WO1992021701A1 - Lignees cellulaires transfectees de mammifere, exprimant le recepteur d'adenosine a¿1? - Google Patents

Lignees cellulaires transfectees de mammifere, exprimant le recepteur d'adenosine a¿1? Download PDF

Info

Publication number
WO1992021701A1
WO1992021701A1 PCT/US1992/004626 US9204626W WO9221701A1 WO 1992021701 A1 WO1992021701 A1 WO 1992021701A1 US 9204626 W US9204626 W US 9204626W WO 9221701 A1 WO9221701 A1 WO 9221701A1
Authority
WO
WIPO (PCT)
Prior art keywords
receptor
dna segment
host cell
cell
dna
Prior art date
Application number
PCT/US1992/004626
Other languages
English (en)
Inventor
David R. Sibley
Lawrence C. Mahan
Elizabeth M. Smyk-Randall
Frederick J. Monsma, Jr.
Original Assignee
The United States Of America Represented By The Secretary, Department Of Health And Human Services
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by The United States Of America Represented By The Secretary, Department Of Health And Human Services filed Critical The United States Of America Represented By The Secretary, Department Of Health And Human Services
Publication of WO1992021701A1 publication Critical patent/WO1992021701A1/fr

Links

Classifications

    • 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

Definitions

  • the subject invention relates to mammalian cell lines which express the A,, adenosine receptor and methods of use thereof.
  • the cell lines may be utilized in the screening of drugs which affect the activity of the A, receptor.
  • Adenosine is a ubiquitous modulator of numerous physiological activities, particularly within the cardiovascular and nervous systems. The effects of adenosine appear to be mediated by specific membrane-bound receptor proteins. Adenosine receptors belong to a large class of neurotransmitter and hormone receptors which are linked to their signal transduction pathways via guanine nucleotide binding regulatory (G) proteins. Biochemical and pharmacological criteria have been used to divide adenosine receptors into two major subtypes referred to as A, and A 2 which either inhibit or stimulate, respectively, the enzyme adenylyl cyclase (Stiles, G.L., Trends Pharmacol. 7:486-49 (1986) and Williams, M. , Ann Rev. Pharmacol.
  • Adenosine receptors are important from a clinical therapeutic viewpoint as drugs which interact with these receptors may be used to treat Alzheimer's disease, epilepsy and disorders of memory, cognition and affect.
  • One problem with currently available adenosine agonist and antagonist drugs is that they have many side effects, like many other drugs which work through interacting with receptors, despite their clinical utility. These side effects are predominantly due to a lack of receptor specificity in that the drug in use interacts not only with adenosine receptors but other neurotrans itter receptors as well.
  • a major goal of clinical neuropharmacology and the pharmaceutical industry is the development of more highly selective drugs with even greater efficacy that those currently in use.
  • a novel approach to the solution of this problem is to clone cDNAs encoding adenosine receptors, construct eukaryotic expression vectors containing these cDNAs and create a series of stably transfected mammalian cell lines which express functional adenosine receptors in high abundance.
  • cell lines which express a homogeneous population of adenosine receptors, can be used by the pharmaceutical industry or others to screen drugs, and study adenosine receptors, using a variety of biochemical, physiological, and pharmacological techiniques.
  • a canine cDNA has been cloned (i.e., clone RDC7) which may represent a species homolog of the rat receptor A, cDNA of the present invention; however, the scientists carrying out this work did not disclose the identity of the protein encoded by their cDNA (Libert et al., Science 244:569-72 (1989) & Libert et al. , Nuc. Acids Res. 18:1916 (1990)).
  • the subject invention relates to mammalian cell lines which express an adenosine receptor referred to as A,.
  • the cell lines may be utilized to screen for drugs which affect the activity of the A, receptor.
  • the cell lines are created by initially isolating a cDNA encoding the A, adenosine receptor subtype, inserting the cDNA into a eukaryotic expression vector and transfecting a host cell with the vector.
  • the present invention relates to a substantially pure form of an A, adenosine receptor wherein the receptor has an amino acid sequence as shown in Figure 1.
  • the invention also relates to antibodies produced against this receptor.
  • the present invention also relates to a DNA segment encoding the receptor described-above.
  • the DNA segment has a nucleotide sequence as shown in Figure 7.
  • the present invention also encompasses a portion of the segment, as well as a DNA segment which encodes an amino acid sequence having the adenylyl cyclase inhibition properties, pharmacological properties, and G regulatory protein coupling properties of a receptor having the amino acid sequence shown in Figure l.
  • the invention further relates to a recombinant DNA molecule comprising: a) the DNA segment discussed above and b) a vector for introducing this DNA molecule into eukaryotic or prokaryotic host cells.
  • the vector may be a eukaryotic expression vector such as pCD-SR ⁇ .
  • the DNA segment which is part of the recombinant molecule may encode the amino acid sequence shown in Figure 1.
  • the present invention relates to a host cell stably transformed or transfected with the recombinant DNA molecule discussed above in a manner allowing expression of the receptor encoded by said recombinant DNA molecule.
  • the host cell may be a prokaryotic or eukaryotic cell. Mammalian cells are particularly useful for transformation. Suitable mammalian host cells include, for example, Chinese hamster ovary cells or a A9-L cells.
  • the present invention further relates to a method of producing an adenosine receptor protein comprising culture the above-transformed host cell, under conditions such that the DNA segment is expressed and the receptor thereby produced.
  • the receptor is isolated in the last step of the method.
  • the invention also includes a method of screening drugs that interact with the A, receptor comprising contacting the transformed host cell with a drug under conditions such that the function of the receptor is either activated or blocked, and detecting the presence or absence of A, receptor activity.
  • Figure 1 shows the deduced amino acid sequence of the rat A, receptor protein. Transmembrane spanning domains are defined on the basis of hydropathy analysis. A potential N- 1inked glycosylation site is indicated by CHO.
  • the cDNA encoding the A, receptor has been inserted into a eukaryotic expression vector and used in the construction of various mammalian cell lines expressing this functional protein for the purpose of, for example. A, receptor drug development.
  • These A, receptor-expressing cell lines can be used to investigate the affinities and efficacies of agonist and antagonist drugs with the A, receptor using various techniques such as radioligland binding and second messenger assays.
  • Figure 2 shows a [ ]DPCPX saturation binding experiment in transfected A9-L cell membranes.
  • Figure 3 represents [ ]DPCPX competition binding assays in transfected A9-L cell membranes. Membranes were prepared and incubated with 0.5 nM [ ]DPCPX and the indicated concentrations of drugs as described in Example IV. The data points respresent the mean +/- SD from two independent experiments. The computer-derived affinity constants are given in the text.
  • A Antagonist competition curves: DPCPX ( ⁇ ) , XAC ( ) , and theophylline ( ⁇ ) .
  • B Agonist competition curves: CCPA ( ) , R-PIA ( ⁇ ),NECA ( ⁇ ) , and S-PIA ( ⁇ ) .
  • FIG. 4 represents adenylyl cyclase assays in transfected CHO cell membranes. Membranes were prepared and adenylyl cyclase assays performed as described in Example V. A, 10 " * M concentrations of each of the indicated agonists were examined for their ability to inhibit forskolin-stimulated adenylyl cyclase activity. B, R-PIA was tested alone (10 "6 ) or in
  • RNA samples were run on denaturing 1% formaldehyde-agarose gels, electrophoretically transferred to GeneScreen (NEN) nitrocellulose, hybridized with a cDNA- specific, [ M P]-labeled oligodeoxynucleotide probe, washed under high stringency and exposed to Kodak X-AR film as described in the Examples.
  • RNA molecular size markers are indicated on the left.
  • CNS tissues mRNA of approximately 3.1 and 5.6 kb (arrows) in length were detected and located predominantly in the cortex (CTX) , cerebellum (CB) , and hippocampus (HIP) .
  • tissue indicated are heart (HT) , lung (LNG) , liver (LIV) , kidney (KID) , spleen (SPL) , stomach (STM) , intestine (INT) , uterus (UT) and testis (TST) .
  • Figure 6 represents in situ hybridization histochemical analysis of the distribution of mRNA for the A, adenosine receptor.
  • D High magnification of the CA4 region of the hippocampus indicating specific localization to neuronal (N) nuclei as opposed to glial (G) nuclei.
  • Fi g ur es 1A -1 J s h ow the genetic sequence of the cDNA of the present invention.
  • This cDNA encodes an A, receptor linked to the inhibition of adenylyl cyclase activity.
  • the present invention relates to mammalian cell lines which express the A, receptor. These cell lines are homogeneous, can be grown in large quantities and are easily manipulated. Most importantly, such cells result in the production of large quantities of adenosine receptor A,. Thus, using the cell lines, one may readily screen drugs that activate or block the receptor and thus affect the production of adenylyl cyclase.
  • the A, receptor of the present invention is linked to the inhibition of adenylyl cyclase activity and is coupled with the guanine nucleotide binding regulatory protein.
  • the receptor can have an amino acid sequence corresponding to the sequence shown in Figure 1 or can, of course, have the sequence of a molecule having substantially the same adenylyl cyclase activation properties, pharmacological properties and G regulatory protein coupling properties of the molecule corresponding to Figure l.
  • the receptor protein may also have an amino acid sequence corresponding to any portion of the protein shown in Figure 1, allelic variations of
  • the receptor may be present in a substantially pure form.
  • the receptor may be present in a form that is substantially free of proteins and nucleic acids with which it is normally associated.
  • the receptor may be purified using methods commonly known in the receptor.
  • the A, receptor may be used as an antigen, according to methods known in the art, in order to produce antibodies thereto. Such antibodies may be monoclonal as well as polyclonal.
  • the present invention relates to cell lines which express the A, receptor.
  • the invention also relates to genetic sequences, in particular, cDNA sequences, which encode the amino acid sequence given in Figure 1. More specifically.
  • Figure 7 shows the sequence of a cDNA clone which encodes the A, receptor of the present invention.
  • the present invention encompasses the genetic sequence shown in Figure 7 as well as allelic variations thereof or portions thereof.
  • the present invention also relates to a recombinant construct or recombinant DNA molecule comprising the DNA segment or sequence shown in Figure 7 (or a portion thereof) and a vector for introducing the sequence into a host cell.
  • the DNA sequence utilized may encode either the receptor shown in Figure 1 or a receptor having the adenylyl cyclase inhibiting properties, pharmacological, and G regulatory protein coupling properties of the A, receptor.
  • the cDNA of the present invention has been inserted into a bacterial cell and deposited with the American Type Culture Collection in Rockville, Maryland. The accession number of the deposit is .
  • the vector which is used in creating the recombinant construct may either be a prokaryotic or eukaryotic expression vector.
  • a plasmid, bacteriophage, or virus such as pCD- SR ⁇
  • the DNA sequence can be present in the vector operably linked to regulatory elements including, but not limited to, a promoter.
  • the recombinant construct may be utilized to transform or transfect either prokaryotic or eukaryotic host cells.
  • the present invention relates to host cells which may be transformed with the recombinant construct discussed directly above.
  • the host cell may be prokaryotic (e.g., a bacterial cell), lower eukaryotic (e.g., fungus, such as yeast cells), or higher eukaryotic (e.g., all mammalian cells, for example, rat or human cells) .
  • Mammalian cells are preferred.
  • both Chinese hamster ovary cells (CHO) as well as A9-L cells may be stably transformed with the recombinant construct of the present invention. Transformation may be accomplished by any method commonly utilized in the art.
  • the transformed cells may be used as a source for the DNA sequence shown in Figure 7 (or an allelic variation or portion thereof) .
  • the transformed cell is used as a source for the A, receptor.
  • the A, receptor protein produced by the transformed cells may be detected in a sample, for example, a cell or tissue culture, by contacting the sample with an antibody which binds to the receptor.
  • the detection of the presence or the absence of the resulting antibody-receptor complex may be achieved by methods known in the art.
  • the presence or absence of a DNA segment encoding the A, receptor protein may be detected in a sample, such as a cell or tissue culture, by contacting the sample with a DNA probe that is comprised of the DNA segment of interest.
  • a complex can form which consists of the probe and the DNA segment from the sample. Again, detection of the presence or absence of the complex can be carried out using conventional methods.
  • the cell lines of the present invention can be used for many purposes related to the expression of the protein.
  • such cell lines can be used in the study and elucidation of receptor proteins and the production thereof.
  • the cell lines may also be used in a clinical setting in order to determine which drugs enhance or interfere with the activity of the receptor and thus affect the regulation of adenylyl cyclase.
  • activation of the receptor by an agonist will inhibit the production of cAMP whereas blockage of the receptor will prevent this effect.
  • activation of the receptor by an agonist will inhibit adenylyl cyclase activity whereas inhibition or blockage of the receptor by an antagonist will increase adenylyl cyclase activity.
  • the cell line of the present invention can be utilized to investigate the affinities and efficacies of A, agonist and antagonist drugs using several techniques, for example, radioligand binding (see Example IV) and second messenger assays.
  • the activity of the drug-treated cell can be compared to a control cell in order to evaluate the activation or blockage of the receptor.
  • expression of the A, receptor cDNA can be measured for diagnostic purposes using conventional methods. This is carried out by utilizing antibodies to the receptor protein. Such antibodies are prepared by injecting all (or a portion) of the receptor protein into a mammal in order to elicit an immune response.
  • Example I The present invention can be illustrated by the use of the following non-limiting examples: Example I
  • RNA was prepared from rat striatum and fractionated according to size by sucrose gradient centrifugation as previously described (Mahan et al., Proc. Natl. Acad. Sci. USA 87:2196-2200 (1990)). RNA of -3 kb was used as a template for first strand synthesis of cDNA.
  • RNA was denatured at 65°C for 5 minutes and reverse transcription was performed at 39- 40°C in PCR buffer (GeneAmp, Perkin Elmer-Cetus) with AMV reverse transcriptase (Promega) and 1.2 ⁇ M of a 64-fold degenerate consensus primer to the sixth transmembrane region of G protein-coupled receptors described previously (Mahan et al. , Proc. Natl. Acad. Sci. USA 87: 2196-220 (1990)). Second strand synthesis and subsequent amplification was performed by the addition of 1.2 ⁇ M of a 256-fold degenerate consensus primer to the third transmembrane region (Libert et al..
  • Nucleotide sequence analysis was performed using the Sanger dideoxy nucleotide chain termination method with Sequenase (US Biochemical Corp.) on denatured doubled-stranded plasmid templates. Primers were synthetic oligonucleotides which were either vector-specific or derived from prior sequence information.
  • CCCGTAGTACTTCrTGGGGGT ⁇ _ ⁇ CCGGAGGAGGCrGACACCTTTTTGTT- 15 3' was synthesized from sequence specific to the putative third intracellular loop.
  • This probe was radiolabelled using terminal deoxynucleotidlytransferase (Boehringer Mannheim) with either [ ⁇ -"P]ATP (Northern blots) or [ ⁇ - 3S S]ATP (in situ hybridization) .
  • Hybridization reactions were carried out on 2 ⁇ g samples of poly (A) * RNA or serial 12 ⁇ m sagittal and coronal sections of adult rat brain.
  • RNA markers (0.24- 9.5kb) were purchased from Bethesda Research Labs.
  • Example III Transfection of the Vector. Transfection of Eukaryotic Cells and Expression of the Receptor Protect! A full-length cDNA insert was subcloned into the pCD-SR ⁇ expression vector (Takebe et al., Molec. Cell. Biol. 8:466-72 (1988)) containing a modified polylinker. Competent DH5 cells were transformed and clones containing the appropriate cDNA insert were used for large-scale plasmid preparations via the CsCl 2 gradient purification method. DNA from the resulting plasmid construct (30 ⁇ g) , along with 3 ⁇ g of pMAMneo (Clontech) for a selectable marker, was used to transfect CHO and A9-L cells by the
  • Radioligand Binding Assay Host cell membranes were prepared as follows:
  • Cells were detached from 150 cm 2 flasks with 1 mM EDTA in Ca * /Mg 2* free Earle's balanced salt solution (EBSS) and were washed by centrifugation at 300 x g and resuspension with cold EBSS (complete) .
  • the cells were then suspended in ice-cold lysis buffer (5 mM Tris HC1, pH 7.4, 5 mM MgClj and transferred to a Dounce homogenizer on ice and homogenized using 10 strokes with an A pestle.
  • EBSS Free Earle's balanced salt solution
  • the homogenate was suspended in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 5 mM KC1, 1.5 mM CaCl 2 , 4 mM MgCl 2 , 120 mM NaCl and centrifuged for 10 min at 43,500 x g.
  • the crude membrane pellet was resuspended in 50 mM Tris- HCl, pH 7.2, lmM EDTA, 10 mM MgCl 2 containing 2 U/ml of adenosine deaminase (Sigma, St. Louis, MO) and incubated for 30 min at 30°C.
  • the membrane homogenate was subsequently centrifuged for 10 min at 43,500 g and the resulting membrane pellet resuspended in the appropriate assay buffer. Protein concentrations were determined using the bicinchoninic acid (BCA) protein reagent (Pierce; Rockford, IL) as described in Smith et al, Anal Biochem. 150:76-85 (1985).
  • BCA bicinchoninic acid
  • receptor binding assays using [ 3 H]DPCPX were then performed as previously described ((Nakata, H., J. Biol. Chem. 264:16545-51 (1989) and Nakata, H., J. Biol. Chem. 265: 671-77 (1990)). Briefly, the membrane preparation was suspended with the appropriate ligands in 40 mM Tris-acetate, pH 7.5, 0.8 mM EDTA, and 4 mM MgCI 2 for a final assay concentration of 150 ⁇ M DPCPX.
  • Competition curves were analyzed using models for competition of radioligand and competitor to one or two independent sites. Results from fits using a two site model were retained only when the two site model fit the data significantly better than a one site model as determined by the partial F test at a significance level of p ⁇ 0.05.
  • the inventors turned to the CHO cells which had been transfected with the A, receptor cDNA and preliminarly characterized as expressing >1 pmol/mg protein specific [ 3 H]DPCPX binding sites (data not shown) .
  • the experiment was carried out as follows: 50 ⁇ l of cell membranes (100 ⁇ g protein) suspended in AC buffer (75 mM Tris-HCl, pH 7.4, 250 mM sucrose, 12.5 mM MgCl 2 , 1.5 mM EDTA) containing 1 mM DTT and 200 ⁇ M sodium metabisulfite and supplemented with 2.75 mM phosphoenolpyruvate, 53 ⁇ M GTP, 0.12 mM ATP, 1.0 U myokinase, 0.2 U pyruvate kinase and 100 ⁇ M RO- 20-1724 (a phosphodiesterase inhibitor, Biomol, Plymouth Meeting, PA) were added to tubes on ice containing 10 ⁇ l H 2 0 or 10 ⁇
  • the membranes were incubated for 5 min at 37°C to generate cAMP and the reaction was stopped by a 3 min incubation in boiling H 2 0.
  • the cAMP generated was assayed by the method of Brown et al. by incubation with cAMP binding protein, prepared from bovine adrenal gland, in the presence of [ 3 H]cAMP (45 Ci/mmol, Amersham Corp., Arlington Heights, IL) at 4°C for 2-16 hr as previously described ((Brown et al., Biochem. J. 121:561-67 (1971) and Barton et al., Molec. Pharmacol. 38:581-41 (1990)).
  • Fig. 4A shows the effect of various adenosine agonists on inhibiting adenylyl cyclase activity.
  • the potent Al selective agonists, CCPA and R-PIA both significantly inhibited forskolin cAMP production.
  • NECA exhibited less of a response while S-PIA was relatively ineffective at 10 nM (Fig. 4A) .
  • 10 nM concentrations of the A 2 -selective agonists, CGS21680 and CV1808, did not affect adenylyl cyclase activity (Fig. 4A) .
  • Fig. 4B shows that the R-PIA inhibitory response could be completely blocked by the A,-selective anatagonists, DPCPX and XAC.
  • This functional agonist and antagonist pharmacology agrees well with that observed for the radioligand binding analyses in Figs. 2 and 3.
  • the cloned Al receptor thus appears to be capable of functional coupling to the inhibition of adenyl cyclase activity.
  • the present inventors investigated the tissue distribution of its corresponding mRNA.
  • Northern blot analysis of a variety of rat brain tissue revealed two species of mRNA for this A, adenosine receptor; one -3.1 kb and a less prominent, higher molecular weight species of -5.6 kb (Fig. 5A) .
  • Highest expression was observed in the cortex, cerebellum, and hippocampus.
  • Olfactory bulb, esencephalon, and striatum also exhibited moderate levels of both messages, while the retina appeared to predominantly express the 5.6 kb species. No expression was apparent in the pituitary.
  • Fig. 6A-D In situ hybridization histochemical studies confirmed the tissue distribution of mRNA expression seen with the Northern blot analyses. In addition, these studies revealed marked expression of A, receptor mRNA in thalamic nuclei, the medial geniculate nucleus and the ventral tegmental area (Fig. 6A- B) . High levels of expression observed in the cerebellum were confined predominantly to the granule cell layer (Fig. 6C) . In most cases, autoradiographic labeling was restricted to neuronal, not glial, nuclei (Fig. 6D) . The expression of A, receptor mRNA and A, receptor protein co-localized in a number of brain tissues.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention se rapporte à des lignées cellulaires de mammifères, lesquelles expriment un récepteur d'adénosine appelé A1. Les lignées cellulaires peuvent être utilisées pour détecter des médicamments affectant l'activité du récepteur A1. On obtient ces lignées cellulaires en isolant initialement un ADNc codant le sous-type du récepteur d'adénosine A1, en introduisant l'ADNc dans un vecteur d'expression eucaryotique et en transfectant une cellule hôte avec ce vecteur.
PCT/US1992/004626 1991-06-05 1992-06-05 Lignees cellulaires transfectees de mammifere, exprimant le recepteur d'adenosine a¿1? WO1992021701A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US71018091A 1991-06-05 1991-06-05
US710,180 1991-06-05

Publications (1)

Publication Number Publication Date
WO1992021701A1 true WO1992021701A1 (fr) 1992-12-10

Family

ID=24852952

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/004626 WO1992021701A1 (fr) 1991-06-05 1992-06-05 Lignees cellulaires transfectees de mammifere, exprimant le recepteur d'adenosine a¿1?

Country Status (2)

Country Link
AU (1) AU2179192A (fr)
WO (1) WO1992021701A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08500967A (ja) * 1992-06-12 1996-02-06 ガーヴァン インスティチュート オブ メディカル リサーチ ヒトＡ１，Ａ２ａおよびＡ２ｂアデノシン受容体をエンコードするＤＮＡ配列

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4859609A (en) * 1986-04-30 1989-08-22 Genentech, Inc. Novel receptors for efficient determination of ligands and their antagonists or agonists

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4859609A (en) * 1986-04-30 1989-08-22 Genentech, Inc. Novel receptors for efficient determination of ligands and their antagonists or agonists

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NATURE, Vol. 311, issued 18 October 1984, W. LEONARD et al., "Molecular cloning and expression of cDNAs for human interleukin-2 receptor", pages 626-631. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 264, issued 05 October 1989, H. NAKATA, "Purification of A1 Adenosine Receptor from Rat Brain Membranes", pages 16545-16551. *

Also Published As

Publication number Publication date
AU2179192A (en) 1993-01-08

Similar Documents

Publication Publication Date Title
US6383762B1 (en) Methods of obtaining compounds that interact with a human serotonin (5-HT2) receptor
Schwinn et al. Molecular cloning and expression of the cDNA for a novel alpha 1-adrenergic receptor subtype.
US5652113A (en) DNA encoding a human 5-HT 1F receptor and uses thereof
US6331401B1 (en) Uses of the 5-HT4 receptor
JP4216326B2 (ja) ヒト神経ペプチドｙ／ペプチドｙｙ／膵臓ポリペプチド受容体（ｙ４）をコードしたｄｎａ、及び該ｄｎａの使用
US5654139A (en) Allelic variation of the serotonin 5HT2c receptor
US5786155A (en) Methods for identifying a compound that binds to a human 5-HT 1E receptor
US6280973B1 (en) Mammalian methadone-specific opioid receptor gene and uses
JPH11508135A (ja) ヒトｅｄｇ−２受容体ホモログ
Oksenberg et al. The third intracellular loop of the 5‐hydroxytryptamine2A receptor determines effector coupling specificity
AU645609B2 (en) Cannabinoid receptor
US5610282A (en) cDNA encoding a rat D1 dopamine receptor linked to adenylyl cyclase activation and expression of the receptor protein in plasmid-transfected cell lines
WO1992021701A1 (fr) Lignees cellulaires transfectees de mammifere, exprimant le recepteur d'adenosine a¿1?
US6783973B1 (en) Mammalian catecholamine receptor genes and uses
US20020155444A1 (en) Human VNO cDNA libraries
US20020123138A1 (en) Human VNO receptor (R1)
AU667510C (en) DNA encoding a human 5-HT-1F receptor and uses thereof
US20030196212A1 (en) Novel pheromone receptor
Lappalainen et al. Allelic variation of the serotonin 5HT 2c receptor
US20030143541A1 (en) Human pheromone receptors
US20020081661A1 (en) DNA encoding 5-HT4 serotonin receptors and uses thereof
Marchese Orphan G protein-coupled receptors
US20030139589A1 (en) G protein coupled receptor A4
JP2004501629A (ja) Ｇタンパク質共役受容体ｏｒｇ１０

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA