WO2001009313A1 - Recepteur couple g-proteine et sequences adn de celui-ci - Google Patents

Recepteur couple g-proteine et sequences adn de celui-ci Download PDF

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Publication number
WO2001009313A1
WO2001009313A1 PCT/EP2000/006854 EP0006854W WO0109313A1 WO 2001009313 A1 WO2001009313 A1 WO 2001009313A1 EP 0006854 W EP0006854 W EP 0006854W WO 0109313 A1 WO0109313 A1 WO 0109313A1
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Prior art keywords
seq
polypeptide
sequence
polynucleotide
isolated
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PCT/EP2000/006854
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English (en)
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Klaus DÜCKER
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Merck Patent Gmbh
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Priority to JP2001514105A priority Critical patent/JP2003506040A/ja
Priority to CA002381437A priority patent/CA2381437A1/fr
Priority to EP00956200A priority patent/EP1198569A1/fr
Publication of WO2001009313A1 publication Critical patent/WO2001009313A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in identifying compounds that may be agonists, antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides, sometimes hereinafter referred to as high affinity lysophosphatidic acid receptor (HA-LPA-R) HA-LPA-R belong to the class of G-protein coupled receptors
  • proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e g , cAMP (Lefkowitz, Nature, 1991 , 351 353-354)
  • these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins
  • GPC receptors such as those for adrenergic agents and dopamine (Kobilka B K , et al , Proc Natl Acad Sci , USA, 1987, 84 46-50, Kobilka, B K , et al Science, 1987, 238 650- 656, Bunzow, J R , et al , Nature, 1988 336 783-787
  • G-proteins themselves, effector proteins, e g , phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e g., protein kinase A and protein kin
  • the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell
  • Enzyme activation by hormones is dependent on the presence of the nucleotide GTP
  • GTP also influences hormone binding
  • a G-protein connects the hormone receptor to adenylate cyclase G-protein was shown to exchange GTP for bound GDP when activated by a hormone receptor
  • the GTP-carrying form then binds to activated adenylate cyclase Hydrolysis of GTP to GDP, catalyzed by the G-protein itself returns the G- protein to its basal, inactive form
  • the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal
  • G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors
  • G-protein coupled receptors (otherwise known as 7TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 ammo acids, connecting at least eight divergent hydrophilic loops
  • the G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders
  • Other examples of members of this family include, but are not limited to, calcitonin, adrenergic, endothelin, cAMP, adenosine, musca ⁇ nic, acetylcho ne, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1 , rhodopsins, odorant, and cytomegalovirus receptors
  • TM1 TM2 Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure
  • TM3 TM4 TM5 TM6 Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure
  • TM7 TM3 The 7 transmembrane regions are designated as TM1 TM2, TM3 TM4 TM5 TM6 and TM7 TM3 has been implicated in signal transduction
  • G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus
  • G-protein coupled receptors such as the b- adrenoreceptor
  • phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization
  • the ligand binding sites of G-protein coupled receptors are believed to comprise hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains said socket being surrounded by hydrophobic residues of the G-protein coupled receptors
  • the hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face inward and form polar ligand binding site TM3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as the TM3 aspartate residue TM5 se ⁇ nes, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in ligand binding
  • G-protein coupled receptors can be intracellularly coupled by heterotrime ⁇ c
  • G-proteins to various intracellular enzymes, ion channels and transporters see, Johnson et al , Endoc Rev 1989 10 317-331 .
  • Different G-protein a-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell Phosphorylation of cytoplasmic residues of G- protein coupled receptors have been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors G-protein coupled receptors are found in numerous sites within a mammalian host
  • Lysophosphatidic acid is a bioactive phosphoiipid with diverse biological activities (Moolenaar, W H , Curr Opin Cell Biol 7(2) 203-10,
  • LPA LPA-induced cellular proliferation
  • alterations in differentiation and survival and suppression of apoptosis LPA also evokes cellular effector functions, which are dependent on cytoskeletal responses such as contraction, secretion, adhesion and chemotaxis (Goetzl, E J and An, S , FASEB J 12(15) 1 589-1598, 1998)
  • cytoskeletal responses such as contraction, secretion, adhesion and chemotaxis
  • LPA functions as a potent survival factor (Weiner, J A , and Chun, J , Proc Natl Acad Sci USA 96(9) 5233-5238, 1999) Additionally, among other processes LPA has been implicated in the pathological processes of mesangial pro ferative glomeruloneph ⁇ tis (Inoue, C N , Epstein, M , Forster, H G , Hotta, 0 , Kondo, Y and linuma, K , C n Sci 96(4) 431 -436, 1999) and atherosclerosis (Siess, W .
  • the present invention relates to HA-LPA-R, in particular HA-LPA-R polypeptides and HA-LPA-R polynucleotides, recombinant materials and methods for their production
  • Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to, infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2, pain, cancers, diabetes, obesity, anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, glomeruloneph ⁇ tis urinary retention, atherosclerosis, osteoporosis, angina pecto ⁇ s, myocardial infarction, stroke, ulcers, asthma, allergies, benign prostatic hypertrophy, migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression depression, delirium, dementia, and severe mental retardation, and dyskinesias such as Huntington's disease
  • the present invention relates to HA-LPA-R polypeptides
  • polypeptides include amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids
  • Polypeptides of the present invention are believed to be members of the G protein-coupled receptors family of polypeptides
  • the biological properties of the HA-LPA-R are hereinafter referred to as "biological activity of HA-LPA-R" or "HA-LPA-R activity”
  • a polypeptide of the present invention exhibits at least one biological activity of HA-LPA-R
  • Polypeptides of the present invention also includes variants of the aforementioned polypeptides, including all allelic forms and splice variants Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof Particularly preferred variants are those in which several, for instance from 50 to 30, from 30 to 20 from 20 to 10, from
  • Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an am o acid sequence having at least 30, 50 or 100 contiguous ammo acids from the ammo acid sequence of
  • Preferred fragments are biologically active fragments that mediate the biological activity of HA-LPA-R, including those with a similar activity or an improved activity, or with a decreased undesirable activity
  • polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention
  • the polypeptides of the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein It is often advantageous to include an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histidme residues, or an additional sequence for stability during recombinant production
  • Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occu ⁇ ng sources, from genetically engineered host cells comprising expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesisers, or a combination of such methods Means for preparing such polypeptides are well understood in the art
  • the present invention relates to HA-LPA-R polynucleotides
  • Such polynucleotides include
  • an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO 2 and/or SEQ ID NO 4 and/or SEQ ID NO 6,
  • polynucleotides that are fragments and variants of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof
  • Preferred fragments of polynucleotides of the present invention include an isolated polynucleotide comprising an nucleotide sequence having at least
  • polynucleotides of the present invention include splice variants, allelic variants and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs)
  • Polynucleotides of the present invention also include polynucleotides encoding polypeptide variants that comprise the am o acid sequence of SEQ ID NO 2 and/or SEQ ID NO 4 and/or SEQ ID NO 6 and in which several, for instance from 50 to 30, from 30 to 20 from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acid residues are substituted, deleted or added in any combination
  • the present invention provides polynucleotides that are RNA transcripts of the DNA sequences of the present invention Accordingly, there is provided an RNA polynucleotide that (a) comprises an RNA transcript of the DNA sequence encoding the polypeptide of SEQ ID NO 2 and/or SEQ ID NO 4 and/or SEQ ID NO 6,
  • (b) is the RNA transcript of the DNA sequence encoding the polyoeptide of SEQ ID NO 2 and/or SEQ ID NO 4 and/or SEQ ID NO 6,
  • (c) comprises an RNA transcript of the DNA sequence of SEQ ID NO 1 and/or SEQ ID NO 3 and/or SEQ ID NO 5 or
  • (d) is the RNA transcript of the DNA sequence of SEQ ID NO 1 and/or SEQ ID NO 3 and/or SEQ ID NO"5
  • the polynucleotide sequence of SEQ ID NO 1 and/or SEQ ID NO 3 and/or SEQ ID NO 5 shows homology with U76385 (Guo, Z ⁇ liom, K , Fischer, D J , Bathurst, I C , Tomei, L D , Kiefer M C and Tigyi, G Proc Natl Acad Sci USA 1996 Dec 10,93(25) 14367-72)
  • the polynucleotide sequence of SEQ ID NO 1 and/or SEQ ID NO 3 and/or SEQ ID NO 5 is a cDNA sequence that encodes the polypeptide of SEQ ID NO 2 and/or SEQ ID NO 4 and/or SEQ ID NO 6
  • the polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 and/or SEQ ID NO 4 and/or SEQ ID NO 6 may be identical to the polypeptide encoding sequence of SEQ ID NO 1 and/or SEQ ID NO 3 and/or SEQ ID NO 5 or it may
  • Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one HA-LPA-R activity
  • Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library derived from mRNA in cells of human brain, kidney blood lung colon lymphe nodes, liver or placenta (see for instance Sambrook er al , Molecular Cloning A Laboratory Manual, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989))
  • Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques
  • the polynucleotide may include the coding sequence for the mature polypeptide, by itself or the coding sequence for the mature polypeptide in reading frame with other coding sequences such as those encoding a leader or secretory sequence a pre- or pro- or prepro- protein sequence, or other fusion peptide portions
  • a marker sequence that facilitates purification of the fused polypeptide can be encoded
  • the marker sequence is a hexa-histid e peptide, as provided in the pQE vector (Qiagen, Inc ) and described in Gentz er al Proc Natl Acad Sci USA (1989) 86 821 -824 or is an HA tag
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, s
  • Polynucleotides that are identical or have sufficient identity to a polynucleotide sequence of SEQ ID NO 1 and/or SEQ ID NO 3 and/or SEQ ID NO 5, may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification reaction (for instance, PCR)
  • probes and primers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similarity to SEQ ID NO 1 and/or SEQ I D NO 3 and/or SEQ I D NO 5 typically at least 95% identity
  • Preferred probes and primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides Particularly preferred probes will have between 30 and 50 nucleotides
  • Particularly preferred primers will have between 20 and 25 nucleotides
  • a polynucleotide encoding a polypeptide of the present invention, including homologs from species other than human may be obtained by a process comprising the steps of screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO 1 and/or SEQ ID NO 3 and/or SEQ ID NO 5 or a fragment thereof, preferably of at least 15 nucleotides, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence
  • Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising 50% formamide 5xSSC (1 50mM NaCl 1 5mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7 6) 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0 1
  • PCR Nucleic acid amplification
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems Accordingly, in a further aspect, the
  • present invention relates to expression systems comprising a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be employed to produce such
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention Polynucleotides may be introduced into host cells by
  • Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated
  • bacterial cells such as Streptococci, Staphylococci E coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • a great variety of expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e g vectors derived from bacterial plasmids, from bacte ⁇ ophage from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof such as those derived from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids
  • the expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used The appropriate polynucleotide sequence may be inserted into an expression system by any of a
  • a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested prior to use in the screening assay If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide If produced mtracellularly the cells must first be lysed before the polypeptide is recovered
  • Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation acid extraction anion or cation exchange chromatography, phosphocellulose chromatography hydrophobic - . 4 -
  • Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene Detection of a mutated form of the gene characterised by the polynucleotide of SEQ ID NO 1 and/or SEQ ID NO 3 and/or SEQ ID NO 5 in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over- expression or altered spatial or temporal expression of the gene Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques well known in the art
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material
  • the genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques prior to analysis RNA or cDNA may also be used in similar fashion
  • Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype
  • Point mutations can be identified by hybridizing amplified DNA to labeled HA-LPA-R nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures
  • DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (see, for instance, Myers et al , Science (1985) 230 1242) Sequence changes at specific locations may also be revealed by nucle
  • An array of oligonucleotides probes comprising HA-LPA-R polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g , genetic mutations
  • Such arrays are preferably high density arrays or grids
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability, see, for example M Chee et al Science 274 610- 5 613 (1996) and other references cited therein
  • Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention Decreased or increased expression can be measured at the RNA level using any of the l o methods well known in the art for the quantitation of polynucleotides such as, for example, nucleic acid amplification, for instance PCR RT- PCR, RNase protection, Northern blotting and other hybridization methods Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived
  • Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays
  • the present invention relates to a diagonostic kit comprising
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO 1 and/or SEQ ID NO 3 and/or SEQ ID NO 5, or a fragment or an RNA transcript thereof
  • polypeptide of the present invention preferably the polypeptide of 25 SEQ ID NO 2 and/or SEQ I D NO 4 and/or SEQ I D NO 6 or a fragment thereof, or
  • an antibody to a polypeptide of the present invention preferably to the polypeptide of SEQ I D NO 2 and/or SEQ ID NO 4 and/or SEQ ID NO 6
  • kits may comprise 30 a substantial component
  • a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others
  • the polynucleotide sequences of the present invention are valuable for chromosome localisation studies
  • the sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data Such data are found in, for example, V McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical
  • DNAs Each of these DNAs contains random human genomic fragments maintained in a hamster background (human / hamster hybrid cell lines) These PCRs result in 93 scores indicating the presence or absence of the PCR product of the gene of interest These scores are compared with scores created using PCR products from genomic sequences of known location This comparison is conducted at http //www genome wi mit edu/ The gene of the present invention maps to human chromosome 6q16 1 -16 3
  • polynucleotide sequences of the present invention are also valuable tools for tissue expression studies Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them
  • the techniques used are well known in the art and include in situ hydndisation techniques to clones arrayed on a grid such as cDNA microarray hybridisation (Schena et al, Science, 270, 467-470, 1995 and Shalon et al.
  • polypeptides of the present invention are expressed in human brain, kidney, blood, lung, colon, lymphe nodes, liver or placenta
  • a further aspect of the present invention relates to antibodies
  • the polypeptides of the invention or their fragments, or cells expressing them, can be used as immunogens to produce antibodies that are immunospecific for polypeptides of the present invention
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art
  • Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bea ⁇ ng fragments, or cells to an animal preferably a non-human animal, using routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hyb ⁇ doma technique (Kohler, G and Milstem, C , Nature (1975) 256 495-497), the t ⁇ oma technique, the human B-cell hybndoma technique (Kozbor et al Immunology Today (1983) 4 72) and Q _
  • antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography
  • Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst others
  • polypeptides and polynucleotides of the present invention may also be used as vaccines Accordingly in a further aspect the present invention relates to a method for inducing an immunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine-producmg T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established within the individual or not
  • An immunological response in a mammal may also be induced by a method comprises delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention
  • One way of administering the vector is by accelerating it into the desired cells as a coating on particles or otherwise
  • Such nucleic acid vector may comprise DNA, RNA, a modified nu
  • Polypeptides of the present invention have one or more biological functions that are of relevance in one or more disease states in particular the diseases of the invention hereinbefore mentioned It is therefore useful to to identify compounds that stimulate or inhibit the function or level of the polypeptide Accordingly, in a further aspect the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function or level of the polypeptide Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the invention as hereinbefore mentioned Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries collections of chemical compounds, and natural product mixtures Such agonists or antagonists so-identified may be natural or modified substrates, gands, receptors, enzymes etc , as the case may be of the polypeptide a structural or functional mimetic thereof (see Coligan et al , Current Protocols in Immunology 1 (2) Chapter 5 (1991 )) or a small molecule
  • the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound
  • the screening method may involve measuring or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e g agonist or antagonist)
  • these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed
  • the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture measuring a HA-LPA-R activity in the mixture, and comparing the HA-LPA-R activity of the mixture to a control mixture
  • Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening
  • HTS HTS formats
  • HTS formats include not only the well-established use of 96- and, more recently 384-well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al Anal Biochem , 246, 20-29 (1997)
  • Fusion proteins such as those made from Fc portion and HA-LPA-R polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D Bennett et al , J Mol Recognition, 8 52-58 (1995) and K Johanson et al , J Biol Chem, 270(16) 9459-9471 (1995))
  • One screening technique includes the use of cells which express receptor of this invention (for example transfected CHO cells) in a system which measures extracellular pH or mtracellular calcium changes caused by receptor activation
  • compounds may be contacted with cells expressing the receptor polypeptide of the present invention
  • a second messenger response e g signal transduction, pH changes, or changes in calcium level is then measured to determine whether the potential compound activates or inhibits the receptor
  • Another method involves screening for receptor inhibitors by determining inhibition or stimulation of receptor-mediated cAMP and/or adenylate cyclase accumulation
  • a method involves transfectmg a eukaryotic cell with the receptor of this invention to express the receptor on the cell surface The cell is then exposed to potential antagonists in the presence of the receptor of this invention The amount of cAMP accumulation is then measured If the potential antagonist binds the receptor, and thus inhibits receptor binding, the levels of receptor-mediated cAMP, or adenylate cyclase, activity will be reduced or increased
  • Another methods for detecting agonists or antagonists for the receptor of the present invention is the yeast based technology as described in U S Patent 5,482,835
  • polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • a polypeptide of the present invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art These include, but are not limited to, ligand binding and crossl king assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ 25
  • antagonists of polypeptides of the present invention include antibodies or, in some cases, oligonucleotides or proteins that are closely related to the gands substrates, receptors, enzymes, etc , as the case may be, of the polypeptide e g , a fragment of the gands, substrates, receptors, enzymes, etc , or a small molecule that bind to the polypeptide of the present invention but do not elicit a response so that the activity of the polypeptide is prevented
  • Screening methods may also involve the use of transgenic technology and HA-LPA-R gene
  • transgenic technology and HA-LPA-R gene
  • the HA-LPA-R gene may be introduced through microinjection into the male pronucleus of fertilized oocytes, retroviral transfer into pre- or post-implantation embryos, or injection of genetically modified , such as by electroporation embryonic stem cells into host blastocysts Particularly useful transgenic animals are so-called
  • Knock-in transgenic animals in which an animal gene is replaced by the human equivalent within the genome of that animal Knock-in transgenic animals are useful in the drug discovery process for target validation, where the compound is specific for the human target
  • Other useful transgenic animals are so-called “knock-out” animals in which the expression of the i s animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence in a cell is partially or completely annulled
  • the gene knock-out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all,
  • Screening kits for use in the above described methods form a further aspect of the present invention
  • Such screening kits comprise
  • polypeptide is preferably that of SEQ ID NO 2 and/or SEQ ID NO 4 30 and/or SEQ ID NO 6
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as
  • Isolated means altered “by the hand of man” from its natural state, / e , if it occurs in nature, it has been changed or removed from its original environment, or both
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living
  • Polynucleotide generally refers to any poly ⁇ bonucleotide (RNA) or polydeox ⁇ bonucleotide (DNA) which may be unmodified or modified RNA or DNA
  • Polynucleotides include without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA
  • the term “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include for example t ⁇ tyl
  • Polypeptide refers to any polypeptide comprising two or more am o acids joined to each other by peptide bonds or modified peptide bonds, i e , peptide isosteres
  • Polypeptide refers to both short chains commonly referred to as peptides, ohgopeptides or oligomers, and to longer chains, generally referred to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded ammo acids
  • Polypeptides include ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature Modifications may occur anywhere in a polypeptide, including the peptide backbone the ammo acid side-chains and the ammo or carboxyl termini
  • polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods Modifications include acetylation, acylation, ADP- ⁇ bosylation, amidation , biotmylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative covalent attachment of a pid or hpid derivative, covalent attachment of phosphotidy nositol, cross-linking, cyc zation, disulfide bond formation demethylation, formation of covalent cross-links, formation of cystme, formation of pyroglutamate
  • “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide
  • “Fragment” of a polynucleotide sequence refers to a polynucioetide sequence that is shorter than the reference sequence of SEQ ID NO 1 and/or SEQ ID NO 3 and/or SEQ ID NO 5
  • Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains the essential properties thereof
  • a typical variant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs in ammo acid sequence from the reference polypeptide Generally, alterations are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in am o acid sequence by one or more substitutions insertions deletions in any combination A
  • Polymorphism refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome within a population.
  • SNP Single Nucleotide Polymorphism
  • SNP Single Nucleotide Polymorphism
  • An SNP may occur within a gene or within intergenic regions of the genome SNPs can be assayed using Allele Specific Amplification (ASA)
  • ASA Allele Specific Amplification
  • a common primer is used in reverse complement to the polymorphism being assayed This common primer can be between 50 and 1500 bps from the polymorphic base
  • the other two (or more) primers are identical to each other except that the final 3' base wobbles to match one of the two (or more) alleles that make up the polymorphism Two (or more)
  • RNA molecules produced from RNA molecules initially transcribed from the same genomic DNA sequence but which have undergone alternative RNA splicing
  • RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of that may encode different ammo acid sequences.
  • the term splice variant also refers to the proteins encoded by the above cDNA molecules
  • Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or ammo acid to ammo acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared
  • a "% identity” may be determined in general, the two sequences to be compared are aligned to give a maximum correlation between the sequences This may include inserting "gaps" in either one or both sequences to enhance the degree of alignment
  • a % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment) that is particularly suitable for sequences of the same or very similar length, or over shorter, defined 5 lengths (so-called local alignment) that is more suitable for sequences of unequal length
  • Similarity is a furtner, more sophisticated measure of the relationship between two polypeptide sequences
  • similarity means a comparison between the ammo acids of two polypeptide chains, on a
  • GAP aligns two sequences, finding a "maximum similarity", according to the algorithm of Neddleman and Wunsch (J Mol Biol, 48, 443-453, 1970) GAP is more suited to comparing sequences that are approximately the same length and an alignment is expected over the entire length
  • the parameters "Gap Weight” ana “Length Weight” used in each program are 50 and 3 for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively es being compared are optimally aligned
  • the BLOSUM62 am o acid substitution matrix (Henikoff S and Henikoff J G, Proc Nat Acad Sci USA, 89, 10915-10919, 1992) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into am o acid sequences before comparison
  • the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a reference polynucleotide or a polypeptide sequence, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore described
  • Identity Index is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence
  • a candidate polynucleotide sequence having, for example, an Identity Index of 0 95 compared to a reference polynucleotide sequence is identical to the reference sequence except that the candidate polynucleotide sequence may include on average up to five differences per each 100 nucleotides of the reference sequence Such differences are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion These differences may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence In other words, to obtain a polynucleotide sequence having an Identity Index of 0 95 compared to a reference polynucleotide sequence, an average of up
  • a candidate polypeptide sequence having, for example, an Identity Index of 0 95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 ammo acids of the reference sequence Such differences are selected from the group consisting of at least one ammo acid deletion, substitution including conservative and non-conservative substitution, or insertion These differences may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between these terminal positions interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • an average of up to 5 in every 100 of the ammo acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof as hereinbefore described The same applies mutatis mutandis for other values of the Identity Index, for instance 0
  • n a is the number of nucleotide or ammo acid differences
  • x a is the total number of nucleotides or am o acids in SEQ ID NO 1 and/or SEQ ID NO 3 and/or SEQ ID NO 5 or SEQ ID NO 2 and/or SEQ ID NO 4 and/or SEQ ID NO 6 respectively
  • I is the Identity Index
  • “Homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereinbefore defined Falling within this generic term are the terms “ortholog”, and “paralog” “Ortholog” refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species "Paralog” refers to a polynucleotideor polypeptide that within the same species which is functionally similar
  • Fusion protein refers to a protein encoded by two, unrelated, fused genes or fragments thereof Examples have been disclosed in US 5541087, 5726044
  • Fc-HA-LPA-R employing an immunoglobu n Fc region as a part of a fusion protein is advantageous for performing the functional expression of Fc-HA-LPA-R or fragments of HA-LPA-R, to improve pharmacokinetic properties of such a fusion protein when used for therapy and to generate a dime ⁇ c Fc-HA-LPA-R
  • the Fc- HA-LPA-R DNA construct comprises in 5' to 3' direction, a secretion cassette, i e a signal sequence that triggers export from a mammalian cell, DNA encoding an immunoglobuhn Fc region fragment, as a fusion partner, and a DNA encoding Fc-HA-LPA-R or fragments thereof
  • the receptors of the present invention are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells To maximize receptor expression typically all 5' and 3' untranslated regions (UTRs) are removed from the receptor cDNA prior to insertion into a pCDN or pCDNA3 vector The cells are transfected with individual receptor cDNAs by pofectin and selected in the presence of 400 mg/ml G418 After
  • a bank of over 600 putative receptor hgands has been assembled for screening
  • the bank comprises transmitters, hormones and chemokines known to act via a human seven transmembrane (7TM) receptor, naturally occurring compounds which may be putative agonists for a human 7TM receptor, non-mammalian, biologically active peptides for which a mammalian counterpart has not yet been identified, and compounds not found in nature, but which activate 7TM receptors with unknown natural hgands
  • This bank is used to initially screen the receptor for known hgands, using both functional (i e calcium cAMP microphysiometer oocyte electrophysiology, etc, see below) as well as binding assays J -
  • Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format.
  • the purified ligand for a receptor is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards its receptor.
  • Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell receptor sources.
  • specific receptor binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.
  • RNA transcripts from linearized plasmid templates encoding the receptor cDNAs of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures.
  • In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml.
  • Ovarian lobes are removed from adult female toads.
  • Stage V defolliculated oocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected in a 50 nl bolus using a microinjection apparatus.
  • Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in response to agonist exposure. Recordings are made in Ca2+ free Barth's medium at room temperature.
  • the Xenopus system can be used to screen known ligands and tissue/cell extracts for activating ligands.
  • Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell
  • the acid formed is largely as a result of the increased metabolic activity required to fuel the mtracellular signaling process
  • the pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd , Menlo Park CA)
  • the CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing mtracellular signaling pathway such as the G-protein coupled receptor of the present invention
  • the 7TM receptor of the invention is also functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc , functional screens) against tissue extracts to identify natural hgands
  • Extracts that produce positive functional responses can be sequencially subfractionated until an activating ligand is isolated identified
  • HEK 293 cells which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimuation or inhibition
  • Basal calcium levels in the HEK 293 cells in receptor-transfected or vector control cells were observed to be in the normal, 100 nM to 200 nM, range HEK 293 cells expressing recombinant receptors are loaded with fura 2 and in a single day > 150 selected hgands or tissue/cell extracts are evaluated for agonist induced calcium mobilization
  • HEK 293 cells expressing recombinant receptors are evaluated for the stimulation or inhibition of cAMP production using standard cAMP quantitation assays
  • Agonists presenting a calcium transient or cAMP flucuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor

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Abstract

L'invention concerne des polypeptides et des polynucléotides HA-LPA-R, ainsi que des procédés de production de ces polypeptides par des techniques recombinantes. L'invention concerne également des procédés utilisant ces polypeptides et ces polynucléotides HA-LPA-R dans des dosages de diagnostic.
PCT/EP2000/006854 1999-07-30 2000-07-18 Recepteur couple g-proteine et sequences adn de celui-ci WO2001009313A1 (fr)

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WO2001085935A2 (fr) * 2000-05-09 2001-11-15 Bayer Aktiengesellschaft Recepteur couple a une proteine g du type du gene 6 de differenciation endotheliale

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WO1997047316A1 (fr) * 1996-06-10 1997-12-18 Millennium Pharmaceuticals, Inc. Procedes de selection pour des composes utiles dans la regulation du poids corporel
WO1999024569A1 (fr) * 1997-11-11 1999-05-20 Ono Pharmaceutical Co., Ltd. Recepteur d'acide lysophosphatidique humain et utilisation dudit recepteur
WO2000022131A2 (fr) * 1998-10-13 2000-04-20 Arena Pharmaceuticals, Inc. Recepteurs non-endogenes de la proteine g humaine ayant une activite constitutive
WO2000031258A2 (fr) * 1998-11-20 2000-06-02 Arena Pharmaceuticals, Inc. Recepteurs humains couples a la proteine g orphan
WO2000031104A1 (fr) * 1998-11-19 2000-06-02 Smithkline Beecham Corporation Nouveau recepteur de 7tm couple a une proteine g (axor15)

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WO1997047316A1 (fr) * 1996-06-10 1997-12-18 Millennium Pharmaceuticals, Inc. Procedes de selection pour des composes utiles dans la regulation du poids corporel
WO1999024569A1 (fr) * 1997-11-11 1999-05-20 Ono Pharmaceutical Co., Ltd. Recepteur d'acide lysophosphatidique humain et utilisation dudit recepteur
EP1029916A1 (fr) * 1997-11-11 2000-08-23 Ono Pharmaceutical Co., Ltd. Recepteur d'acide lysophosphatidique humain et utilisation dudit recepteur
WO2000022131A2 (fr) * 1998-10-13 2000-04-20 Arena Pharmaceuticals, Inc. Recepteurs non-endogenes de la proteine g humaine ayant une activite constitutive
WO2000031104A1 (fr) * 1998-11-19 2000-06-02 Smithkline Beecham Corporation Nouveau recepteur de 7tm couple a une proteine g (axor15)
WO2000031258A2 (fr) * 1998-11-20 2000-06-02 Arena Pharmaceuticals, Inc. Recepteurs humains couples a la proteine g orphan

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085935A2 (fr) * 2000-05-09 2001-11-15 Bayer Aktiengesellschaft Recepteur couple a une proteine g du type du gene 6 de differenciation endotheliale
WO2001085935A3 (fr) * 2000-05-09 2002-04-11 Bayer Ag Recepteur couple a une proteine g du type du gene 6 de differenciation endotheliale

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