolate was identified and designated pcDNA3HYx-RI, which contained the 1.6 kb in the correct orientation for expression from the CMV promoter.
In an analagous fashion, 2 μg of pcDNA3 DNA (Invitrogen Corp., San Diego, CA) was digested with restriction enzymes EcoRI and Hind III according to the manufacturer's directions (Bethesda Research Labs, Bethesda, MD) in a reaction mixture of 20 μl. The large fragment of doubly digested vector was purified by Gene Clean and resuspended in 50 μl of TE. Approximately 2 μg of one of the isolated human NPYx receptor clones, pCD13, was digested with restriction enzymes£cO RI and Hind III according to the manufacturer's directions (Bethesda Research Labs, Bethesda, MD) in a 20 μl reaction. The approximately 1.6kb fragment containing the receptor coding sequences was purified by Gene Clean and resuspended in 50 μl of TE. The 1.6 kb fragment was ligated to the pcDNA3 vector DNA in a 10 μl reaction containing 1 μl of purified vector, 3 μl of purified 1.6 kb fragment, 1 μl of lOx T4 DNA ligation buffer (Boehringer Mannheim, Indianapolis, IN), 5 μl of water, and 1 μl of T4 DNA ligase (1U, Boehringer Mannheim, Indianapolis, IN). After ligation for 2 hr at 20°C, 1 μl of the ligation mixture was transformed into 50 μl of Max Efficiency DH5 alpha competent E. coli cells (Bethesda Research Labs, Bethesda, MD), according to the manufacturer's directions. Transformants were isolated as ampicillin-resistant colonies and plasmid DNA was isolated from transformants using the WIZARD miniprep system (Promega Corporation, Madison WI). Among the transformants containing the 1.6 kb insert, one isolate was identified and designated pcDNA3HYx-Hind RI, which contained the 1.6 kb in the correct orientation for expression from the CMV promoter. The two expression constructs, pcDNA3HYx-RI and pcDNA3HYx- Hind/RI, differ in the amount of cDNA sequences 5 prime to the ATG translational start codon which remain in the final expression construct.
EXAMPLE 9 Cloning and Expression of NPY Yx Receptor DNA into Bacterial Expression Vectors
Recombinant receptor is produced in a bacterial expression system such as coli. The receptor expression cassette is transferred into an coli expression vector; expression vectors include but are not limited to, the pET series (Novagen). The pET vectors place receptor expression under control of the tightly regulated bacteriophage T7 promoter. Following transfer of this construct into an coli host which contains a chromosomal copy of the T7 RNA polymerase gene driven by the inducible lac promoter, expression of receptor is induced by addition of an appropriate lac substrate (IPTG) added to the culture. The levels of expressed receptor are determined by the assays described herein.
EXAMPLE 10 Cloning and Expression of NPY Yx Receptor DNA into a Vector for Expression in Insect Cells
Baculovirus vectors derived from the genome of the AcNPV virus are designed to provide high level expression of DNA in the Sf9 line of insect cells (ATCC CRL# 171 1). Recombinant baculovirus expressing receptor DNA is produced by the following standard methods (I Vitrogen Maxbac Manual): the receptor DNA constructs are ligated into the polyhedrin gene in a variety of baculovirus transfer vectors, including the pAC360 and the BlueBac vector (InVitrogen). Recombinant baculoviruses are generated by homologous recombination following co-transfection of the baculovirus transfer vector and linearized AcNPV genomic DNA [Kitts, P.A., Nuc. Acid. Res. 18, 5667 (1990)] into Sf9 cells. Recombinant pAC360 viruses are identified by the absence of inclusion bodies in infected cells and recombinant pBlueBac viruses are identified on the basis of B-galactosidase expression (Summers, M. D. and Smith, G. E., Texas Agriculture Exp. Station Bulletin No. 1555). Following plaque purification, receptor expression is measured by assays described herein. Authentic receptor is found in association with the infected cells. Active receptor is extracted from infected cells by hypotonic or detergent lysis.
Alternatively, the receptor is expressed in the Drosophila Schneider 2 cell line by cotransfection of the Schneider 2 cells with a vector containing the receptor DNA downstream and under control of an inducible metallothionin promoter, and a vector encoding the G418 resistant neomycin gene. Following growth in the presence of G418, resistant cells are obtained and induced to express receptor by the addition of Q1SO4. Identification of modulators of the receptor is accomplished by assays using either whole cells or membrane preparations.
EXAMPLE 1 1 Cloning of NPY Yx Receptor DNA into a Yeast Expression Vector Recombinant receptor is produced in the yeast cerevisiae following the insertion of the receptor DNA cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins. In the case of intracellular expression, vectors such as EmBLyex4 or the like are ligated to the receptor cistron [Rinas, U. et al., Biotechnology 8, 543-545 (1990); Horowitz B. et al., J. Biol. Chem. 265, 4189-4192 (1989)]. For extracellular expression, the receptor cistron is ligated into yeast expression vectors which fuse a secretion signal. The levels of expressed receptor are determined by the assays described herein. EXAMPLE 12 Purification of Recombinant NPY Yx Receptor
Recombinantly produced receptor may be purified by a variety of procedures, including but not limited to antibody affinity chromatography.
Receptor antibody affinity columns are made by adding the anti-receptor antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1 M ethanolamine HC1 (pH 8). The column is washed with water followed by 0.23 M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) together with appropriate membrane solubilizing agents such as detergents, and the cell culture supernatants or cell extracts containing solubilized receptor or receptor subunits are slowly passed through the column. The column is then washed with phosphate-buffered saline (PBS) supplemented with detergents until the optical density (A280) falls to background; then the protein is eluted with 0.23 M glycine-HCl (pH 2.6) supplemented with detergents. The purified receptor protein is then dialyzed against PBS.
EXAMPLE 13
Screening Assays
Recombinant cells containing DNA encoding the novel murine or human NPY Yx receptor, membranes derived from the recombinant cells, or recombinant receptor preparations derived from the cells or membranes may be used to identify compounds that modulate the murine or human NPY Yx receptor activity. Modulation of such activity may occur at the level of DNA, RNA, protein or combinations thereof. One method of identifying compounds that modulate NPY Yx receptor activity or modulate binding of ligands to the protein, comprises:
(a) mixing a test compound with a solution containing NPY Yx receptor, or a functional derivative thereof, to form a mixture;
(b) measuring NPY Yx receptor activity in the mixture; and
(c) comparing the NPY Yx receptor activity of the mixture to a standard. Preferably, the neuropeptide Y Yx receptor, or functional derivative thereof, is characterized by a pharmacological binding profile with affinities of PYY ≡ NPY = [Leu31Pro34]NPY ≡ NPY(2-36) > NPY( 13-36).
This screening assay will detect a compound that has affinity for the NPY Yx receptor. Such compounds may be either agonists or antagonists and may be peptides, proteins, or non- proteinaceous organic molecules.
Another method of identifying compounds that modulate the NPY Yx receptor, comprises:
(a) mixing a test compound with cells which express the NPY Yx receptor to form a mixture;
(b) culturing the cells in the presence of the test compound;
(c) measuring NPY Yx receptor or second messenger activity in the mixture; and (d) comparing the NPY Yx receptor activity of the mixture to a standard. In a preferred embodiment, the cells are transformed by an expression vector containing a sequence selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3, a fragment of the sequence of substantial homology to the sequence shown in Figure 3, a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5.
In one example of the method, the expression vector contains a DNA sequence selected from a DNA sequence having a sequence selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3 or a fragment of the sequence of substantial homology to the sequence shown in Figure 3. Preferably, the expression vector contains a DNA sequence selected from the sequence shown in Figure 3, the sequence of substantial homology to the sequence shown in Figure 3 or the fragment of the sequence shown in Figure 3 comprising bases 822 to 1934.
In another example of the method, the expression vector contains a DNA sequence selected from a DNA sequence having a sequence selected from a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5. Preferably, the expression vector contains a DNA sequence selected from the sequence shown in Figure 5, the sequence of substantial homology to the sequence shown in Figure 5 or the fragment of the sequence shown in Figure 5 comprising bases 182 to 1291.
This screening assay will detect a compound which modulates NPY Yx receptor activity. Such compounds may be either agonists, antagonists, suppressors or inducers. Such compounds may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.
While the invention has been described and illustrated with reference to certain preferred embodiments thereof, those skilled in the art will appreciate that various changes, modifications and substitutions can be made therein without departing from the spirit and scope of the invention. It is intended, therefore, that the invention be limited only by the scope of the claims which follow and that such claims be interpreted as broadly as is reasonable. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Cascieri, Margaret A. Linemeyer, David L. MacNeil, Douglas J. Shiao, Lin-Lin Strader, Catherine D. Tan, Carina P. Weinberg, David H.
(ii) TITLE OF INVENTION: NEUROPEPTIDE Y RECEPTOR
(iii) NUMBER OF SEQUENCES: 14
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Mary A. Appollina
(B) STREET: P.O. Box 2000, 126 E. Lincoln Ave.
(C) CITY: Rahway
(D) STATE: NJ
(E) COUNTRY: USA
(F) ZIP: 07065
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/383,746
(B) FILING DATE: 03-FEB-1995
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/415,818
(B) FILING DATE: 03-APR-1995
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Appollina, Mary A.
(B) REGISTRATION NUMBER: 34,087
(C) REFERENCE/DOCKET NUMBER: 19390Y
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-594-3462
(B) TELEFAX: 908-594-4720
(2) INFORMATION FOR SEQ ID NO:1: - 45
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CACTGGGTCT TTGGTGAGGC GATGTG 26
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : CCCATAAAAT ATGGGGTTGA CACAAGTGG 29
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : TTGGCCATGA TATTTACCTT AGCT 24
(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : GCATCAAGTG TTACATTTTG GAAC 24
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2280 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 822..1937
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CTGCAGTCTA TTGGATGAAG AGTGTACATA TTCATATAAT TCTTAAAGTA GGCAGAAATT 60
AAAGGGGATG GAAATATATA CTTGTACTGC CTTAGATAGT CACC GGATG TTGTTACAGT 120
CTTCGTTTAC TGCTTCTGAA GCCTATACTG ATAGAATTAA TAAAATACTG AGAGAGAGAG 180
AGAGGGACAG AGAGAGAGAG GGGGAGAGAG AGAGAGAGAG AGAGAGAGAG AGAGAGAGAG 240
AGAGAGAGAG AGAAGAGAAG AAAACAAGGT AAGCCATCTG CTTAACTTAT GTCCACATTC 300
TCTCAAGAGC ATTGTCCTAT TTGTAGAATT ATCTATATTG TTAAGAATCA TCTCCATTGT 360
TAAGATTTTG TGGGCTGGAG ATCCAGCTCT GTTGATAAAG TGCTTGCCTA ACATGCATGA 420
AGTCCTAGGT TCTATTCCCA AGGCTACATA AAACCTTGTG TTGTGATGAA TGCCTGTAAT 480 CCCAGTACGC AGCAAGGAGA GACAAGGAGG ATCAGAAGCT TAAGGACATC ATTTTGTACA 540
TAGTGAGTTT GAGGAAAGCT GAGGTTACAT GGAACTCTCT CTCTCTCAAA AACAAAACAA 600
AACAAAACAA AACCTTCTAC TAATATTCTG GATTCTGTTT GATTTTTAGG ATCTCAAGAG 660
CATGCTGACG TCATTTATGT GTTTCCATCA GATACAGACA GAGATCATAA ACATTTAACT 720
CATTGATTAT ATGTTGAGAG TTGTCCCTCA AGAACCAATG GCCAAACATC CACTGAGGAT 780
ACACGGAAGC TTAGAAAATC TCTAATTAAA ATCCTGACAT A ATG GAA GTG CTC 833
Met Glu Val Leu 1
ACA AAC CAG CCA ACA CCT AAT AAA ACC AGT GGC AAG AGC AAC AAC TCG 881 Thr Asn Gin Pro Thr Pro Asn Lys Thr Ser Gly Lys Ser Asn Asn Ser 5 10 15 20
GCA TTT TTC TAC TTT GAA TCC TGC CAA CCC CCT TTT CTA GCC ATA CTC 929 Ala Phe Phe Tyr Phe Glu Ser Cys Gin Pro Pro Phe Leu Ala lie Leu 25 30 35
TTG CTA CTC ATA GCA TAT ACT GTG ATC CTA ATC ATG GGC ATT TTT GGA 977 Leu Leu Leu lie Ala Tyr Thr Val lie Leu lie Met Gly lie Phe Gly 40 45 50
AAC CTC TCT CTT ATC ATC ATC ATC TTT AAG AAA CAG AGA GAA GCT CAA 1025 Asn Leu Ser Leu lie lie lie lie Phe Lys Lys Gin Arg Glu Ala Gin 55 60 65
AAT GTT ACC AAC ATA CTG ATT GCC AAC CTG TCC CTC TCT GAC ATC TTG 1073 Asn Val Thr Asn lie Leu lie Ala Asn Leu Ser Leu Ser Asp lie Leu 70 75 80
GTG TGT GTC ATG TGC ATC CCT TTT ACG GTC ATC TAC ACT CTG ATG GAC 1121 Val Cys Val Met Cys lie Pro Phe Thr Val lie Tyr Thr Leu Met Asp 85 90 95 100
CAC TGG GTA TTT GGG AAC ACT ATG TGT AAA CTC ACT TCC TAC GTG CAA 1169 His Trp Val Phe Gly Asn Thr Met Cys Lys Leu Thr Ser Tyr Val Gin 105 110 115
AGT GTC TCA GTT TCT GTG TCC ATA TTC TCC CTT GTG TTG ATT GCT ATT 1217 Ser Val Ser Val Ser Val Ser He Phe Ser Leu Val Leu He Ala He 120 125 130
GAA CGA TAT CAG CTG ATT GTG AAC CCC CGT GGC TGG AAA CCC AGA GTA 1265 Glu Arg Tyr Gin Leu He Val Asn Pro Arg Gly Trp Lys Pro Arg Val 135 140 145
GCT CAT GCC TAT TGG GGG ATC ATC TTG ATT TGG CTC ATT TCT CTG ACA 1313 Ala His Ala Tyr Trp Gly He He Leu He Trp Leu He Ser Leu Thr 150 155 160
TTG TCT ATT CCC TTA TTC CTG TCC TAC CAC CTC ACC AAT GAG CCC TTT 1361 Leu Ser He Pro Leu Phe Leu Ser Tyr His Leu Thr Asn Glu Pro Phe 165 170 175 180
CAT AAT CTC TCT CTC CCT ACT GAC ATC TAC ACC CAC CAG GTA GCT TGT 1409 His Asn Leu Ser Leu Pro Thr Asp He Tyr Thr His Gin Val Ala Cys 185 190 195
GTG GAG ATT TGG CCT TCT AAA CTG AAC CAA CTC CTC TTT TCT ACA TCA 1457 Val Glu He Trp Pro Ser Lys Leu Asn Gin Leu Leu Phe Ser Thr Ser 200 205 210
TTA TTT ATG CTC CAG TAT TTT GTC CCT CTG GGT TTC ATT CTT ATC TGC 1505 Leu Phe Met Leu Gin Tyr Phe Val Pro Leu Gly Phe He Leu He Cys 215 220 225
TAC CTG AAG ATC GTT CTC TGC CTC CGA AAA AGA ACT AGG CAG GTG GAC 1553 Tyr Leu Lys He Val Leu Cys Leu Arg Lys Arg Thr Arg Gin Val Asp 230 235 240
AGG AGA AAG GAA AAT AAG AGC CGT CTC AAT GAG AAC AAG AGG GTA AAT 1601 Arg Arg Lys Glu Asn Lys Ser Arg Leu Asn Glu Asn Lys Arg Val Asn 245 250 255 260
GTG ATG TTG ATT TCC ATC GTA GTG ACT TTT GGA GCC TGC TGG TTG CCC 1649 Val Met Leu He Ser He Val Val Thr Phe Gly Ala Cys Trp Leu Pro 265 270 275
TTG AAC ATT TTC AAT GTC ATC TTC GAC TGG TAT CAT GAG ATG CTG ATG 1697 Leu Asn He Phe Asn Val He Phe Asp Trp Tyr His Glu Met Leu Met 280 285 290
AGC TGC CAC CAC GAC CTG GTA TTT GTA GTT TGC CAC TTG ATT GCT ATG 1745 Ser Cys His His Asp Leu Val Phe Val Val Cys His Leu He Ala Met 295 300 305
GTT TCT ACT TGC ATA AAT CCT CTC TTT TAT GGA TTT CTC AAC AAA AAC 1793 Val Ser Thr Cys He Asn Pro Leu Phe Tyr Gly Phe Leu Asn Lys Asn 310 315 320
TTC CAG AAG GAT CTA ATG ATG CTT ATT CAC CAC TGT TGG TGT GGT GAA 1841 Phe Gin Lys Asp Leu Met Met Leu He His His Cys Trp Cys Gly Glu 325 330 335 340
CCT CAG GAA AGT TAT GAA AAT ATT GCC ATG TCT ACT ATG CAC ACA GAT 1889 Pro Gin Glu Ser Tyr Glu Asn He Ala Met Ser Thr Met His Thr Asp 345 350 355
GAA TCC AAG GGA TCA TTA AAA CTG GCT CAC ATA CCA ACA GGC ATA TAG 1937 Glu Ser Lys Gly Ser Leu Lys Leu Ala His He Pro Thr Gly He * 360 365 370
AAACTGGTAA GCAAAATCAA AGCCCTTCTG TTATGAAAGA AAGAGAAGAA ATAGTATGGA 1997
ATAGGGCAAG GTGCAGAGGA AGCCAGACTT AAACACATAA TATCTTTGGG CCCAGTTTTG 2057
CTTTAAGTTA AGCATGTCTA CTCCATTCAG CCATAGAACA CACAGAGATT TATCCCTACC 2117 - 49 -
CTTTCTTTTT TTCCTTTGGA AGAATAATAA CTTAAACAAC CTAGACATCA TTACTGAGGA 2177 AGAGAACAAA AATGAGAGAG CATACAAGGA CAGCAGAGAT GTCTGGGGTA CAAAATTCAC 2237 GTTATTCGCT GGAATAGCTA GAAAGTTATT AGTTGTGCTG CAG 2280
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 372 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Glu Val Leu Thr Asn Gin Pro Thr Pro Asn Lys Thr Ser Gly Lys 1 5 10 15
Ser Asn Asn Ser Ala Phe Phe Tyr Phe Glu Ser Cys Gin Pro Pro Phe 20 25 30
Leu Ala He Leu Leu Leu Leu He Ala Tyr Thr Val He Leu He Met 35 40 45
Gly He Phe Gly Asn Leu Ser Leu He He He He Phe Lys Lys Gin 50 55 60
Arg Glu Ala Gin Asn Val Thr Asn He Leu He Ala Asn Leu Ser Leu 65 70 75 80
Ser Asp He Leu Val Cys Val Met Cys He Pro Phe Thr Val He Tyr 85 90 95
Thr Leu Met Asp His Trp Val Phe Gly Asn Thr Met Cys Lys Leu Thr 100 105 110
Ser Tyr Val Gin Ser Val Ser Val Ser Val Ser He Phe Ser Leu Val 115 120 125
Leu He Ala He Glu Arg Tyr Gin Leu He Val Asn Pro Arg Gly Trp 130 135 140
Lys Pro Arg Val Ala His Ala Tyr Trp Gly He He Leu He Trp Leu 145 150 155 160
He Ser Leu Thr Leu Ser He Pro Leu Phe Leu Ser Tyr His Leu Thr 165 170 175
Asn Glu Pro Phe His Asn Leu Ser Leu Pro Thr Asp He Tyr Thr His 180 185 190
Gin Val Ala Cys Val Glu He Trp Pro Ser Lys Leu Asn Gin Leu Leu 195 200 205 Phe Ser Thr Ser Leu Phe Met Leu Gin Tyr Phe Val Pro Leu Gly Phe 210 215 220
He Leu He Cys Tyr Leu Lys He Val Leu Cys Leu Arg Lys Arg Thr 225 230 235 240
Arg Gin Val Asp Arg Arg Lys Glu Asn Lys Ser Arg Leu Asn Glu Asn 245 250 255
Lys Arg Val Asn Val Met Leu He Ser He Val Val Thr Phe Gly Ala 260 265 270
Cys Trp Leu Pro Leu Asn He Phe Asn Val He Phe Asp Trp Tyr His 275 280 285
Glu Met Leu Met Ser Cys His His Asp Leu Val Phe Val Val Cys His 290 295 300
Leu He Ala Met Val Ser Thr Cys He Asn Pro Leu Phe Tyr Gly Phe 305 310 315 320
Leu Asn Lys Asn Phe Gin Lys Asp Leu Met Met Leu He His His Cys 325 330 335
Trp Cys Gly Glu Pro Gin Glu Ser Tyr Glu Asn He Ala Met Ser Thr 340 345 350
Met His Thr Asp Glu Ser Lys Gly Ser Leu Lys Leu Ala His He Pro 355 360 365
Thr Gly He * 370
(2) INFORMATION FOR SEQ ID NO:7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 382 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met Asn Ser Thr Leu Phe Ser Lys Val Glu Asn His Ser He His Tyr 1 5 10 15
Asn Ala Ser Glu Asn Ser Pro Leu Leu Ala Phe Glu Asn Asp Asp Cys 20 25 30
His Leu Pro Leu Ala Val He Phe Thr Leu Ala Leu Ala Tyr Gly Ala 35 40 45
Val He He Leu Gly Val Ser Gly Asn Leu Ala Leu He He He He 50 55 60
Leu Lys Gin Lys Glu Met Arg Asn Val Thr Asn He Leu He Val Asn 65 70 75 80
Leu Ser Phe Ser Asp Leu Leu Val Ala Val Met Cys Leu Pro Phe Thr 85 90 95
Phe Val Tyr Thr Leu Met Asp His Trp Val Phe Gly Glu Thr Met Cys 100 105 110
Lys Leu Asn Pro Phe Val Gin Cys Val Ser He Thr Val Ser He Phe 115 120 125
Ser Leu Val Leu He Ala Val Glu Arg His Gin Leu He He Asn Pro 130 135 140
Arg Gly Trp Arg Pro Asn Asn Arg His Ala Tyr He Gly He Thr Val 145 150 155 160
He Trp Val Leu Ala Val Ala Ser Ser Leu Pro Phe Val He Tyr Gin 165 170 175
He Leu Thr Asp Glu Pro Phe Gin Asn Val Ser Leu Ala Ala Phe Lys 180 185 190
Asp Lys Tyr Val Cys Phe Asp Lys Phe Pro Ser Asp Ser His Arg Leu 195 200 205
Ser Tyr Thr Thr Leu Leu Leu Val Leu Gin Tyr Phe Gly Pro Leu Cys 210 215 220
Phe He Phe He Cys Tyr Phe Lys He Tyr He Arg Leu Lys Arg Arg 225 230 235 240
Asn Asn Met Met Asp Lys He Arg Asp Ser Lys Tyr Arg Ser Ser Glu 245 250 255
Thr Lys Arg He Asn He Met Leu Leu Ser He Val Val Ala Phe Ala 260 265 270
Val Cys Trp Leu Pro Leu Thr He Phe Asn Thr Val Phe Asp Trp Asn 275 280 285
His Gin He He Ala Thr Cys Asn His Asn Leu Leu Phe Leu Leu Cys 290 295 300
His Leu Thr Ala Met He Ser Thr Cys Val Asn Pro He Phe Tyr Gly 305 310 315 320 - 52 -
Phe Leu Asn Lys Asn Phe Gin Arg Asp Leu Gin Phe Phe Phe Asn Phe 325 330 335
Cys Asp Phe Arg Ser Arg Asp Asp Asp Tyr Glu Thr He Ala Met Ser 340 345 350
Thr Met His Thr Asp Val Ser Lys Thr Ser Leu Lys Gin Ala Ser Pro 355 360 365
Val Ala Phe Lys Lys He Ser Met Asn Asp Asn Glu Lys Val 370 375 380
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ACCAGTGGCA AGAGCAACAA C 21
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9 :
CTCATTGGTG AGGTGGTAGG AC 22
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GGGCATTTTT GGAAACCTCT C 21
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1499 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CCCCGGGCTG CAGGAATTCC CACATGTTTC CATCAAATAC AGACACAGAT CAGGGAAGAT 60
TAAACCCTAC TAATTTCTCG TCGGATGCCT CACAACAAGG TGCCTTCCAA GAACTAATGG 120
CCAAAATATC CACCCACAAC ACAAATAAGC TTAGAAAATC TCTTCTTACA ATCCTGACAC 180
AATGGAAGTT TCCCTAAACC ACCCAGCATC TAATACAACC AGCACAAAGA ACAACAACTC 240
GGCATTTTTT TACTTTGAGT CCTGTCAACC CCCTTCTCCA GCTTTACTCC TATTATGCAT 300
AGCCTATACT GTGGTCTTAA TTGTGGGCCT TTTTGGAAAC CTCTCTCTCA TCATCATCAT 360
CTTTAAGAAG CAGAGAAAAG CTCAGAATTT CACCAGCATA CTGATTGCCA ATCTCTCCCT 420
CTCTGATACC TTGGTGTGTG TCATGTGCAT CCATTTTACT ATCATCTACA CTCTGATGGA 480
CCACTGGATA TTTGGGGATA CCATGTGCAG ACTCACATCC TATGTGCAGA GTGTCTCAAT 540
CTCTGTGTCC ATATTCTCAC TTGTATTCAC TGCTGTCGAA AGATATCAGC TAATTGTGAA 600
CCCCCGTGGC TGGAAGCCCA GTGTGACTCA TGCCTACTGG GGCATCACAC TGATTTGGCT 660 GTTTTCCCTT CTGCTGTCTA TTCCCTTCTT CCTGTCCTAC CACCTCACTG ATGAGCCCTT 720
CCACAACCTC TCTCTCCCCA CTGACCTCTA CACCCACCAG GTGGCCTGTG TGGAGAACTG 780
GCCCTCCAAA AAGGACCGGC TGCTCTTCAC CACCTCCCTT TTTCTGCTGC AGTATTTTGT 840
TCCTCTAGGC TTCATCCTCA TCTGCTACTT GAAGATTGTT ATCTGCCTCC GCAGGAGAAA 900
TGCAAAGGTA GATAAGAAGA AGGAAAATGA GGGCCGGCTC AATGAGAACA AGAGGATCAA 960
CACAATGTTG ATTTCCATCG TGGTGACCTT TGGAGCCTGC TGGCTGCCCC CGAATATCTT 1020
CAATGTCATC TTTGACTGGT ATCATGAGGT GCTGATGAGC TGCCACCACG ACCTGGTATT 1080
TGTAGTTTGC CACTTGGTTG CTATGGTTTC CACATGTATA AACCCTCTCT TTTATGGCTT 1140
TCTCAACAAA AATTTCCAAA AGGACCTGGT AGTGCTTATT CACCACTGCT GGTGCTTCAC 1200
ACCTCAGGAA AGATGTGAAA ATATTGCCAT CTCCACTATG CACACAGACT CCAAGAGGTC 1260
TT AGATTG GCTCGTATAA CAACAGGTAT ATGAAAATTG ATAATGCTGA AGCTCTTCTT 1320
GAATGGGAGC TGGACAGGTA ATGGTGGGAA TAGGGCAAGA TGCAGAAAGA AGAAACCAGA 1380
ACCAAAAATA GCAACTTTAT ACCCACTTTT CCTTTAGGCT AAGACTGCCT GTCTCATATG 1440
TCTATCCAAC ACACCCTCCG GAATTCGATA TCAAGCTTAT CGATACCGTC GACCTCGAG 1499 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 370 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Glu Val Ser Leu Asn His Pro Ala Ser Asn Thr Thr Ser Thr Lys 1 5 10 15
Asn Asn Asn Ser Ala Phe Phe Tyr Phe Glu Ser Cys Gin Pro Pro Ser 20 25 30
Pro Ala Leu Leu Leu Leu Cys He Ala Tyr Thr Val Val Leu He Val
35 40 45
Gly Leu Phe Gly Asn Leu Ser Leu He He He He Phe Lys Lys Gin
50 55 60 Arg Lys Ala Gin Asn Phe Thr Ser He Leu He Ala Asn Leu Ser Leu 65 70 75 80
Ser Asp Thr Leu Val Cys Val Met Cys He His Phe Thr He He Tyr 85 90 95
Thr Leu Met Asp His Trp He Phe Gly Asp Thr Met Cys Arg Leu Thr 100 105 110
Ser Tyr Val Gin Ser Val Ser He Ser Val Ser He Phe Ser Leu Val 115 120 125
Phe Thr Ala Val Glu Arg Tyr Gin Leu He Val Asn Pro Arg Gly Trp 130 135 140
Lys Pro Ser Val Thr His Ala Tyr Trp Gly He Thr Leu He Trp Leu 145 150 155 160
Phe Ser Leu Leu Leu Ser He Pro Phe Phe Leu Ser Tyr His Leu Thr 165 170 175
Asp Glu Pro Phe His Asn Leu Ser Leu Pro Thr Asp Leu Tyr Thr His 180 185 190
Gin Val Ala Cys Val Glu Asn Trp Pro Ser Lys Lys Asp Arg Leu Leu 195 200 205
Phe Thr Thr Ser Leu Phe Leu Leu Gin Tyr Phe Val Pro Leu Gly Phe 210 215 220
He Leu He Cys Tyr Leu Lys He Val He Cys Leu Arg Arg Arg Asn 225 230 235 240
Ala Lys Val Asp Lys Lys Lys Glu Asn Glu 'Gly Arg Leu Asn Glu Asn 245 250 255
Lys Arg He Asn Thr Met Leu He Ser He Val Val Thr Phe Gly Ala 260 265 270
Cys Trp Leu Pro Pro Asn He Phe Asn Val He Phe Asp Trp Tyr His 275 280 285
Glu Val Leu Met Ser Cys His His Asp Leu Val Phe Val Val Cys His 290 295 300
Leu Val Ala Met Val Ser Thr Cys He Asn Pro Leu Phe Tyr Gly Phe 305 310 315 320
Leu Asn Lys Asn Phe Gin Lys Asp Leu Val Val Leu He His His Cys 325 330 335
Trp Cys Phe Thr Pro Gin Glu Arg Cys Glu Asn He Ala He Ser Thr 340 345 350
Met His Thr Asp Ser Lys Arg Ser Leu Arg Leu Ala Arg He Thr Thr 355 360 365 -56-
Gly He 370
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GATCGAATTC GCCATGGAAG TGCTCACAAA C 31
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GATCTCTAGA CTATATGCCT GTTGGTATGT G 31

Claims

WHAT IS CLAIMED IS:
1. An isolated DNA sequence encoding a neuropeptide Y Yx receptor, the DNA sequence having a sequence selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3, a fragment of the sequence of substantial homology to the sequence shown in Figure 3, a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5.
2. The isolated DNA sequence of Claim 1 , wherein the DNA sequence has a sequence selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3 or a fragment of the sequence of substantial homology to the sequence shown in Figure 3.
3. The DNA sequence of Claim 2, which encodes a murine NPY Yx receptor.
4. The DNA sequence of Claim 2, having a sequence selected from the sequence shown in Figure 3, the sequence of substantial homology to the sequence shown in Figure 3 or the fragment of the sequence shown in Figure 3 comprising bases 822 to 1934.
5. An expression vector containing the DNA sequence of Claim 2.
6. A cell transformed by the expression vector of Claim 5.
7. A method of producing a neuropeptide Y Yx receptor, comprising culturing the cell of Claim 6 under conditions which allow the production of the neuropeptide Y Yx receptor and optionally recovering the neuropeptide Y Yx receptor.
8. The method of Claim 7, wherein the cell is a mammalian cell.
9. The method of Claim 8, wherein the cell is a COS-7 cell.
10. A neuropeptide Y Yx receptor produced by the process of Claim 7.
11. The neuropeptide Y Yx receptor of Claim 10, which is characterized by a pharmacological binding profile with affinities of PYY = NPY = [Leu31Pro34]NPY = NPY(2-36) > NPY( 13-36).
12. A neuropeptide Y Yx receptor, or a functional derivative thereof, in substantially pure form, which is characterized by a pharmacological binding profile with affinities of PYY ≡ NPY = [Leu 1Pro34]NPY = NPY(2-36) > NPY(13-36).
13. Antibody immunologically reactive with the receptor of Claim 12.
14. Isolated RNA encoded by the DNA sequence of Claim 2 or its complementary sequence.
15. A single-stranded DNA, or fragment thereof, with a sequence complementary to the RNA of Claim 14.
16. The DNA of Claim 15, wherein the DNA, or fragment thereof, modulates the expression of the receptor.
17. The isolated DNA sequence of Claim 1 , wherein the DNA sequence has a sequence selected from a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5.
18. The DNA sequence of Claim 17, which encodes a human NPY Yx receptor.
19. The DNA sequence of Claim 17, having a sequence selected from the sequence shown in Figure 5, the sequence of substantial homology to the sequence shown in Figure 5 or the fragment of the sequence shown in Figure 5 comprising bases 182 to 1291.
20. An expression vector containing the DNA sequence of Claim 17.
21. A cell transformed by the expression vector of
Claim 20.
22. A method of producing a neuropeptide Y Yx receptor, comprising culturing the cell of Claim 21 under conditions which allow the production of the neuropeptide Y Yx receptor and optionally recovering the neuropeptide Y Yx receptor.
23. The method of Claim 22, wherein the cell is a mammalian cell.
24. The method of Claim 23, wherein the cell is selected from a COS-7 or a CHO cell.
25. A neuropeptide Y Yx receptor produced by the process of Claim 22.
26. The neuropeptide Y Yx receptor of Claim 25, which is characterized by a pharmacological binding profile with affinities of
PYY ≡ NPY ≡ [Leu31Pro34]NPY = NPY(2-36) > NPY( 13-36).
27. Isolated RNA encoded by the DNA sequence of Claim 17 or its complementary sequence.
28. A single-stranded DNA, or fragment thereof, with a sequence complementary to the RNA of Claim 27.
29. The DNA of Claim 28, wherein the DNA, or fragment thereof, modulates the expression of the receptor.
30. A method of identifying compounds that modulate neuropeptide Y Yx receptor activity or modulate binding of ligands to the NPY Yx receptor, comprising: (a) mixing a test compound with a solution containing the neuropeptide Y Yx receptor, or functional derivative thereof, to form a mixture;
(b) measuring NPY Yx receptor activity in the mixture; and (c) comparing the receptor activity of the mixture to a standard.
31. The method of Claim 30, wherein the neuropeptide Y Yx receptor, or functional derivative thereof, is characterized by a pharmacological binding profile with affinities of PYY ≡ NPY ___ [Leu31pr034]NPY ≡ NPY(2-36) > NPY( 13-36).
32. A method of identifying compounds that modulate the NPY Yx receptor, comprising: (a) mixing a test compound with cells which express the NPY Yx receptor to form a mixture;
(b) culturing the cells in the presence of the test compound; (c) measuring NPY Yx receptor or second messenger activity in the mixture; and
(d) comparing the NPY Yx receptor activity of the mixture to a standard.
33. The method of Claim 32, wherein the cells are transformed by an expression vector containing a sequence selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3, a fragment of the sequence of substantial homology to the sequence shown in Figure 3, a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5.
34. The method of Claim 33, wherein the expression vector contains a DNA sequence selected from a DNA sequence having a sequence selected from a sequence as shown in Figure 3, a sequence of substantial homology to the sequence shown in Figure 3, a fragment of the sequence shown in Figure 3 or a fragment of the sequence of substantial homology to the sequence shown in Figure 3.
35. The method of Claim 34, wherein the expression vector contains a DNA sequence selected from the sequence shown in Figure 3, the sequence of substantial homology to the sequence shown in Figure 3 or the fragment of the sequence shown in Figure 3 comprising bases 822 to 1934.
36. The method of Claim 33, wherein the expression vector contains a DNA sequence selected from a DNA sequence having a sequence selected from a sequence as shown in Figure 5, a sequence of substantial homology to the sequence shown in Figure 5, a fragment of the sequence shown in Figure 5 or a fragment of the sequence of substantial homology to the sequence shown in Figure 5.
37. The method of Claim 36, wherein the expression vector contains a DNA sequence selected from the sequence shown in Figure 5, the sequence of substantial homology to the sequence shown in Figure 5 or the fragment of the sequence shown in Figure 5 comprising bases 182 to 1291.
38. A neuropeptide Y Yx receptor in substantially pure form comprising an amino acid sequence selected from a sequence as shown in Figure 3 (SEQ. I.D. NO. 6), a sequence as shown in Figure 6 (SEQ. I.D. NO. 12), or a functional derivative thereof.
39. The neuropeptide Y Yx receptor of Claim 38, wherein the receptor has an amino acid sequence as shown in Figure 3 (SEQ. I.D. NO. 6) or a functional derivative thereof.
40. The neuropeptide Y Yx receptor of Claim 38, wherein the receptor has an amino acid sequence as shown in Figure 6 (SEQ. I.D. NO. 12) or a functional derivative thereof.
41. A method of identifying compounds that bind to a NPY Yx receptor comprising: a) cloning DNA which encodes a neuropeptide Y Yx receptor; b) splicing the DNA into an expression vector to produce a construct such that the NPY Yx receptor is operably linked to transcription and translation signals sufficient to induce expression of the NPY Yx receptor upon introduction of the construct into a prokaryotic or eukaryotic cell; c) introducing the construct into a prokaryotic or eukaryotic cell which does not express the NPY Yx receptor in the absence of the introduced construct; and d) incubating cells or membranes isolated from cells produced in step c with a quantifiable compound known to bind to the NPY Yx receptor, and subsequently adding test compounds at a range of concentrations so that the test compounds compete with the quantifiable compound for the NPY Yx receptor, such that an IC50 for the test compound is obtained as the concentration of test compound at which 50% of the quantifiable compound becomes displaced from the NPY Yx receptor.
PCT/US1996/001444 1995-02-03 1996-01-30 Neuropeptide y receptor Ceased WO1996023809A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US08/894,236 US5939263A (en) 1995-02-03 1996-01-30 Neuropeptide Y receptor
EP96904553A EP0809648A4 (en) 1995-02-03 1996-01-30 NEUROPEPTIDE Y RECEPTOR
JP8523748A JPH11500610A (en) 1995-02-03 1996-01-30 Neuropeptide Y receptor

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US38374695A 1995-02-03 1995-02-03
US383,746 1995-02-03
US08/415,818 US5621079A (en) 1995-02-03 1995-04-03 Neuropeptide Y receptor
US415,818 1995-04-03

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JP (1) JPH11500610A (en)
CA (1) CA2212225A1 (en)
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US5968819A (en) * 1994-12-02 1999-10-19 Synaptic Pharmaceutical Corporation DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5)
US5989920A (en) * 1994-12-02 1999-11-23 Synaptic Pharmaceutical Corporation Methods of modifying feeding behavior compounds useful in such methods and DNA encoding a hypothalmic atypical neuropeptide Y/peptide YY receptor Y5
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US5958709A (en) * 1993-12-28 1999-09-28 Synaptic Pharmaceutical Corporation Processes for identifying compounds that bind to the human Y4 receptor
US6913892B1 (en) 1993-12-28 2005-07-05 H. Lundbeck A/S Method of obtaining compositions comprising Y4 specific compounds
US5976814A (en) * 1993-12-28 1999-11-02 Synaptic Pharmaceutical Corporation DNA encoding a human neuropeptide Y/peptide YY/pancreatic polypeptide receptor (Y4) and uses thereof
US5989834A (en) * 1994-02-03 1999-11-23 Synaptic Pharmaceutical Corporation Uses of nucleic acid encoding neuropeptide Y/peptide YY (Y2) receptors nucleic acid encoding
US6420532B1 (en) 1994-02-03 2002-07-16 Synaptic Pharmaceutical Corporation Method of obtaining compositions comprising Y2 specific compounds
US6316203B1 (en) 1994-12-02 2001-11-13 Synaptic Pharmaceutical Corporation Methods of screening and preparing a composition using DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5)
US5989920A (en) * 1994-12-02 1999-11-23 Synaptic Pharmaceutical Corporation Methods of modifying feeding behavior compounds useful in such methods and DNA encoding a hypothalmic atypical neuropeptide Y/peptide YY receptor Y5
US6645774B1 (en) 1994-12-02 2003-11-11 Synaptic Pharmaceutical Corporation Methods of modifying feeding behavior using compounds with afinity for the human hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5)
US6818445B2 (en) 1994-12-02 2004-11-16 Synaptic Pharmaceutical Corporation Methods of modifying feeding behavior, compounds useful in such methods, and DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5)
US5968819A (en) * 1994-12-02 1999-10-19 Synaptic Pharmaceutical Corporation DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5)
WO1997017440A1 (en) * 1995-11-09 1997-05-15 Garvan Institute Of Medical Research Neuropeptide y-y5 receptor
US6528303B1 (en) 1995-11-09 2003-03-04 Garvan Institute Of Medical Research Neuropeptide Y-Y5 receptor
US6713265B1 (en) 1997-06-04 2004-03-30 Synaptic Pharmaceutical Corporation Methods of modifying feeding behavior, compounds useful in such methods, and DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5)
WO2001057085A3 (en) * 2000-02-02 2002-03-14 Incyte Genomics Inc G-protein coupled receptors

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CA2212225A1 (en) 1996-08-08
US5621079A (en) 1997-04-15
EP0809648A4 (en) 2000-05-03
JPH11500610A (en) 1999-01-19
US5939263A (en) 1999-08-17
EP0809648A1 (en) 1997-12-03

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