WO2000034333A1 - Nouveau recepteur couple aux proteines g - Google Patents

Nouveau recepteur couple aux proteines g Download PDF

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Publication number
WO2000034333A1
WO2000034333A1 PCT/SE1999/002302 SE9902302W WO0034333A1 WO 2000034333 A1 WO2000034333 A1 WO 2000034333A1 SE 9902302 W SE9902302 W SE 9902302W WO 0034333 A1 WO0034333 A1 WO 0034333A1
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receptor
test compound
cells
protein
seq
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PCT/SE1999/002302
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English (en)
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Sultan Ahmad
Cyrla Hoffert
Paola Lembo
Dajan O'donnell
Philippe Walker
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Astrazeneca Ab
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Priority to AU20181/00A priority Critical patent/AU2018100A/en
Priority to CA002352569A priority patent/CA2352569A1/fr
Priority to EP99963815A priority patent/EP1147135A1/fr
Publication of WO2000034333A1 publication Critical patent/WO2000034333A1/fr

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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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Definitions

  • the present invention relates to nucleic acids encoding a novel G protein-coupled receptor and to the receptor itself.
  • G protein-coupled receptors constitute a family of proteins sharing a common structural organization characterized by an extracellular N-terminal end, seven hydrophobic alpha helices putatively constituting transmembrane domains and an intracellular C-terminal domain. GPCRs bind a wide variety of ligands that trigger intracellular signals through the activation of transducing G proteins (Caron et al, Rec. Prog. Horm. Res. 48:211-290 (1993); Freedman et al., Rec. Prog. Horm. Res. 57:319-353 (1996)).
  • G protein-coupled receptors located in the central nervous system. G protein-coupled receptors in this region are known to be involved in the transmission, modulation and sensation of pain. Thus, G protein-coupled receptors derived from the brain and spinal column may be used in assays for the identification of new anesthetic and analgesic agents.
  • the present invention is based upon the discovery of a novel G protein-coupled receptor which is distinct from previously reported receptors in terms of structure and distribution. It is referred to herein as the "MP-10 receptor.”
  • the invention is directed to a protein, except as existing in nature, comprising an amino acid sequence consisting functionally of SEQ ID NO: 1.
  • consisting functionally of refers to proteins in which the sequence of SEQ LD NO: 1 has undergone additions, deletions or substitutions which do not substantially alter the functional characteristics of the receptor.
  • the term is intended to encompass proteins having exactly the same amino acid sequence as that of SEQ ID NO: l, as well as proteins with sequence differences that are not substantial as evidenced by their retaining the basic, qualitative ligand binding and physiological properties of the MP-10 receptor.
  • the term "except as existing in nature” refers to a compound that is either expressed by recombinant means or that is in a purified (preferably substantially purified) state.
  • the protein has an amino acid sequence consisting essentially of the sequence of SEQ LD NO: 1.
  • the invention includes antibodies that bind preferentially to such a protein (i. e.. antibodies having at least a 100-fold greater affinity for MP- 10 than any other protein): and antibodies made by a process involving the injection of a pharmaceutically acceptable preparation of MP- 10 into an animal capable of antibody production.
  • monoclonal antibody to MP-10 is produced by administering MP-10 to a mouse and then fusing the mouse's spleen cells with myeloma cells.
  • the invention is also directed to a polynucleotide, except as existing in nature, encoding a protein with an amino acid sequence consisting functionally of SEQ LD NO: l .
  • This aspect of the invention encompasses polynucleotides encoding proteins consisting essentially of the amino acid sequence of SEQ LD NO: l , expression vectors comprising such polynucleotides, and host cells transformed with such vectors. Also included is recombinant MP- 10 receptor produced by host cells made in this manner.
  • the polynucleotide encoding the MP-10 receptor has a sequence consisting essentially of nucleotides 61 1-1772 of SEQ LD NO:2, and the vectors and host cells used for expression of the receptor also use this particular polynucleotide.
  • the present invention is directed to a method for assaying a test compound for its ability to bind to the MP-10 receptor.
  • the method is performed by incubating a source of MP-10 with a ligand known to bind to the receptor and with the test compound.
  • the source of receptor should, preferably, express a large amount of MP-10 relative to other G protein-coupled receptors.
  • the ability of the test compound to bind to MP-10 is determined by the extent to which ligand binding has been displaced.
  • the receptor present should have the sequence shown in SEQ LD NO: l .
  • the binding assay can be accompanied by an assay to determine whether a second messenger pathway, e.g. , the adenyl cyclase pathway, has become activated. This should help to determine whether a particular compound binding to MP-10 is acting as an agonist or antagonist.
  • An alternative method for determining if a test compound is an MP- 10 agonist is to use a cell signaling assay, e.g., an assay measuring either intracellular adenyl cyclase activity or intracellular calcium concentration.
  • the test compound should generally be incubated with cells expressing high amounts of MP- 10 relative to other G protein-coupled receptors, typically a cell transfected with an expression vector encoding the MP- 10 of SEQ LD NO: l .
  • Test compounds that are agonists are identified by their causing a statistically significant change in the results obtained from the cell signaling assay when compared to control cells not exposed to test compound.
  • control cells may be either cells that have not been transfected or cells that have been mock transfected with a vector that does not produce active receptor.
  • MP- 10-expressing cells exposed to test compounds that are agonists would typically be expected to show significant signaling responses, for example changes in adenylate cyclase activity or an increase in intracellular calcium concentration relative to control cells.
  • the invention also encompasses a method for determining if a test compound is an antagonist of MP-10 which relies upon the known constitutive activation of G protein- coupled receptors that occurs when such receptors are expressed in large amounts.
  • This method requires that DNA encoding the receptor be incorporated into an expression vector so that it is operably linked to a promoter and that the vector then be used to transfect an appropriate host.
  • expression systems capable of copious protein production are preferred, e.g., the MP-10 DNA may be operably linked to a CMV promoter and expressed in COS or HEK293 cells. After transfection.
  • cells with activated receptors are selected based upon their showing increased activity in a cell signaling assay relative to comparable cells that have either not been transfected or that have been transfected with a vector that is incapable of expressing functional MP-10.
  • cells will be selected either because they show a statistically significant change in intracellular adenyl cyclase activity or in intracellular calcium concentration.
  • the selected cells are contacted with the test compound and the cell signaling assay is repeated to determine if this results in a decrease in activity relative to selected cells that have not been contacted with the test compound.
  • a statistically significant decrease in either adenyl cyclase activity or calcium concentration relative to control cells would indicate that the test compound is an antagonist of MP-10.
  • the MP-10 used in assays has the sequence of SEQ LD NO: 1.
  • Assays for compounds interacting with MP-10 may be performed by incubating a source containing the receptor (e.g., a stably transformed cell) with a ligand specific for MP-10 both in the presence and absence of test compound and measuring the modulation of intracellular calcium concentration.
  • a source containing the receptor e.g., a stably transformed cell
  • a significant increase or decrease in ligand-stimulated calcium signaling in response to test compound is indicative of an interaction occurring at the MP-10 receptor.
  • the preferred receptor is that having the amino acid sequence of SEQ LD NO: l.
  • the present invention is directed to a method for assaying a test compound for its ability to alter the expression of MP-10.
  • This method is performed by growing cells expressing MP-10 in the presence of the test compound. Cells are then collected and the expression of MP-10 is compared with expression in control cells grown under essentially identical conditions but in the absence of test compound.
  • the preferred receptor is one having the amino acid sequence of SEQ LD NO: l.
  • a preferred test compound is an oligonucleotide at least 15 nucleotides in length comprising a sequence complementary to the sequence of the MP-10 mRNA used in the assay.
  • Figure 1 contains the complete nucleotide sequence of a clone constructed by the methods described in the Examples section.
  • the clone was deposited with the International Depository Authority Deutsche Sammlung Von Mikroorganismen Und Zellkulturen GmbH at the address Mascheroder Weg 1 B, D-3300 Braunschweig, Germany. The deposit was made on November 13, 1998 and was given the accession number DSM 12499.
  • Figure 2 shows the deduced amino acid sequence of rat MP-10.
  • the polynucleotide of Figure 1 codes for a protein 387 amino acids in length.
  • Cloning vector A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell and which is characterized by one or a small number of restriction endonuclease recognition sites. A foreign DNA fragment may be spliced into the vector at these sites in order to bring about the replication and cloning of the fragment.
  • the vector may contain a marker suitable for use in the identification of transformed cells.
  • a marker may provide tetracycline resistance or ampicillin resistance.
  • Expression vector A vector similar to a cloning vector but which is capable of inducing the expression of the DNA that has been cloned into it, after transformation into a host.
  • the cloned DNA is usually placed under the control of (i.e., operably linked to) certain regulatory sequences such as promoters or enhancers. Promoter sequences may be constitutive, inducible or repressible.
  • substantially pure means that the desired product is essentially free from contaminating cellular components.
  • a "substantially pure" protein or nucleic acid will typically comprise at least 85% of a sample, with greater percentages being preferred. Contaminants may include proteins, carbohydrates or lipids.
  • One method for determining the purity of a protein or nucleic acid is by electrophoresing a preparation in a matrix such as polyacrylamide or agarose. Purity is evidenced by the appearance of a single band after staining. Other methods for assessing purity include chromatography and analytical centrifugation.
  • Recombinant protein A recombinant protein or recombinant receptor is a non-endogenous protein produced by the introduction of an expression vector into host cells.
  • Any prokaryotic or eukaryotic cell that is the recipient of a replicable expression vector or cloning vector is the "host" for that vector.
  • the term encompasses prokaryotic or eukaryotic cells that have been engineered to incorporate a desired gene on its chromosome or in its genome. Examples of cells that can serve as hosts are well known in the art, as are techniques for cellular transformation (see, e.g.. Sambrook et al.. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor (1989)).
  • Promoter A DNA sequence typically found in the 5 ' region of a gene, located proximal to the start codon. Transcription is initiated at the promoter. If the promoter is of the inducible type, then the rate of transcription increases in response to an inducing agent.
  • a complementary nucleotide sequence refers to the sequence that would arise by normal base pairing. For example, the nucleotide sequence 5 '-AGAC-3 ' would have the complementary sequence 5 '- GTCT-3 '.
  • Expression is the process by which a polypeptide is produced from DNA. The process involves the transcription of the gene into mRNA and the translation of this mRNA into a polypeptide.
  • the present invention is directed to an MP-10 receptor protein, genetic sequences coding for the protein, a method for assaying compounds binding to MP-10 receptors and a method for assaying compounds for their ability to alter receptor expression.
  • the receptor and the nucleic acid encoding the receptor are defined by the structures shown in figures 1 and 2 and by SEQ LD NOs: l and 2.
  • the invention encompasses not only sequences identical to those shown in the figures and sequence listing, but also sequences that are essentially the same and sequences that are otherwise substantially the same and which result in a receptor retaining the basic binding characteristics of MP-10.
  • techniques such as site-directed mutagenesis may be used to introduce variations into a protein's structure.
  • Variations in the MP-10 receptor introduced by this or some similar method are encompassed by the invention provided that the resulting receptor retains the basic qualitative binding and physiological characteristics of unaltered MP- 10.
  • the invention relates to proteins comprising amino acid sequences consisting functionally of SEQ LD NO: 1.
  • DNA sequences coding for rat MP-10 are expressed throughout the adult rat central nervous system, as well as in the spleen. Tissue from either of these areas may be used as a source for the isolation of nucleic acids coding for the receptor.
  • cells and cell lines that express MP-10 may be used. These may either be cultured cells that have not undergone transformation or cell lines specifically engineered to express recombinant
  • MP- 10 Most preferred are the cells deposited as DSM No. 12499.
  • the deposited cells, DNA encoding MP- 10 may be obtained as the result of standard restriction digestions.
  • poly A + mRNA may be isolated from tissue or cells, reverse transcribed and cloned.
  • the cDNA library thus formed may then be screened using probes derived from
  • Probes should typically be at least 14 bases in length and should, preferably, not be obtained from regions of the DNA corresponding to highly conserved transmembrane domains of MP-10.
  • MP-10 can also be obtained from recombinant cells containing the full-length MP- 10 sequence or from cDNA libraries by performing PCR amplifications using primers located at either end of the MP- 10 gene. These primers can be selected from the sequences shown in SEQ LD NO:2.
  • the present invention is also directed to antibodies that bind specifically to MP-10 and to a process for producing such antibodies.
  • Antibodies that "bind specifically" are defined as those that have at least a one hundred fold greater affinity for MP- 10 than for any other protein.
  • the process for producing such antibodies may involve either injecting the MP-10 protein itself into an appropriate animal or. alternatively, injecting short peptides made to correspond to different regions of the receptor.
  • the peptides should be at least five amino acids in length and should be selected from regions believed to be unique to MP- 10. Thus, highly conserved transmembrane regions should generally be avoided in selecting peptides for the generation of antibodies.
  • Antibody is meant to include intact molecules as well as fragments which retain their ability to bind to antigen (e.g. , Fab and F(ab 2 fragments). These fragments are typically produced by proteolytically cleaving intact antibodies using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • the term “antibody” also refers to both monoclonal antibodies and polyclonal antibodies. Polyclonal antibodies are derived from the sera of animals immunized with the antigen. Monoclonal antibodies can be prepared using hybridoma technology (Kohler et al, Nature 256:495 (1975); Hammerling et al, in: Monoclonal Antibodies and T-Cell
  • Hybridomas Elsevier, M.Y., pp. 563-681 (1981)).
  • this technology involves immunizing an animal, usually a mouse, with either intact MP-10 or a fragment derived from MP-10.
  • the splenocytes of the immunized animals are extracted and fused with suitable myeloma cells, e.g., SP 2 O cells. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then cloned by limiting dilution (Wands et al. ,
  • the antibodies, or fragments of antibodies, of the present invention may be used to detect the presence of MP-10 using any of a variety of immunoassays.
  • the antibodies may be used in radioimmunoassays or in immunometric assays, also known as "two-site” or “sandwich” assays (see Chard, T., "An Introduction to Radioimmune Assay and Related Techniques," in Laboratory Techniques in Biochemistry and Molecular Biology, North Holland Publishing Co., N.Y. (1978)).
  • a quantity of unlabeled antibody is bound to a solid support that is insoluble in the fluid being tested, e.g., blood, lymph, cellular extracts, etc.
  • a quantity of detectably labeled second antibody (which may or may not be the same as the first) is added to permit detection and/or quantitation of bound antigen (see, e.g., Radioimmune Assay Method, Kirkham et al, ed., pp. 199-206, E & S. Livingstone, Edinburgh (1970)).
  • detectably labeled second antibody (which may or may not be the same as the first) is added to permit detection and/or quantitation of bound antigen (see, e.g., Radioimmune Assay Method, Kirkham et al, ed., pp. 199-206, E & S. Livingstone, Edinburgh (1970)).
  • Assays are known in the art and may be employed for the detection of MP-10.
  • Antibodies to MP-10 may also be used in the purification of either intact receptor or fragments of the receptor (see generally. Dean et al., Affinity Chromatographv. A Practical Approach, LRL Press (1986)).
  • antibody is immobilized on a chromatographic matrix such as Sepharose 4B.
  • the matrix is then packed into a column and the preparation containing MP-10 is passed through under conditions that promote binding, e.g., under conditions of low salt.
  • the column is then washed and bound MP-10 is eluted using a buffer that promotes dissociation from antibody, e.g., buffer having an altered pH or salt concentration.
  • the eluted MP-10 may be transferred into a buffer of choice, e.g., by dialysis, and either stored or used directly.
  • MP-10 nucleic acids and recombinant proteins are in assays designed to identify agents capable of binding to the receptor. Such agents may either be agonists, mimicking the normal effects of receptor binding, or antagonists, inhibiting the normal effects of receptor binding. Of particular interest is the identification of agents which bind to the MP-10 receptor and modulate intracellular signaling, such as adenyl cyclase activity or intracellular calcium. These agents have potential therapeutic application as either analgesics or anesthetics.
  • a source of MP-10 is incubated together with a ligand known to bind to the receptor and with the compound being tested for binding activity.
  • the preferred source of MP-10 is cells, preferably mammalian cells, transformed to recombinantly express the receptor.
  • the cells selected should not express a substantial amount of any other G protein-coupled receptor that might bind to ligand and distort results. This can easily be determined by performing binding assays on cells derived from the same tissue or cell line as those recombinantly expressing MP-10 but which have not undergone transformation. The assay may be performed either with intact cells or with membranes prepared from the cells (see, e.g., Wang et al, Proc. Natl Acad. Sci. U.S.A. 90: 10230-10234 (1993)).
  • the membranes, or cells are incubated with a ligand specific for the MP-10 receptor and with a preparation of the compound being tested. After binding is complete, receptor is separated from the solution containing ligand and test compound, e.g. , by filtration, and the amount of binding that has occurred is determined.
  • the ligand used is detectably labeled with a radioisotope such as " I.
  • fluorescent or chemiluminescent labels can be used instead.
  • fluorescent labeling compounds are fluorescein isothiocynate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • Useful chemiluminescent compounds include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester. Any of these agents can be used to produce a ligand suitable for use in the assay.
  • Nonspecific binding may be determined by carrying out the binding reaction in the presence of a large excess of unlabeled ligand.
  • labeled ligand may be incubated with receptor and test compound in the presence of a thousandfold excess of unlabeled ligand.
  • Nonspecific binding should be subtracted from total binding, i.e. binding in the absence of unlabeled ligand, to arrive at the specific binding for each sample tested.
  • Other steps such as washing, stirring, shaking, filtering and the like may be included in the assays as necessary.
  • wash steps are included after the separation of membrane- bound ligand from ligand remaining in solution and prior to quantitation of the amount of ligand bound, e.g., by counting radioactive isotope.
  • the specific binding obtained in the presence of test compound is compared with that obtained in the presence of labeled ligand alone to determine the extent to which the test compound has displaced receptor binding.
  • the compound being tested should be in a buffer which does not itself substantially inhibit the binding of ligand to
  • MP-10 and should, preferably, be tested at several different concentrations. Preparations of test compound should also be examined for proteolytic activity and it is desirable that antiproteases be included in assays. Finally, it is highly desirable that compounds identified as displacing the binding of ligand to MP-10 receptor be reexamined in a concentration range sufficient to perform a Scatchard analysis of the results. This type of analysis is well known in the art and can be used for determining the affinity of a test compounds for receptor (see, e.g., Ausubel et al, Current Protocols in Molecular Biology. 11.2.1-1 1.2.19 (1993): Laboratory Techniques in Biochemistry and Molecular Biology. Work et al, ed., N.Y. (1978) etc.).
  • the activation of receptor by the binding of ligand may be monitored using a number of different assays.
  • adenyl cyclase assays may be performed by growing cells in wells of a microtiter plate and then incubating the wells in the presence or absence of test compound.
  • cAMP may then be extracted in ethanol, lyophilized and resuspended in assay buffer.
  • Assay of cAMP thus recovered may be carried out using any method for determining cAMP concentration, e.g. the Biotrack cAMP Enzyme-immunoassay System (Amersham) or the Cyclic AMP [ 3 H] Assay System (Amersham).
  • adenyl cyclase assays will be performed separately from binding assays, but it may also be possible to perform binding and adenyl cyclase assays on a single preparation of cells.
  • Other "cell signaling assays” that can be used to monitor receptor activity are described below.
  • MP-10 receptors may also be used to screen for drug candidates using cell signaling assays.
  • the DNA encoding a receptor is incorporated into an expression vector and then transfected into an appropriate host.
  • the transformed cells are then contacted with a series of test compounds and the effect of each is monitored.
  • assays that can be used are assays measuring cAMP production (see discussion above), assays measuring the activation of reporter gene activity, assays measuring the modulation of the binding of ligand, e.g. , GTP-gamma-S, or assays measuring changes in intracellular calcium concentration.
  • Cell signaling assays may also be used to identify MP-10 antagonists.
  • G protein-coupled receptors can be put into their active state even in the absence of their cognate ligand by expressing them at very high concentration in a heterologous system.
  • receptor may be overexpressed using the baculovirus infection of insect Sf9 cells or the MP- 10 gene may be operably linked to a CMV promoter and expressed in COS or HEK293 cells. In this activated constitutive state, antagonists of the receptor can be identified in the absence of ligand by measuring the ability of a test compound to inhibit constitutive cell signaling activity.
  • Appropriate assays for this are, again, cAMP assays, reporter gene activation assays or assays measuring the binding of GTP-gamma-S.
  • One preferred cell signaling assay is based upon cells stably transfected with MP-10 showing a change in intracellular calcium levels in response to incubation in the presence of ligand.
  • a procedure can be used to identify MP-10 agonists or antagonists that is similar to the radioreceptor assays discussed above except that calcium concentration is measured instead of bound radioactivity.
  • the concentration of calcium in the presence of test compound and ligand is compared with that in the presence of ligand alone to determine whether the test compound is interacting at the MP-10 receptor.
  • a statistically significant increase in intracellular calcium in response to test compound indicates that the test compound is acting as an agonist whereas a statistically significant decrease in intracellular calcium indicates that it is acting as an antagonist.
  • Assays may also be performed that measure the activation of a reporter gene.
  • a reporter gene e.g., a chloramphenicol acetyltransferase or luciferase gene
  • the cells are then incubated with test compounds and the expression of the reporter gene is compared to expression in control cells that do not express recombinant MP-10 but that are essentially identical in other respects.
  • a statistically significant change in reporter gene expression in the MP-10-expressing cells is indicative of a test compound that interacts with the MP-10 receptor.
  • MP-10 One way to either increase or decrease the biological effects of MP-10 is to alter the extent to which the receptor is expressed in cells. Therefore, assays for the identification of compounds that either inhibit or enhance expression are of considerable interest. These assays are carried out by growing cells expressing MP-10 in the presence of a test compound and then comparing receptor expression in these cells with expression in cells grown under essentially identical conditions but in the absence of test compound. As in the binding assays discussed above, it is desirable that the cells used be substantially free of competing G protein-coupled receptors.
  • One way to measure receptor expression is to fuse the MP- 10 sequence to a sequence encoding a peptide or protein that can be readily quantitated.
  • the MP-10 sequence may be ligated to a sequence encoding hemagglutinin and used to stably transfect cells. After incubation with test compound, the hemagglutinin/receptor complex can be immuno-precipitated and Western blots performed using anti-hemagglutinin antibody.
  • Scatchard analysis of binding assays may be performed with labeled ligand to determine receptor number.
  • the binding assays may be carried out as discussed above and will preferably utilize cells that have been engineered to recombinantly express MP-10.
  • a preferred group of test compounds for inclusion in the MP-10 expression assay consists of oligonucleotides complementary to various segments of the MP-10 nucleic acid sequence shown in SEQ LD NO:2. These oligonucleotides should be at least 15 bases in length and should be derived from non-conserved regions of the receptor nucleic acid sequence.
  • Oligonucleotides which are found to reduce receptor expression may be derivatized or conjugated in order to increase their effectiveness. For example, nucleoside phosphorothioates may be substituted for their natural counterparts (see Cohen, J., Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, CRC Press (1989)).
  • the oligonucleotides may also be delivered to a patient in vivo for the purpose of inhibiting MP-10 expression. When this is done, it is preferred that the oligonucleotide be administered in a form that enhances its uptake by cells.
  • the oligonucleotide may be delivered by means of a liposome or conjugated to a peptide that is ingested by cells (see, e.g., U.S. Patent Nos. 4,897,355 and 4,394,448; see also non-U.S. patent documents WO 8903849 and EP 0263740).
  • Other methods for enhancing the efficiency of oligonucleotide delivery are well known in the art and are also compatible with the present invention.
  • Degenerate polymer ase chain reaction One method of successfully obtaining MP-10 DNA involves performing a PCR amplification followed by the screening of a cDNA library. The following oligonucleotides were used as PCR primers:
  • oligonucleotides were used in a polymerase chain reaction mixture (total volume 50 ⁇ l) which contained about 250 ⁇ g of rat genomic DNA (NOVAGEN), IX PCR buffer (50 mM KC1, 1.5 mM MgCl 2 , 10 mM Tris-HCl (pH 8.9), Pharmacia), 400 ⁇ M dNTPs (Pharmacia); 50 pmol each of the above primers and 5U Taq polymerase
  • the 500bp rat fragment was identified as a probable GPCR partial sequence since it contained several structural features characteristic of a GPCR.
  • cDNA library screening The rat MP-10 fragment was used as a tool to isolate the full length rat clone. Several cDNA libraries were screened by PCR using the aforementioned oligonucleotides. One rat cDNA library, Stratagene rat brainstem/spinal cord (cat # 936521), was found to be positive. One million plaques were plated, transferred to Hybond N+ filters (Amersham), and denatured for 7 minutes (1.5M NaCl and 0.5M NaOH), followed by two neutralization treatments of 5 minutes each ( 1.5M NaCl, 0.5M Tris-HCl pH 7 and 0.001 EDTA). The filters were rinsed in 2X SSC and air-dried for one hour at room temperature.
  • the membranes were hybridized overnight at 42°C in 50% formamide, 10% dextran sulphate (from a 50% solution), 1% SDS, 5XSSC, 5X Denhardt's, lOmM Tris and lOO ⁇ g/ml of sonicated salmon sperm DNA.
  • the rat MP-10 fragment was randomly labeled using T7 Quick Prime (Pharmacia) and added to the hybridization solution at 10X10 6 cpm/ml. Filters were washed with 2XSSC/0.1%SDS at room temperature followed by a high stringency wash with 0.1% SDS/0.2XSSC at 65°C.
  • Riboprobe synthesis The plasmid pGEMT-MP-10 (containing a 501 bp fragment) was linearized using either Pstl or SacLI restriction enzymes (Pharmacia Biotech) which cut in the polylinker on either side of the inserted cDNA.
  • Antisense and sense MP-10 riboprobes were transcribed in vitro using either T7 or SP6 RNA polymerases (Promega), respectively in the presence of [ 35 S]UTP (approximately 800 Ci/mmol; Amersham, Oakville, Ontario).
  • DNA template was digested with DNAse I (Pharmacia). Riboprobes were subsequently purified on ProbeQuant G-50 microcolumns (Pharmacia Biotech, USA) according to the manufacturer's specifications. Quality of labeled riboprobes was verified by polyacrylamide -urea gel electrophoresis.
  • Hybridization was performed in a buffer containing 75% formamide (Sigma, St-Louis, Mo), 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, IX Denhardt's solution (Sigma), 50 mg/ml denatured salmon sperm DNA (Sigma), 10% dextran sulfate (Sigma), 20 mM dithiothreitol and [ 35 S]UTP-labeled cRNA probes (10 X10 6 cpm/ml) at 55°C for 18 hours in humidified chambers.
  • Example 3 Development of an Assay for the MP-1 Endogenous Ligand
  • a fluorescent probe e.g. the green fluorescent protein (GFP).
  • GPCRs internalize (Ashworth et al, Proc. Nat. Acad. Sci. USA 02:512-516 (1995)). If the receptors are coupled to a fluorescent probe, internalization can be measured using conventional techniques (e.g. fluorescent microscopy, confocal microscopy, etc).
  • tissue extracts can be prepared from brain, spinal cord etc., incubated with cells expressing a hybrid MP- 10-GFP construct and internalization monitored. Only those fractions that contain an endogenous ligand for MP-10 will cause the hybrid to internalize. Such active fractions can then be further fractionated until a pure ligand is recovered.
  • a similar procedure can be employed to assay for receptor-activating compounds in a complex mixture of synthetic compounds.

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Abstract

La présente invention concerne un nouveau récepteur couplé aux protéines G exprimé, inter alia, dans le système nerveux central et la rate de rats. L'invention englobe la protéine du récepteur de même que des acides nucléiques codant la protéine. De plus, l'invention concerne des méthodes et des compositions utilisant le récepteur. Le récepteur est appelé récepteur MP-10.
PCT/SE1999/002302 1998-12-10 1999-12-08 Nouveau recepteur couple aux proteines g WO2000034333A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU20181/00A AU2018100A (en) 1998-12-10 1999-12-08 Novel g protein-coupled receptor
CA002352569A CA2352569A1 (fr) 1998-12-10 1999-12-08 Nouveau recepteur couple aux proteines g
EP99963815A EP1147135A1 (fr) 1998-12-10 1999-12-08 Nouveau recepteur couple aux proteines g

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SE9804274A SE9804274D0 (sv) 1998-12-10 1998-12-10 Novel receptor
SE9804274-0 1998-12-10

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WO2000034333A1 true WO2000034333A1 (fr) 2000-06-15

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EP (1) EP1147135A1 (fr)
AU (1) AU2018100A (fr)
CA (1) CA2352569A1 (fr)
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002002599A2 (fr) * 2000-06-30 2002-01-10 Ingenium Pharmaceuticals Ag Recepteur couple aux proteines g humaines igpcr17, et son utilisation
WO2002036631A1 (fr) * 2000-11-01 2002-05-10 Yamanouchi Pharmaceutical Co., Ltd. Procede de criblage d'anti-plaquettes
EP1240350A1 (fr) * 1999-12-23 2002-09-18 Cor Therapeutics, Inc. Recepteur p2y12

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998050549A2 (fr) * 1997-05-07 1998-11-12 Human Genome Sciences, Inc. Deux recepteurs couples par des proteines g: gpcr2 induit par veb (ebi-2) et gpcr de type gde-1

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998050549A2 (fr) * 1997-05-07 1998-11-12 Human Genome Sciences, Inc. Deux recepteurs couples par des proteines g: gpcr2 induit par veb (ebi-2) et gpcr de type gde-1

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1240350A1 (fr) * 1999-12-23 2002-09-18 Cor Therapeutics, Inc. Recepteur p2y12
EP1240350A4 (fr) * 1999-12-23 2003-11-19 Millennium Pharm Inc Recepteur p2y12
US6762029B2 (en) 1999-12-23 2004-07-13 Portola Pharmaceuticals, Inc. Methods of identifying agents that modulate P2Y12 receptor activity
WO2002002599A2 (fr) * 2000-06-30 2002-01-10 Ingenium Pharmaceuticals Ag Recepteur couple aux proteines g humaines igpcr17, et son utilisation
WO2002002599A3 (fr) * 2000-06-30 2002-10-10 Ingenium Pharmaceuticals Ag Recepteur couple aux proteines g humaines igpcr17, et son utilisation
WO2002036631A1 (fr) * 2000-11-01 2002-05-10 Yamanouchi Pharmaceutical Co., Ltd. Procede de criblage d'anti-plaquettes
US7405051B2 (en) 2000-11-01 2008-07-29 Astellas Pharma Inc. Method of screening antiplatelet agents

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CA2352569A1 (fr) 2000-06-15
EP1147135A1 (fr) 2001-10-24
SE9804274D0 (sv) 1998-12-10
AU2018100A (en) 2000-06-26

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