EP0644935A1 - SEQUENCES D'ADN CODANT LES RECEPTEURS HUMAINS DE L'ADENOSINE A1, A2a ET A2b - Google Patents

SEQUENCES D'ADN CODANT LES RECEPTEURS HUMAINS DE L'ADENOSINE A1, A2a ET A2b

Info

Publication number
EP0644935A1
EP0644935A1 EP93912427A EP93912427A EP0644935A1 EP 0644935 A1 EP0644935 A1 EP 0644935A1 EP 93912427 A EP93912427 A EP 93912427A EP 93912427 A EP93912427 A EP 93912427A EP 0644935 A1 EP0644935 A1 EP 0644935A1
Authority
EP
European Patent Office
Prior art keywords
human
dna
adenosine
adenosine receptor
receptor
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Withdrawn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
EP93912427A
Other languages
German (de)
English (en)
Inventor
Kerrie Diane Pierce
Constance Andrea Townsend-Nicholson
John Shine
Timothy Furlong
Lisa Selbie
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.)
Garvan Institute of Medical Research
Original Assignee
Garvan Institute of Medical Research
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 Garvan Institute of Medical Research filed Critical Garvan Institute of Medical Research
Publication of EP0644935A1 publication Critical patent/EP0644935A1/fr
Withdrawn legal-status Critical Current

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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to DNA sequences encoding the human Al, A2a and A2b adenosine receptors.
  • the present invention relates to the use of these DNA sequences in the production of the human Al, A2a and A2b adenosine receptors using recombinant DNA technology.
  • Adenosine influences cardiovascular function (by- slowing heart rate and decreasing blood pressure) and also influences nervous system function (through sedative and anti-epileptic effects). In addition, adenosine can induce bronchoconstriction. Adenosine binds specifically to at least three receptors, Al and A2a and A2b. Adenosine receptors have been shown to couple to a number of second messenger systems. Additional adenosine receptor subtypes may exist. As adenosine receptor agonists and antagonists may have commercial value as anti-hypertensive agents, hypnotics, anti-psychotics c d bronchodilators, the ability to produce adenosine receptors by recombinant DNA technology is advantageous.
  • the present inventors have isolated three related cDNA fragments encoding the human Al, A2a and A2b adenosine receptors from human hippocampal cDNA by using either the. polymerase chain reaction and unique degenerate oligonucleotides to generate specific probes or by using specific consensus oligonucleotide probes for cDNA library screening.
  • Full-length cDNA clones for each of the three receptors were isolated from a human hippocampal cDNA library.
  • the receptor sequences were identified as the human Al, A2a and A2b adenosine receptors by expression in mammalian cells and both measurement of the affinity of the encoded receptors for various adenosine analogues and
  • the receptors have homology to cDNA's encoding the dog Al and A2a adenosine receptors (MAENHAUT, C, VAN SANDE, J., LIBERT, F., ADRAMO IC, . , PARMENTIER, M., VANDERHAEGEN, J. , DUMONT, D. , VASSART, G. AND
  • SCHIFFMANN S. (1990); LIBERT, F., SCHUFFMANN, S.M., LEFORT, A., PARMENTIER, M. , GERARD, C, DUMONT, J.E., VANDERHAEGHEN J.J., VASSART, G. (1991)) and the rat A2b adenosine receptor (STEHLE, J.H., RIVKEES, S.A., LEE, J.J., WEAVER, D.R., DEEDS, J.D. AND REPPERT, S.M.
  • the present invention consists in a DNA molecule encoding the human Al adenosine receptor, the DNA molecule having a sequence substantially as shown in Figure 1 or a functionally equivalent sequence.
  • the present invention consists in a DNA molecule encoding the human A2a receptor subtype, the DNA molecule having a sequence substantially as shown in Figure 2 or a functionally equivalent sequence.
  • the present invention consists in a DNA molecule encoding the human A2b adenosine receptor subtype, the DNA molecule having a sequence substantially as shown in Figure 3 or a functionally equivalent sequence.
  • the term “functionally equivalent sequence” is intended to cover variations in the DNA sequence which, due to degeneracy of the DNA code, do not result in the sequence encoding a different polypeptide. Further, this term is intended to cover alterations in the DNA code which lead to changes in the encoded polypeptide, but in which such changes do not affect the biological activity of the polypeptide.
  • DNA molecule is intended to
  • SUBSTITUTE SHEET cover both genomic DNA and cDNA.
  • the present invention consists in a method of producing the human Al adenosine receptor comprising culturing a cell transformed with the DNA molecule of the first aspect of the present invention under conditions which allow expression of the DNA sequence such that the human Al adenosine receptor is expressed on the cell surface and optionally recovering the human Al adenosine receptor.
  • the present invention consists of a method of producing a human A2a adenosine receptor comprising culturing a cell transformed with the DNA molecule of the second aspect of the present invention under conditions which allow expression of the DNA sequence such that the human A2 adenosine receptor is expressed on the cell surface and optionally recovering the human A2a adenosine receptor.
  • the present invention consists of a method of producing a human A2b adenosine receptor comprising culturing a cell transformed with the DNA molecule of the third aspect of the present invention under conditions which allow expression of the DNA sequence such that the human A2 adenosine receptor is expressed on the cell surface and optionally recovering the human A2b adenosine receptor.
  • the present invention consists of a method of screening a molecule for adenosine agonist or antagonist activity, comprising contacting the molecule with the human Al, A2a or A2b adenosine receptors produced by the method of the fourth, fifth or sixth aspect of the present invention.
  • the present invention consists in oligonucleotides 305, 377 and 376 as hereinafter described.
  • the DNA molecules of the present invention represent
  • SUBSTITUTE SHEET novel human receptors These receptors may be of interest both clinically and commercially as they are expressed in many regions of the body and as adenosine affects a wide number of systems.
  • the isolated full-length DNA clones containing the complete coding region for these receptors can be used to establish mammalian cell lines producing the receptors for use in agonist and antagonist screening.
  • the receptor DNA sequence can be used for additional homology screening to identify novel members of this receptor family.
  • Figure 2 shows the nucleotide and amino acid sequence of the human A2a adenosine receptor cDNA.
  • Figure 3 shows the nucleotide and amino acid sequence of the human A2b adenosine receptor cDNA.
  • Figure 4A shows saturation isotherms of the total (unfilled triangle), specific (filled circle) and non-specific (unfilled square) binding of the Al adenosine receptor antagonist DPCPX (8-cyclopentyl-l,3 dipropylxanthine) to mammalian CHO.Kl cells expressing the human Al adenosine receptor.
  • DPCPX 8-cyclopentyl-l,3 dipropylxanthine
  • NECA 5'-N-ethylcarboxamido adenosine
  • CA 2-chloroadenosine
  • CPA N -cyclopentyladenosine
  • XAC xanthine amine con
  • SUBSTITUTE SHEET Figure 5 shows the effects of the different adenosine receptor subtypes, Al, A2a and A2b upon cyclic AMP production.
  • Al adenosine receptor activation leads to inhibition of forskolin stimulated cAMP levels.
  • Activation of both the A2a and A2b adenosine receptors leads to stimulation of cAMP levels.
  • oligonucleotides corresponding to the transmembrane II (TM II) and IV (TM IV) regions of G protein-coupled receptors and containing either a 5' EcoRI restriction enzyme site (TM II oligonucleotide 377) or a 3' Hind III restriction enzyme site (TM IV oligonucleotides 305 and 376) were synthesized on an Applied Biosystems automated DNA synthesiser.
  • the sequences of the oligonucleotides are as follows:-
  • DNA sequences included inosine (I) residues. Crude oligonucleotides were then used in the polymerase chain reaction. PCR Amplification Sequences homologous to the G protein-coupled
  • SUBSTITUTE SHEET receptor oligonucleotides were amplified from human cDNA using PCR and the Hybaid thermocycler.
  • DNA was prepared from a human neuroblastoma (Clontech) cDNA library in lambda gtlO and from a hippocampal (Stratagene) cDNA library in lambda ZapII.
  • DNA was prepared by phenol and o chloroform extraction of approximately 10 library phage and ethanol precipitation to recover the DNA.
  • DNA from the cDNA libraries (l-5 ⁇ g) was incubated with 200 ⁇ M of each dNTP, 0.5 ⁇ M oligonucleotide, 0.5 units Tth enzyme (Toyobo) in 50mM KCl, 50mM Tris-HCl pH9.0, 1.5mM MgCl 2
  • Amplified DNA (20 ⁇ l) was removed and analysed by gel electropheresis in 1% agarose and 3% NuSieve (SeaKem) .
  • Amplification products 260bp-330bp in length were excised from the gel and purified with Geneclean. DNA fragments were then digested with Hind III for one hour at 37°C and EcoRI for one hour at 37°C, the DNA again purified with Geneclean and eluted into 10 ⁇ l H 2 0. Digested DNA fragments were then subcloned into M13mpl9 and sequenced by the Sanger dideoxy chain-termination method using the Pharmacia or the
  • PCR amplification of hums f t hippocampal cDNA with the degenerate oligonucleotides 377 and 376 produced a cDNA fragment with a sequence that was 76% homologous at the nucleotide level to sequence 3.1 and was designated 3.2
  • the DNA sequences were searched on the GenBank and EMBL databases for comparison to known sequences and were confirmed to be novel sequences with a high level of homology to dog adenosine Al and A2 receptors. Isolation of Full-Length cDNA Clones
  • oligonucleotides corresponding to the second extracellular loop (679) and to the third intracellular loop (678) were synthesised on an Applied Biosystems automated DNA synthesiser.
  • the sequences of the oligonucleotides are as follows:-
  • Hybond-N+nylon filters (0.45 ⁇ M, 137mm, Amersham) . DNA was denatured on the filters with a 3 minute incubation on
  • SUBSTITUTE SHEET was fixed to the filters with a 15 minute exposure to 0.4M NaOH. Filters were then rinsed in 2 x SSC (3M NaCl, 0.3M sodiuih citrate) and allowed to dry before a 30 minute prehybridisation in 40% formamide, 5 x SSC, 5 x Denhardt's, 50mM NaPO., 0.5% sodium dodecyl sulphate (SDS), O.lmg/ml salmon sperm DNA at room temperature. Oligonucleotides 678 and 679 were pooled and 50 pmoles total were radiolabelled using ⁇ T>-ATP and the DNA 5' end-labelling system (Promega) .
  • the filters were hybridised with this radiolabelled probe overnight at 42°C, after which time they were washed once briefly in 2 x SSC at room temperature then twice for 10 minutes each wash in 2 x SSC, 0.1SDS at room temperature with a final wash in 0.1 x SSC, 0.1%SDS for 15 minutes at 50°C.
  • the filters were then exposed to Kodak X-0MAT AR5 film overnight at -70 C. Over twenty pure phage isolates which hybridised to the radiolabelled 678 and 679 oligonucleotides were obtained.
  • Several of these different cDNAs were sequenced. The sequence of one such cDNA (together with the deduced amino acid sequence) which encodes the human Al adenosine receptor is shown in Figure 1.
  • A2a and A2b adenosine receptor cDNA isolation
  • Hybond-N nylon filters (0.45 ⁇ M, 137mm, Amersham) .
  • DNA was denatured on the filters with a 3 minute incubation on 0.5M NaOH, 1.5M NaCl and neutralised with a 7 minute incubation in 0.5M Tris pH 7.2, ImM EDTA and 1.5M NaCl.
  • Filters were rinsed in 2 x SSC (20 x SSC is 3M NaCl, 0.3M sodium citrate) and DNA fixed to the filters with a 5 minute exposure to ultraviolet light (312nm).
  • Each cloned full-length cDNA was subcloned into a mammalian cell expression vector (pcDNAlneo for A2a and A2b and pRc/CMV for Al (Invitrogen)) in such a way as to direct expression of the encoded receptor portion.
  • Mammalian cell lines (Chinese Hamster Ovary - CHO Kl or Human Embryonic Kidney - HEK 293) were independently transfeeted with the recombinant expression vectors and cell lines established which had stably integrated the
  • cDNA clone 3.1 encodes an adenosine A2a receptor
  • cDNA clone 3.2 encodes an adenosine A2b receptor
  • Al cDNA encodes an adenosine Al receptor.

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)
  • Endocrinology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des séquences d'ADN codant les récepteurs humains de l'adénosine A1, A2a et A2b. De plus, la présente invention concerne l'utilisation de ces séquences d'ADN dans la production des récepteurs humains de l'adénosine A1, A2a et A2b en utilisant la technologie résultant de la recombinaison d'ADN.
EP93912427A 1992-06-12 1993-06-11 SEQUENCES D'ADN CODANT LES RECEPTEURS HUMAINS DE L'ADENOSINE A1, A2a ET A2b Withdrawn EP0644935A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AUPL2936/92 1992-06-12
AUPL293692 1992-06-12
PCT/AU1993/000277 WO1993025677A1 (fr) 1992-06-12 1993-06-11 SEQUENCES D'ADN CODANT LES RECEPTEURS HUMAINS DE L'ADENOSINE A1, A2a ET A2b

Publications (1)

Publication Number Publication Date
EP0644935A1 true EP0644935A1 (fr) 1995-03-29

Family

ID=3776217

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93912427A Withdrawn EP0644935A1 (fr) 1992-06-12 1993-06-11 SEQUENCES D'ADN CODANT LES RECEPTEURS HUMAINS DE L'ADENOSINE A1, A2a ET A2b

Country Status (3)

Country Link
EP (1) EP0644935A1 (fr)
JP (1) JPH08500967A (fr)
WO (1) WO1993025677A1 (fr)

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2264948B (en) * 1992-03-13 1996-10-16 Merck & Co Inc Human adenosine receptors
US5646156A (en) * 1994-04-25 1997-07-08 Merck & Co., Inc. Inhibition of eosinophil activation through A3 adenosine receptor antagonism
GB2289218A (en) * 1994-05-06 1995-11-15 Merck & Co Inc Inhibition of TNFalpha production with agonists of the A2b subtype of the adenosine receptor
US6025339A (en) * 1995-06-07 2000-02-15 East Carolina University Composition, kit and method for treatment of disorders associated with bronchoconstriction and lung inflammation
US6040296A (en) * 1995-06-07 2000-03-21 East Carolina University Specific antisense oligonucleotide composition & method for treatment of disorders associated with bronchoconstriction and lung inflammation
US7034007B1 (en) 1995-06-07 2006-04-25 East Carolina University Low adenosine anti-sense oligonucleotide, compositions, kit & method for treatment of airway disorders associated with bronchoconstriction, lung inflammation, allergy(ies) & surfactant depletion
US20020015967A1 (en) * 1998-06-02 2002-02-07 Lauren Silverman Functional expression of adenosine receptors in yeast
US20030017528A1 (en) 1998-11-20 2003-01-23 Ruoping Chen Human orphan G protein-coupled receptors
USRE42190E1 (en) 1998-11-20 2011-03-01 Arena Pharmaceuticals, Inc. Method of identifying a compound for inhibiting or stimulating human G protein-coupled receptors
US7816492B2 (en) 1998-11-20 2010-10-19 Arena Pharmaceuticals, Inc. Human G protein-coupled receptors
US6221660B1 (en) 1999-02-22 2001-04-24 Synaptic Pharmaceutical Corporation DNA encoding SNORF25 receptor
US6214807B1 (en) 1999-06-22 2001-04-10 Cv Therapeutics, Inc. C-pyrazole 2A A receptor agonists
US6403567B1 (en) 1999-06-22 2002-06-11 Cv Therapeutics, Inc. N-pyrazole A2A adenosine receptor agonists
USRE47351E1 (en) 1999-06-22 2019-04-16 Gilead Sciences, Inc. 2-(N-pyrazolo)adenosines with application as adenosine A2A receptor agonists
AU784543B2 (en) * 1999-11-16 2006-04-27 Pharmacia & Upjohn Company Novel G protein-coupled receptors
AU1410801A (en) * 1999-11-17 2001-05-30 Compugen Ltd. Variants of alternative splicing
US20020012946A1 (en) 2000-02-23 2002-01-31 Luiz Belardinelli Method of identifying partial agonists of the A2A receptor
US8470801B2 (en) 2002-07-29 2013-06-25 Gilead Sciences, Inc. Myocardial perfusion imaging methods and compositions
US20050020915A1 (en) 2002-07-29 2005-01-27 Cv Therapeutics, Inc. Myocardial perfusion imaging methods and compositions
NZ537975A (en) 2002-07-29 2007-08-31 Cv Therapeutics Inc Method of producing coronary vasodilation without peripheral vasodilation comprising administering at least 10 mcg of at least one A2A receptor agonist
EP1802317A2 (fr) 2004-10-20 2007-07-04 Cv Therapeutics, Inc. Utilisation d'agonistes du recepteur de l'adenosine a2a
US7732595B2 (en) 2006-02-03 2010-06-08 Gilead Palo Alto, Inc. Process for preparing an A2A-adenosine receptor agonist and its polymorphs
DK2121919T3 (da) * 2007-03-22 2012-05-21 Heptares Therapeutics Ltd Mutante g-proteinkoblede receptorer og fremgangsmåder til udvælgelse heraf

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ZA902280B (en) * 1989-03-29 1990-12-28 Merrell Dow Pharma Selective adenosine receptor agents
WO1991016056A1 (fr) * 1990-04-16 1991-10-31 The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce Emploi d'agonistes de recepteur purinergique utilises comme agents antineoplastiques
WO1992021701A1 (fr) * 1991-06-05 1992-12-10 The United States Of America Represented By The Secretary, Department Of Health And Human Services Lignees cellulaires transfectees de mammifere, exprimant le recepteur d'adenosine a¿1?

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9325677A1 *

Also Published As

Publication number Publication date
JPH08500967A (ja) 1996-02-06
WO1993025677A1 (fr) 1993-12-23

Similar Documents

Publication Publication Date Title
EP0644935A1 (fr) SEQUENCES D'ADN CODANT LES RECEPTEURS HUMAINS DE L'ADENOSINE A1, A2a ET A2b
Zhou et al. Molecular cloning and characterization of an adenosine receptor: the A3 adenosine receptor.
van Galen et al. Adenosine A1 and A2 receptors: structure–function relationships
Simmons et al. Isolation of a cDNA encoding CD33, a differentiation antigen of myeloid progenitor cells.
Ruth et al. Primary structure of the β subunit of the DHP-sensitive calcium channel from skeletal muscle
Roberds et al. Cloning and tissue-specific expression of five voltage-gated potassium channel cDNAs expressed in rat heart.
Sullivan et al. Inhibitory and stimulatory G proteins of adenylate cyclase: cDNA and amino acid sequences of the alpha chains.
Yamaki et al. The sequence of human retinal S-antigen reveals similarities with α-transducin
Lo Ten Foe et al. Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA
Fukuda et al. cDNA cloning and regional distribution of a novel member of the opioid receptor family
Voglmaier et al. Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2
Weinshank et al. Cloning, expression, and pharmacological characterization of a human alpha 2B-adrenergic receptor.
Gitt et al. Evidence that a human soluble beta-galactoside-binding lectin is encoded by a family of genes.
Price et al. Primary sequence and domain structure of chicken vinculin
Kirchloff et al. Cloning and analysis of mRNAs expressed specifically in the human epididymis
Tanaka et al. Molecular cloning of cDNA for proteasomes from rat liver: primary structure of component C3 with a possible tyrosine phosphorylation site
HEIM et al. Microdiversity of human‐plasma‐membrane calcium‐pump isoform 2 generated by alternative RNA splicing in the N‐terminal coding region
US5599671A (en) Human A2B adenosine receptor assay
Meng et al. Cloning and expression of the A 2a adenosine receptor from guinea pig brain
Casella et al. Isolation and characterization of cDNA encoding the alpha subunit of Cap Z (36/32), an actin-capping protein from the Z line of skeletal muscle.
Berg et al. Molecular characterization of rat NKR-P2, a lectin-like receptor expressed by NK cells and resting T cells.
Matsuoka et al. Complete sequence of human cardiac α‐myosin heavy chain gene and amino acid comparison to other myosins based on structural and functional differences
Than et al. Cloning and sequence analysis of cDNAs encoding human placental tissue protein 17 (PP17) variants
Tsuda et al. Structural analysis of mouse S-antigen
Abbott et al. Evidence that the 14 kDa soluble β-galactoside-binding lectin in man is encoded by a single gene

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19950103

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): CH DE FR GB IT LI

RIN1 Information on inventor provided before grant (corrected)

Inventor name: SELBIE, LISA

Inventor name: FURLONG, TIMOTHY

Inventor name: SHINE, JOHN

Inventor name: TOWNSEND-NICHOLSON, CONSTANCE ANDREA

Inventor name: PIERCE, KERRIE DIANE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19970102