EP0907734A2 - RECEPTEUR DE LA GALANINE GalR2 - Google Patents

RECEPTEUR DE LA GALANINE GalR2

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Publication number
EP0907734A2
EP0907734A2 EP97929814A EP97929814A EP0907734A2 EP 0907734 A2 EP0907734 A2 EP 0907734A2 EP 97929814 A EP97929814 A EP 97929814A EP 97929814 A EP97929814 A EP 97929814A EP 0907734 A2 EP0907734 A2 EP 0907734A2
Authority
EP
European Patent Office
Prior art keywords
leu
galr2
galanin
ala
ctg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97929814A
Other languages
German (de)
English (en)
Inventor
Brian T. Bloomquist
Michael L. Mccaleb
Linda J. Cornfield
Heeja Yoo-Warren
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer AG
Bayer Corp
Original Assignee
Bayer AG
Bayer Corp
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Filing date
Publication date
Priority claimed from US08/665,034 external-priority patent/US6410686B1/en
Application filed by Bayer AG, Bayer Corp filed Critical Bayer AG
Publication of EP0907734A2 publication Critical patent/EP0907734A2/fr
Withdrawn legal-status Critical Current

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Definitions

  • This invention relates to a novel neuropeptide galanin receptor and its nucleic acid sequence.
  • Galanin is a 29 amino acid peptide hormone (30 amino acids in human) which is present in a wide range of central and peripheral tissues. Skofitsch, Peptides 6, 509 (1985); Merchenthaler, Prog. Neurobiol. 40, 711 (1993). Galanin is involved in many diverse physiological functions. Galanin is known to regulate the secretion of both endocrine and exocrine hormones. Galanin inhibits insulin secretion from pancreatic beta cells, and can inhibit pancreatic amylase secretion; in the stomach, galanin inhibits gastrin and somatostatin secretion.
  • Galanin stimulates VIP (vasoactive intestinal protein) release from the hypothalamus, prolactin and growth hormone release from the pituitary; and inhibits the secretion of ACTH in the hypothalamus. Furthermore, the secretion of neurotransmitters can be modulated. For example, galanin can inhibit the release of histamine and norepinephrine in the hypothalamus. Other secondary messenger systems are also regulated: galanin can either stimulate or inhibit intracellular cAMP accumulation; is involved in the closure of N- and L-type voltage-sensitive calcium channels, and in the opening of ATP-sensitive and - insensitive potassium channels; and has been shown to stimulate the release of calcium from intracellular stores. Moreover, galanin is involved in the inhibition of acetylcholine release and the inhibition of muscarinic receptor-mediated phosphoinositide turnover. Bartfai, Crit. Rev. Neurobiol. 7, 229 (1993)
  • Galanin is implicated in the modulation of many cognitive and sensory functions. Galanin has potent antinociceptive effects, and can impair performance in one-trial learning, t-maze, and swim maze learning and memory paradigms. Its inhibition of the anoxic release of glutamate, as well as its inhibitory actions on cholinergic function suggest a role in neuroprotection, and in the development of Alzheimer's Disease. Crawley, Regulatory Peptides 59, 1 (1995). Galanin is known to induce feeding in rodents and, in contrast with the effects of Neuropeptide Y on feeding, galanin increases preference for fat intake. Akabayashi, Proc. Natl. Acad. Set. USA 91, 10375 (1994).
  • Galanin is also involved in the regulation of gastrointestinal smooth muscle contraction. Because of the important role of galanin in these many physiological processes, there is a strong need to further develop materials and methods for investigating the mechanistic behavior of the receptors and for treating diseased and other abnormal states associated with these physiological processes.
  • GalRl receptor has been obtained from rat. Burgevin, J. Mol. Neurosci. 6, 33 (1995). The in vivo functions mediated through this cloned GalRl receptor have not yet been elucidated.
  • EP-0711830-A2 disclose a closely-related GalRl sequence, differing in that Cysl5 ⁇ Trp is varied.
  • the present invention provides a novel galanin receptor protein, the GalR2 receptor. Also provided are the nucleic acid sequences encoding this novel receptor protein as well as methods for using this protein and its nucleic acid sequence, and methods useful for developing and identifying compounds for the treatment of diseases and disorders in which galanin is implicated. The importance of this discovery is manifested in the effects of galanin, which include antinociceptive activity, smooth muscle contraction, cardiovascular activity, pituitary hormone release, cognition, and increased food intake. Thus, this receptor protein is useful for screening for galanin agonist and antagonist activity for controlling these conditions.
  • nucleic acid sequences encoding the complete rat receptor and partial sequences of the human receptor.
  • These nucleic acid sequences have a variety of uses. For example, they are useful for making vectors and for transforming cells, both of which are ultimately useful for production of the GalR2 receptor protein. They are also useful as scientific research tools for developing nucleic acid probes for determining receptor expression levels, e.g., to identify diseased or otherwise abnormal states. They are useful for developing analytical tools such as antisense oligonucleotides for selectively inhibiting expression of the receptor gene to determine physiological responses.
  • the present invention can also be used to isolate the homologous nucleic acid sequence of other species, such as human, primate, dog, mouse, etc.
  • a homogeneous composition comprising the receptor GalR2 protein.
  • the protein is useful for screening drugs for agonist and antagonist activity, and, therefore, for screening for drugs useful in regulating physiological responses associated with the GalR2 receptor.
  • antagonists to the GalR2 receptor could be used to treat obesity and diabetes by reducing appetite and food consumption, whereas agonists could be used for the treatment of anorexic conditions.
  • drugs could be used to treat Alzheimer's disease, stroke, neuropathic pain, and/or endocrine disorders.
  • the proteins are also useful for developing antibodies for detection of the protein.
  • vectors such as plasmids, comprising the receptor GalR2 nucleic acid sequence that may further comprise additional regulatory elements, e.g., promotors,
  • additional regulatory elements e.g., promotors
  • transformed cells that express the GalR2 receptor
  • nucleic acid probes e.g., nucleic acid probes
  • antisense oligonucleotides e.g., agonists, (f) antagonists, and (g) transgenic mammals.
  • transgenic mammals e.g., transgenic mammals.
  • Further aspects of the invention comprise methods for making and using the foregoing compounds and compositions.
  • the invention includes polynucleotide molecules coding for a rat or human GalR2, or a galanin binding fragment thereof.
  • a purified and isolated rat or human GalR2 protein The GalR2 protein comprising SEQ ID NO:2.
  • the full-length GalR2 protein the partial sequence of which is indicated in SEQ ID NO:6.
  • SEQ ID NO:3 or a galanin binding fragment thereof.
  • a polynucleotide molecule comprising SEQ ID NO: 3 or a degenerate variant thereof.
  • a purified and isolated variant of rat GalR2 protein comprising SEQ ID NO:4.
  • Figure 1 is the polynucleotide sequence of rat GalR2 of the invention.
  • Figure 2 is the amino acid sequence of rat GalR2.
  • Figures 3-4 are the polynucleotide sequence of the Y107 variant of rat GalR2.
  • Figure 5 is the amino acid sequence of Yl 07(omitting putative intron).
  • Figure 6 is the partial polynucleotide sequence of human GalR2.
  • Figure 7 is the partial amino acid sequence of human GalR2.
  • Figure 8 is the representative saturation isotherm of [ 125 I]hGalanin binding to rat GalR2
  • the present invention comprises, in part, a novel galanin receptor protein, the GalR2 receptor.
  • Particularly preferred embodiments of the GalR2 receptor are those having an amino acid sequence substantially the same as SEQ ID NO: 2, 4 or 6.
  • reference to the GalR2 receptor is meant as a reference to any protein having an amino acid sequence substantially the same as SEQ ID NO: 2, 4 or 6.
  • the present invention also comprises the nucleic acid sequence encoding the GalR2 protein, which nucleic acid sequence is substantially the same as SEQ ID NO: 1, 3 or 5.
  • Receptors SEQ ID NO: 1 and SEQ ID NO: 3 are nucleic acid sequences of rat GalR2 receptors but SEQ ID NO: 3 appears to contain an intronic region; therefore, SEQ ID NO: 1 is the preferred embodiment of the rat GalR2 receptor of this invention.
  • Receptor SEQ ID NO: 5 is the partial nucleic acid sequence of human GalR2.
  • Receptors SEQ ID NO: 2 and 4 are rat GalR2 receptors and appear to be allelic variants.
  • Receptor SEQ ID NO: 6 is the partial amino acid sequence of human GalR2.
  • a protein "having an amino acid sequence substantially the same as SEQ ID NO: x" means a protein whose amino acid sequence is the same as SEQ ID NO: x or differs only in a way such that IC 50 [galanin] as determined according to the method detailed in Example 2, infra, are less than or equal to 1 nM.
  • a molecule having a nucleotide sequence substantially the same as SEQ ID NO: y means a nucleic acid encoding a protein "having an amino acid sequence substantially the same as SEQ ID NO: y*" (wherein “y*” is the number of the amino acid sequence for which nucleotide sequence "y” codes) as defined above.
  • This definition is intended to encompass natural allelic variations in the GalR2 sequence.
  • Cloned nucleic acid provided by the present invention may encode GalR2 protein of any species of origin, including (but not limited to), for example, mouse, rat, rabbbit, cat, dog, primate, and human.
  • the nucleic acid provided by the invention encodes GaIR2 receptors of mammalian, and most preferably, rat or human origin.
  • the invention also includes nucleotide sequences encoding chimeric proteins comprised of parts of the GalR2 receptor and parts of other related seven-transmembrane receptors.
  • the BMB77 clone (SEQ ID NO: 1) (see Example 1, infra) has a 1.7-kb cDNA insert with a open reading frame from nucleotides 279 to 1394 that encodes a 372 amino acid protein (SEQ ID NO: 2).
  • Hydrophobicity plot analysis using the PEPPLOT function of GCG shows that the GalR2 receptor has seven transmembrane-like domains, indicating it might be a G-protein-coupIed receptor.
  • GalR2 is 26 amino acids longer in length than GalRl, the only other published galanin receptor. This extra length of amino acids within GalR2 is due to an extended C-terminal tail sequence. However, the putative N-terminal extracellular domain of GalR2 is about 7 amino acids shorter than the corresponding region in GalRl. It is also important to note that the GaIR2 sequence shows only 38% amino acid sequence identity to the GalRl receptor.
  • the Y107 clone (SEQ ID NO: 3) ⁇ see Example 1, infra) has a 1.9-kb cDNA insert with an open reading frame from nucleotides 17-384, and from nucleotides 874-1621.
  • the sequence between nucleotides 384 and 874 contains multiple STOP codons in all three reading frames.
  • the dinucleotides GT and AG at positions 385-386 and 872- 873, respectively, fulfill the criteria for being a splice donor and acceptor site, respectively.
  • the partial human GalR2 nucleic acid sequence (SEQ ID NO: 5) contains a 337 amino acid opening reading frame.
  • the initial leucine residue of this partial human GalR2 protein (SEQ ID NO: 6) corresponds to amino acid Leu 51 in rat GalR2 (SEQ ID NO: 2 and 4).
  • Nucleic acid hybridization probes provided by the invention are DNAs consisting essentially of the nucleotide sequences complementary to any sequence depicted in SEQ ID NO:s 1 and 3 that is effective in nucleic acid hybridization.
  • Nucleic acid probes are useful for detecting GalR2 gene expression in cells and tissues using techniques well-known in the art, including, but not limited to, Northern blot hybridization, in situ hybridization, and Southern hybridization to reverse transcriptase - polymerase chain reaction product DNAs.
  • the probes provided by the present invention including oligonucleotide probes derived therefrom, are also useful for Southern hybridization of mammalian, preferably human, genomic DNA for screening for restriction fragment length polymorphism (RFLP) associated with certain genetic disorders.
  • RFLP restriction fragment length polymorphism
  • the term complementary means a nucleic acid having a sequence that is sufficiently complementary in the Watson-Crick sense to a target nucleic acid to bind to the target under physiological conditions or experimental conditions which those skilled in the art routinely use when employing probes.
  • a protein having substantially the same affinity profile as the GalR2 receptor means a protein in which the IC 50 of each of the peptides listed in Table 1 , infra, is no more than an order of magnitude greater than those listed in Table 1 for each of the respective peptides as measured according to the methods described in
  • DNA which encodes receptor GalR2 may be obtained, in view of the instant disclosure, by chemical synthesis, by screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by screening genomic libraries from appropriate cells, or by combinations of these procedures, as illustrated below. Screening of mRNA or genomic DNA may be carried out with oligonucleotide probes generated from the GalR2 gene sequence information provided herein.
  • Probes may be labeled with a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with known procedures and used in conventional hybridization assays, as described in greater detail in the Examples below.
  • the GalR2 gene sequence may be obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers being produced from the GalR2 gene sequence provided herein. See U.S. Patent Nos.
  • Receptor GalR2 may be synthesized in host cells transformed with a recombinant expression construct comprising a nucleic acid encoding the receptor GalR2.
  • a recombinant expression construct can also be comprised of a vector that is a replicable DNA construct.
  • Vectors are used herein either to amplify DNA encoding GalR2 and/or to express DNA which encodes GalR2.
  • a recombinant expression construct is a replicable DNA construct in which a DNA sequence encoding GaIR2 is operably linked to suitable control sequences capable of effecting the expression of GalR2 in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen.
  • control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation.
  • Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. See, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Press, New York, 1989).
  • Vectors useful for practicing the present invention include plasmids, viruses
  • the vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself.
  • Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host.
  • the vectors may be self-replicating.
  • Suitable vectors for the purposes of the present invention include pBluescript, pcDNA3, pSV-SPORT, and, for insect cells, baculovirus.
  • a preferred vector is the plasmid pcDNA3 (Invitrogen, San Diego, CA).
  • Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and relegated in the form desired.
  • Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions that are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog.
  • suitable restriction enzyme or enzymes
  • about 1 ⁇ g of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ⁇ l of buffer solution. Often excess of restriction enzyme is used to ensure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37°C are workable, although variations are tolerable. After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction.
  • the nucleic acid may be recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymology 65, 499-560 (1980).
  • Transformed host cells are cells which have been transformed or transfected with recombinant expression constructs made using recombinant DNA techniques and comprising mammalian GalR2-encoding sequences.
  • Preferred host cells for transient transfection are
  • Transformed host cells may ordinarily express GalR2, but host cells transformed for purposes of cloning or amplifying nucleic acid hybridization probe DNA need not express the receptor.
  • the mammalian GalR2 protein When expressed, the mammalian GalR2 protein will typically be located in the host cell membrane. See, Sambrook et al., ibid. Cultures of cells derived from multicellular organisms are desirable hosts for recombinant GalR2 protein synthesis. In principal, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. However, mammalian cells are preferred, as illustrated in the Examples. Propagation of such cells in cell culture has become a routine procedure. See Tissue Culture (Academic Press, Kruse & Patterson, Eds., 1973). Examples of useful host cell lines are bacteria cells, insect cells, yeast cells, human 293 cells,
  • VERO and HeLa cells LM ⁇ C cells, and WI 138, BHK, CHO, COS-7, CV, and MDCK cell lines (American Type Culture Collection, Rockville, MD). CHO cells are preferred.
  • the invention provides homogeneous compositions of mammalian GalR2 produced by transformed eukaryotic cells as provided herein. Such homogeneous compositions are intended to be comprised of mammalian GalR2 protein that comprises at least 90% of the protein in such homogenous composition.
  • the invention also provides membrane preparation from cells expressing GalR2 as the result of transformation with a recombinant expression construct, as described here.
  • Mammalian GalR2 protein made from cloned genes in accordance with the present invention may be used for screening compounds for GalR2 agonist or antagonist activity, or for determining the amount of a GalR2 agonist or antagonist drug in a solution (e.g., blood plasma or serum).
  • host cells may be transformed with a recombinant expression construct of the present invention, GalR2 protein expressed in those host cells, the cells lysed, and the membranes from those cells used to screen compounds for GaIR2 binding activity.
  • Competitive binding assays in which such procedures may be carried out are well known in the art. By selection of host cells which do not ordinarily express GalR2, pure or crude preparations of membranes containing GalR2 can be obtained.
  • GaIR2 agonists and antagonists can be identified by transforming host cells with a recombinant expression construct as provided by the present invention. Membranes obtained from such cells (and membranes of intact cells) can be used in binding studies wherein the drug dissociation activity is monitored.
  • the neurotransmitter galanin is a regulator of appetite, cognition, endocrine function, pain, and smooth muscle control.
  • the various galanin analogs/fragments that induce these physiological responses bind with a high affinity to the GalR2 receptor. It is therefore evident that by modulating the activity of the GalR2 receptor, various physiological activities can be regulated.
  • antagonists to the GalR2 receptor identified by the methods described herein, could be used to treat obesity, diabetes, hyperlipidemia, stroke, neuropathic pain, Alzheimer's disease, and/or endocrine disorders.
  • This invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a drug identified by the method described herein and a pharmaceutically acceptable carrier.
  • Such drugs and carrier can be administered by various routes, for example oral, subcutaneous, intramuscular, intravenous or intracerebral.
  • the preferred route of administration would be oral at daily doses of about 0.01-100 mg/kg.
  • This invention provides a method of treating obesity, diabetes, hyperlipidemia, stroke, neuropathic pain, Alzheimer's disease, or endocrine disorders wherein the abnormality is improved by reducing the activity of GalR2 receptor or blocking the binding of ligands to a GalR2 receptor which comprises administering an effective amount of the pharmaceutical composition described above.
  • the recombinant expression constructs of the present invention are useful in molecular biology to transform cells which do not ordinarily express GalR2 to thereafter express this receptor. Such cells are useful as intermediates for making cell membrane preparations useful for receptor binding assays, which are in turn useful for drug screening. Drugs identified from such receptor assays can be used for the treatment of obesity, diabetes, anorexia, hyperlipidemia, stroke, neuropathic pain, Alzheimer's disease, or endocrine disorders.
  • the recombinant expression constructs of the present invention are also useful in gene therapy.
  • Cloned genes of the present invention, or fragments thereof, may also be used in gene therapy carried out by homologous recombination or site-directed mutagenesis. See generally Thomas & Capecchi, Cell 51, 503-512 (1987); Bertling, Bioscience Reports 7, 107- 112 (1987); Smithies et al., Nature 317, 230-234 (1985).
  • Oligonucleotides of the present invention are useful as diagnostic tools for probing GalR2 gene expression in tissues.
  • tissues are probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiographic techniques, as explained in greater detail in the Examples below, to investigate native expression of this receptor or pathological conditions relating thereto.
  • chromosomes can be probed to investigate the presence or absence of the GalR2 gene, and potential pathological conditions related thereto, as also illustrated by the Examples below.
  • Probes according to the invention should generally be at least about 15 nucleotides in length to prevent binding to random sequences, but, under the appropriate circumstances may be smaller.
  • the invention also provides antibodies that are immunologically reactive to a mammalian GalR2, preferably rat or human GalR2.
  • the antibodies provided by the invention are raised in animals by inoculation with cells that express a mammalian GalR2 or epitopes thereof, using methods well known in the art.
  • Animals that are used for such inoculations include individuals from species comprising cows, sheep, pigs, mice, rats, rabbits, hamsters, goats and primates.
  • Preferred animals for inoculation are rodents (including mice, rats, hamsters) and rabbits. The most preferred animal is the mouse.
  • Cells that can be used for such inoculations, or for any of the other means used in the invention include any cell line which naturally expresses a mammalian GalR2, or any cell or cell line that expresses a mammalian GalR2 or any epitope thereof as a result of molecular or genetic engineering, or that has been treated to increase the expression of a mammalian
  • GaIR2 by physical, biochemical or genetic means.
  • Preferred cells are human cells, most preferably HEK 293 cells that have been transformed with a recombinant expression construct comprising a nucleic acid encoding a mammalian GalR2, preferably a rat or human GalR2, and that express the mammalian GalR2 gene product.
  • the present invention provides monoclonal antibodies that are immunologically reactive with an epitope of mammalian GalR2 or fragment thereof and that is present on the surface of mammalian cells, preferably human or mouse cells. These antibodies are made using methods and techniques well known to those of skill in the art.
  • Monoclonal antibodies provided by the present invention are produced by hybridoma cell lines, that are also provided by the invention and that are made by methods well known in the art.
  • Hybridoma cell lines are made by fusing individual cells of a myeloma cell line with spleen cells derived from animals immunized with cells expressing the GalR2 receptor, preferably rat or human cells, as described above.
  • the myeloma cell lines used in the invention include lines derived from myelomas of mice, rats, hamsters, primates and humans.
  • Preferred myeloma cell lines are from mouse.
  • the animals from whom spleens are obtained after immunization are rats, mice and hamsters, preferably mice, most preferably Balb/c mice.
  • Spleen cells and myeloma cells are fused using a number of methods well known in the art, including but not limited to incubation with inactivated Sendai virus and incubation in the presence of polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the most preferred method for cell fusion is incubation in the presence of a solution of 45% (w/v) PEG- 1450.
  • Monoclonal antibodies produced by hybridoma cell lines can be harvested from cell culture supernatant fluids from in vitro cell growth; alternatively, hybridoma cells can be injected subcutaneously and/or into the peritoneal cavity of an animal, most preferably a mouse, and the monoclonal antibodies obtained from blood and/or ascites fluid.
  • Monoclonal antibodies provided by the present invention are also produced by recombinant genetic methods well known to those of skill in the art, and the present invention encompasses antibodies made by such methods that are immunologically reactive with an epitope of a mammalian GalR2.
  • the present invention encompasses fragments of the antibody that are immunologically reactive with an epitope of a mammalian GalR2. Such fragments are produced by any number of methods, including but not limited to proteolytic cleavage, chemical synthesis or preparation of such fragments by means of genetic engineering technology.
  • the present invention also encompasses single-chain antibodies that are immunologically reactive with an epitope of a mammalian GalR2 made by methods known to those of skill in the art.
  • the present invention also encompasses an epitope of a mammalian GalR2 that is comprised of sequences and/or a conformation of sequences present in the mammalian GalR2 molecule.
  • This epitope may be naturally occurring, or may be the result of proteolytic cleavage of the mammalian GalR2 molecule and isolation of an epitope-containing peptide or may be obtained by synthesis of an epitope-containing peptide using methods well known to those skilled in the art.
  • the present invention also encompasses epitope peptides produced as a result of genetic engineering technology and synthesized by genetically engineered prokaryotic or eukaryotic cells.
  • the invention also includes chimeric antibodies, comprised of light chain and heavy chain peptides immunologically reactive to an epitope that is a mammalian GaIR2.
  • the chimeric antibodies embodied in the present invention include those that are derived from 13 naturally occurring antibodies as well as chimeric antibodies made by means of genetic engineering technology well known to those of skill in the art.
  • non-human transgenic animals grown from germ cells transformed with the GalR2 nucleic acid sequence according to the invention and that express the GalR2 receptor according to the invention and offspring and descendants thereof.
  • transgenic non-human mammals comprising a homologous recombination knockout of the native GalR2 receptor, as well as transgenic non-human mammals grown from germ cells transformed with nucleic acid antisense to the GaIR2 nucleic acid of the invention and offspring and descendants thereof.
  • transgenic animals which the native GalR2 receptor has been replaced with the human homolog.
  • offspring and descendants of all of the foregoing transgenic animals are also encompassed by the invention.
  • Transgenic animals according to the invention can be made using well known techniques with the nucleic acids disclosed herein.
  • transgenic animals are useful for screening for and determining the physiological effects of GalR2 receptor agonists and antagonist. Consequently, such transgenic animals are useful for developing drugs to regulate physiological activities in which galanin participates.
  • oligo(dT) anchor primer was used for reverse transcription, and the library was cloned unidirectionally into pcDNA3 vector which contains a CMV (cytomegalovirus) promoter for eukaryotic expression.
  • the cDNA library had 5.3 x 10 s primary recombinants with an average insert size of 2.59 kb.
  • the probe was prepared by digesting the parent GalRl plasmid with Sad and AccI, separating the fragments by agarose gel electrophoresis, and purifying the 1-kb SacI-AccI fragment from the gel.
  • the probe was labeled with digoxigenin dUTP according to the manufacturer's instructions (GENIUS Kit, Boehringer Mannheim, Indianapolis, IN, PN 1093
  • the filter lifts, denaturation, neutralization, hybridization, and washing were done according to the manufacturer's instructions except that hybridization was done at 30°C and the washes were performed twice for 40 minutes each: once at room temperature; the second 15 at 37°C.
  • Plasmid DNA homologous to the probe was purified and subcloned into pBluescript vector (Stratagene (La Jolla, California) 212206) by standard molecular biological techniques.
  • This clone designated SI2112, was subjected to sequence analysis and was found to contain a sequence which had characteristics of a novel, but truncated member of the G-protein-coupled 7TMD receptor family.
  • This clone was later used as probe to determine the identity of a novel galanin-binding clone (Y107) found in the expression cloning strategy (see below.)
  • the partial human GalR2 cDNA was obtained by screening approximately two million phage of a human small intestine library (Clontech (Palo Alto, CA) HL 1133a) were screened with a human GalR2 probe obtained by PCR amplification from human genomic DNA. The conditions used were essentially as described for the rat GalR2 isolation.
  • the [ l25 IJgalanin binding assay was performed in the fiaskette chamber.
  • the cells were washed once with 25 mM Tris-HCl, 10 mM MgCl 2 , pH 7.4, and blocked for 15 minutes with 1 ml total binding buffer (25 mM Tris-HCl, 10 mM MgCl 2 , 1% bovine serum albumin, pH 7.4) at room temperature.
  • the darkbox was brought to room temperature for 1 hour after which the slides were developed in D- 19 developer (Kodak (Rochester, NY) 1464593) for three minutes at 15°C, placed in fixer (Kodak (Rochester, NY) 197 1746) for three minutes at 15°C, washed in water, and air dried.
  • Cells were stained with Diff-Quik stain set (Baxter (McGaw Park, IL) B4132-1) and air dried. Slides were dipped into xylenes and mounted with DPX mountant (Electron Microscopy Sciences (Fort Washington, PA) 13510). Positive cells were identified using dark field microscopy.
  • SI2112 probe and, 3) DNA from pool Y107, when used to transiently transfect COS-7 cells, conferred the ability upon the cells to bind galanin, it was deduced that the clone within pool Y107, which conferred galanin-binding ability when expressed, was likely to be an expressible version of the SI2112 clone. Therefore, clones from pool Y107 were probed with radiolabeled DNA prepared from SI2112, and a single clone hybridizing to the SI2112 probe was purified from non-homologous clones. This clone was called Y107.
  • the first intronless version of the Y107 was in the pSV-SPORT vector (GIBCO (Gaithersburg, MD) 15386-014); the complete cDNA insert of this clone was subcloned into the pcDNA3 vector (Invitrogen (San Diego, CA) V790-20) in order to maintain, for subsequent pharamacological analyses, common vector and promoter (CMV) backgrounds amongst our clones.
  • the intronless clone in pSV-SPORT was designated BMB77.sv40; the intronless clone contained within pcDNA3 is named BMB77.
  • Clones BMB77 and Y107 differ by one amino acid in sequence. Pharmacological analyses have been performed with both the Y107 (SEQ ID NO: 3 and 4) and BMB77 (SEQ ID NO: 1 and 2) clones.
  • Plasmid DNA was sequenced by Lark Technologies Inc. (Houston, Texas) and Biotechnology Resource Laboratory of Yale University (New Haven, CT) using Sequenase Kit (US Biochemical (Cleveland, OH) 70770) or Applied Biosystems' automatic sequencer system (Model 373 A). The peptide sequence was deduced from the long open-reading-frame of the nucleotide sequence. DNA and peptide sequences were analyzed using the GCG program (Genetics Computer Group, Madison, WI).
  • SEQ ID NO: 1 the nucleic acid sequence of clone BMB77
  • SEQ ID NO: 2 the amino acid sequence of clone BMB77
  • SEQ ID NO: 3 the nucleic acid sequence of clone Y107
  • SEQ ID NO: 4 the amino acid sequence, omitting the putative intronic region, of clone Y107
  • SEQ ID NO: 5 the partial nucleic acid sequence of human GalR2
  • SEQ ID NO: 6 the partial amino acid sequence of human GaIR2
  • Monkey kidney cells were maintained in T-l 75 cm 2 flasks (Nunc, Inc. (Naperville, IL) 171226) at 37°C with 5% CO 2 in a humidified atmosphere.
  • Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (GIBCO (Gaithersburg, MD) 11965- 092) supplemented with 2 mM glutamine, 10% fetal bovine serum, 1 mM sodium pyruvate and a antibiotic/antimycotic comprised of pennicillin streptomycin amphotericinB (GIBCO, Gaithersburg, MD, PN 15240-013).
  • DMEM Dulbecco's Modified Eagle Medium
  • GEBCO Gibco's Modified Eagle Medium
  • a antibiotic/antimycotic comprised of pennicillin streptomycin amphotericinB
  • Cells at 70% confluency were transfected with rat GalR2 DNA using the Lipofectamine reagent (GIBCO (G
  • DNA and 90 ⁇ l of lipofectamine were added to each flask. Media was replaced 24 hours post transfection, and membranes were harvested 24 hours later.
  • Rat GalRl Receptor (clone BMB77) 293 cells (human embryonic kidney, ATCC) were plated onto a T-25 flask one day prior to transfection, such that they were 50-70% confluent at the time of transfection.
  • 15 ug of rat GalR2 (BMB77) DNA were added to 0.3 ml of Optimem I culture media (GIBCO Life Sciences), and 25 ul of lipofectamine were added to 0.3 ml of Optimem I.
  • the DNA and lipofectamine solutions were combined and incubated at room temperature for 20 minutes. An additional 2.4 ml of Optimem I was added to the DNA/lipofectamine mixture.
  • the media was aspirated from T-25 flask containing 293 cells, washed with Optimem I, and the DNA/lipofectamine mixture was added to the 293 cells. After a 5-6 hour incubation in a 37°C incubator (5% CO 2 ) for 5-6 hours, the DNA/lipofectamine mixture was replaced with culture media (DMEM, 10% fetal bovine serum, 2 mM glutamine and antibiotic/antimycotic.)
  • culture media DMEM, 10% fetal bovine serum, 2 mM glutamine and antibiotic/antimycotic.
  • the media was removed from each flask of transfected cells, and the cells were washed twice with 20 ml ice-cold phosphate buffered saline.
  • the cells were scraped from the flask in 5 ml of Tris buffer (20 mM Tris-HCl, 5 mM EDTA, pH 7.7), and then transferred to a centrifuge tube. Each flask was rinsed with an additional 5 ml of Tris buffer, and combined in the centrifuge tube.
  • the cells were homogenized in a Polytron PT-3000 (Brinkman Instruments, Mill Valley, New York) for 2 x 10 seconds (12 mM probe, 7000-8000 rpm) and 19 centrifuged at 20,000 x g for 30 minutes at 4°C. The pellet was resuspended in fresh Tris buffer, and centrifuged again at 20,000 x g for 30 minutes at 4°C. Protein concentration was measured using the Bio-Rad kit according to the standard manufacturer's protocol (Bio-Rad Laboratories (Hercules, CA) 500-0001) using bovine IgG as the standard.
  • the binding assays were performed on 96-well plates (GF/C Millipore Corp., Bedford, MA PN MAFC NOB 50) pretreated with 0.3% polyethylenimine (PEI) for at least 3 hours prior to use.
  • PEI polyethylenimine
  • the PEI was aspirated from the plates on a vacuum manifold, and the wells were rinsed with 200 ⁇ l of ice-cold binding buffer (25 mM Tris, 10 mM MgCl 2 , 0.1%
  • Table 1 summarizes the IC J0 values (50% inhibition of specific binding, as determined using nonlinear regression analysis) of various standard peptides, fragments and chimeras for 20
  • Transiently expressed rat GalRl is included in Table 1 for comparison of its pharmacological profile to this novel rat GalR2 receptor.
  • the preliminary pharmacological binding profile for rat GalR2 differs from rat GalRl such that rat galanin itself has about 10-fold lower affinity for GalR2, no matter how it is expressed.
  • the chimeric peptide C7 also has about 10-fold lower affinity for GalR2; this difference, however, is not observed with rat GalR2 stably expressed in 293 cells.
  • Rat(l-16)galanin has about 5- to 10-fold lower affinity for GalR2 than GalRl (Table 1).
  • the ratio of affinities of (1-12) and (l-15)galanin are markedly different for GalRl (22-fold difference), while these two peptides have more equivalent binding affinities for GalR2.
  • Table 1 summarizes the IC 30 values for various standard peptides for [ l25 I]hGalanin binding to rat GalRl and GalR2 clones.
  • the averages ⁇ standard error of the mean (SEM) represent values from at least three independent experiments. Two independent experiments are represented by the average, followed by the individual values in parentheses. Remaining values without SEM are from a single experiment.
  • SEQ ID NO: 1 Clone BMB77 nucleic acid sequence
  • SEQ ID NO: 2 Clone BMB77 amino acid sequence
  • SEQ ID NO: 3 Clone Y107 nucleic acid sequence
  • SEQ ID NO: 4 Clone Y107 amino acid sequence (omitting putative intron)
  • SEQ ID NO: 5 Human GaIR2 partial nucleic acid sequence
  • SEQ ID NO: 6 Human GalR2 partial amino acid sequence

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract

L'invention concerne une nouvelle protéine récepteur de galanine, appelée récepteur GalR2. On décrit par ailleurs les séquences nucléotidiques codant la protéine, des procédés d'utilisation de cette protéine et de sa séquence nucléotidique, et des procédés de préparation et d'identification de composés propres au traitement des maladies et des troubles faisant intervenir la galanine. L'importance de l'invention réside dans les effets produits par la galanine, à savoir: activité antinociceptive, contraction des muscles lisses, activité cardio-vasculaire, secrétion d'hormones hypophysaires, cognition, et apport alimentaire accru. La protéine est donc utile pour le dépistage des activités agonistes et antagonistes liées à la galanine en vue de contrôler les états correspondants.
EP97929814A 1996-06-05 1997-06-05 RECEPTEUR DE LA GALANINE GalR2 Withdrawn EP0907734A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US665034 1996-06-05
US08/665,034 US6410686B1 (en) 1996-06-05 1996-06-05 Galanin receptor 2 protein
US86803497A 1997-06-03 1997-06-03
PCT/US1997/009787 WO1997046681A2 (fr) 1996-06-05 1997-06-05 RECEPTEUR DE LA GALANINE GalR2
1999-05-06

Publications (1)

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EP0907734A2 true EP0907734A2 (fr) 1999-04-14

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EP97929814A Withdrawn EP0907734A2 (fr) 1996-06-05 1997-06-05 RECEPTEUR DE LA GALANINE GalR2

Country Status (2)

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EP (1) EP0907734A2 (fr)
CA (1) CA2256523A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE9602822D0 (sv) 1996-07-19 1996-07-19 Astra Pharma Inc New receptor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9746681A3 *

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CA2256523A1 (fr) 1997-12-11

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