WO2000068250A1 - Apj du rat de recepteurs 7tm - Google Patents
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- WO2000068250A1 WO2000068250A1 PCT/US2000/013235 US0013235W WO0068250A1 WO 2000068250 A1 WO2000068250 A1 WO 2000068250A1 US 0013235 W US0013235 W US 0013235W WO 0068250 A1 WO0068250 A1 WO 0068250A1
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in identifying compounds that may be agonists and/or antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
- 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.
- Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., Proc. Natl Acad.
- G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802-8).
- effector proteins e.g., phospholipase C, adenyl cyclase, and phosphodiesterase
- actuator proteins e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802-8).
- 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 The membrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane ⁇ -helices connected by extracellular or cytoplasmic loops. 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 amino 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.
- members of this family include, but are not limited to, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorant, and cytomegalo virus receptors.
- 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.
- the 7 transmembrane regions are designated as TMl, TM2, TM3, TM4, TM5, TM6, and TM7.
- TM3 has been implicated in signal transduction.
- Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors.
- Most 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 ⁇ -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 sockets 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 a 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 serines, 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 heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc.
- G-protein coupled receptors are found in numerous sites within a mammalian host.
- the present invention relates to Rattus norvegicus APJ, in particular Rattus norvegicus APJ polypeptides and Rattus norvegicus APJ polynucleotides, recombinant materials and methods for their production.
- the invention relates to methods for identifying agonists and antagonists/inhibitors of the Rattus norvegicus APJ gene.
- This invention further relates to the generation of in vitro and in vivo comparison data relating to the polynucleotides and polypeptides in order to predict oral absorption and pharmacokinetics in man of compounds that either agonize or antagonize the biological activity of such polynucleotides or polypeptides. Such a comparison of data will enable the selection of drugs with optimal pharmacokinetics in man, i.e., good oral bioavailability, blood-brain barrier penetration, plasma half life, and minimum drug interaction.
- the present invention further relates to methods for creating transgenic animals, which overexpress or underexpress or have regulatable expression of a APJ gene and "knock-out" animals, preferably mice, in which an animal no longer expresses a APJ gene. Furthermore, this invention relates to transgenic and knock-out animals obtained by using these methods. Such animal models are expected to provide valuable insight into the potential pharmacological and toxicological effects in humans of compounds that are discovered by the aforementioned screening methods as well as other methods.
- Rattus norvegicus APJ gene functions in these animal models is expected to provide an insight into treating and preventing human 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; urinary retention; osteoporosis; angina pectoris; 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 or Gilles dela Tourett's syndrome, hereinafter referred to as "the Diseases", amongst others.
- the Diseases Huntington's disease or Gilles dela Tourett's syndrome
- the present invention relates to Rattus norvegicus APJ polypeptides.
- Such polypeptides include isolated polypeptides comprising an amino acid sequence having at least a 95% identity, most preferably at least a 97-99% identity, to that of SEQ LD NO:2 over the entire length of SEQ ID NO:2.
- Such polypeptides include those comprising the amino acid of SEQ ID NO:2.
- polypeptides of the present invention are believed to be members of the 7TM receptor family of polypeptides. They are, therefore, of interest, because G-protein coupled receptors, more than any other gene family, are the targets of pharmaceutical intervention. Furthermore, the polypeptides of the present invention can be used to establish assays to predict oral absorbtion and pharmacokinetics in man and thus enhance compound and formulation design, among others.
- a polypeptide of the present invention exhibits at least one biological activity of Rattus norvegicus APJ.
- Polypeptides of the present invention also includes variants of the aforementioned polypeptides, including alleles and splice variants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non- conservative.
- Particularly preferred variants are those 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 amino acids are inserted, substituted, or deleted, in any combination.
- Particularly preferred primers will have between 20 and 25 nucleotides.
- Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ LD NO:2.
- biologically active fragments which are those fragments that mediate activities of APJ, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Rattus norvegicus or the ability to initiate, or maintain cause the Diseases in an individual, particularly a human. Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants maybe employed as intermediates for producing the full-length polypeptides of the invention.
- polypeptides of the present invention may be in the form of a "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence that contains secretory or leader sequences, pro- sequences, sequences that aid in purification, for instance, multiple histidine residues, or an additional sequence for stability during recombinant production.
- the present invention also includes variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.
- Polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
- the present invention relates to Rattus norvegicus APJ polynucleotides.
- Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide having at least a 95 % identity, to the amino acid sequence of SEQ LD NO:2, over the entire length of SEQ LD NO:2.
- polypeptides which have at least a 97% identity are highly preferred, while those with at least a 98-99%o identity are more highly preferred, and those with at least a 99% identity are most highly preferred.
- polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ LD NO:l encoding the polypeptide of SEQ LD NO:2.
- polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence having at least a 95%> identity, to a nucleotide sequence encoding a polypeptide of SEQ LD NO:2, over the entire coding region.
- polynucleotides which have at least a 97% 0 identity are highly preferred, while those with at least a 98-99% identity are more highly preferred, and those with at least a 99% identity are most highly preferred.
- polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence having at least a 95% identity, to SEQ ID NO:l over the entire length of SEQ ID NO: 1.
- polynucleotides which have at least a 97% identity are highly preferred, while those with at least a 98-99%> identify are more highly preferred, and those with at least a 99% identity are most highly preferred.
- Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ LD NO:l, as well as the polynucleotide of SEQ LD NO: 1.
- the invention also provides polynucleotides which are complementary to all the above described polynucleotides.
- the nucleotide sequence of SEQ LD NO: 1 shows homology with human APJ.
- the nucleotide sequence of SEQ LD NO:l is a cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 1 to 1134) encoding a polypeptide of 377 amino acids, the polypeptide of SEQ LD NO:2.
- polypeptide of the SEQ LD NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO:l or it may be a sequence other than SEQ ED NO:l, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
- the polypeptide of the SEQ LD NO:2 is structurally related to other proteins of the 7TM receptor family, having homology and/or structural similarity with human APJ.
- 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 APJ 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 Rattus norvegicus fetal spleen, using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252:1651-1656; Adams, M.D.
- EST expressed sequence tag
- 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-histidine peptide, as provided in the pQE vector (Qiagen, Lnc.) and described in Gentz, et al, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
- the polynucleotide may also comprise non-coding 5' and 3' sequences, such as transcribed, non- translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
- polynucleotides encoding polypeptide variants that comprise the amino acid sequence of SEQ ED NO:2 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 1 to 1 or 1 amino acid residues are substituted, deleted or added, in any combination.
- Particularly preferred probes will have between 30 and 50 nucleotides, but may have between 100 and 200 contiguous nucleotides of the polynucleotide of SEQ ED NO:l.
- TAG set forth in SEQ ED NO:l of Table 1, both of which encode an APJ polypeptide.
- the invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:
- Ri and R3 is independently any nucleic acid residue or modified nucleic acid residue
- m is an integer between 1 and 3000 or zero
- n is an integer between 1 and 3000 or zero
- R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof.
- R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the right, bound to R3.
- Any stretch of nucleic acid residues denoted by either R ⁇ and/or R 2 , where m and/or n is greater than 1 may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
- the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary.
- m and/or n is an integer between 1 and 1000.
- Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
- Polynucleotides that are identical, or are substantially identical to a nucleotide sequence of SEQ ED NO:l maybe used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, 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 homologs and orthologs from species other than Rattus norvegicus) that have a high sequence identity to SEQ ED NO: 1. Typically these nucleotide sequences are 95% identical to that of the referent.
- Preferred probes or primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides, and may even have at least 100 nucleotides. Particularly preferred primers will have between 20 and 25 nucleotides.
- a polynucleotide encoding a polypeptide of the present invention may be obtained by a process comprising the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof, preferably of at least 15 nucleotides in length; and isolating full-length cDNA and genomic clones comprising said polynucleotide sequence.
- a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof, preferably of at least 15 nucleotides in length; and isolating full-length cDNA and genomic clones comprising said polynucleotide sequence.
- Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in O.lx SSC at about 65°C.
- the present invention also includes isolated polynucleotides, preferably of at least 100 nucleotides in length, obtained by screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ED NO:l or a fragment thereof, preferably of at least 15 nucleotides.
- isolated polynucleotides preferably of at least 100 nucleotides in length, obtained by screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ED NO:l or a fragment thereof, preferably of at least 15 nucleotides.
- PCR Nucleic acid amplification
- the products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
- 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 systems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
- host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
- Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis, et al, BASIC METHODS LN MOLECULAR BIOLOGY (1986) and Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
- Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
- 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 include 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
- expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, 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 bacteriophage genetic elements, such as cosmids and phagemids.
- the expression systems may comprise control regions that regulate as well as engender expression.
- 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 nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook, et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).
- 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 intracellularly, 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 interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification. Rattus norvegicus APJ gene products can be expressed in transgenic animals.
- mice Animals of any species, including, but not limited to: mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, chimpanzees, maybe used to generate APJ transgenic animals.
- This invention further relates to a method of producing transgenic animals, preferably Rattus norvegicus, over-expressing APJ, which method may comprise the introduction of several copies of a segment comprising at least the polynucleotide sequence encoding SEQ ED NO:2 with a suitable promotor into the cells of a Rattus norvegicus embryo, or the cells of another species, at an early stage.
- This invention further relates to a method of producing transgenic animals, preferably Rattus norvegicus, under-expressing or regulatably expressing APJ, which method may comprise the introduction of a weak promoter or a regulatable promoter (e.g., an inducible or repressible promoter) respectively, expressibly linked to the polynucleotide sequence of SEQ LD NO:l into the cells of a Rattus norvegicus embryo at an early stage.
- a weak promoter or a regulatable promoter e.g., an inducible or repressible promoter
- This invention also relates to transgenic animals, characterised in that they are obtained by a method, as defined above.
- Any technique known in the art may be used to introduce a Rattus norvegicus APJ transgene into animals to produce a founder line of animals.
- Such techniques include, but are not limited to: pronuclear microinjection (U.S. Patent No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten, et al, Proc. Natl. Acad. Sci., USA 82: 6148-6152 (1985); gene targeting in embryonic stem cells (Thompson, et al, Cell 56: 313-321 (1989); electropolation of embryos (Lo, Mol Cell Biol.
- a further aspect of the present invention involves gene targeting by homologous recombination in embryonic stem cells to produce a transgenic animal with a mutation in a
- APJ gene ("knock-out” mutation).
- knock-out animals there is inactivation of the APJ gene or altered gene expression, such that the animals are useful to study the function of the APJ gene, thus providing animals models of human disease, which are otherwise not readily available through spontaneous, chemical or irradiation mutagenesis.
- Another aspect of the present invention involves the generation of so-called “knock-in” animals in which a portion of a wild-type gene is fused to the cDNA of a heterologous gene.
- This invention further relates to a method of producing "knock-out" animals, preferably mice, no longer expressing APJ.
- a Rattus norvegicus APJ cDNA SEQ ED NO: 1 can be used as a probe to screen suitable libraries to obtain the murine APJ genomic DNA clone.
- the method used to create a knockout mouse is characterised in that: a suitable mutation is produced in the polynucleotide sequence of the murine APJ genomic clone, which inhibits the expression of a gene encoding murine APJ, or inhibits the activity of the gene product; said modified murine APJ polynucleotide is introduced into a homologous segment of murine genomic DNA, combined with an appropriate marker, so as to obtain a labeled sequence comprising said modified murine genomic DNA; said modified murine genomic DNA comprising the modified polynucleotide is transfected into embryonic stem cells and correctly targeted events selected in vitro; then said stem cells are reinjected into a mouse embryo; then said embryo is implanted into a female recipient and brought to term as a chimera which transmits said mutation through the germline; and homozygous recombinant mice are obtained at the F2 generation which are recognisable by the presence of the marker.
- a mutation is generated in a murine APJ allele by the introduction of a DNA construct comprising DNA of a gene encoding murine APJ, which murine gene contains the mutation.
- the mutation is targeted to the allele by way of the DNA construct.
- the DNA of the gene encoding murine APJ comprised in the construct may be foreign to the species of which the recipient is a member, may be native to the species and foreign only to the individual recipient, may be a construct comprised of synthetic or natural genetic components, or a mixture of these.
- the mutation may constitute an insertion, deletion, substitution, or combination thereof.
- the DNA construct can be introduced into cells by, for example, calcium-phosphate DNA co-precipitation. It is prefe ⁇ ed that a mutation be introduced into cells using electroporation, microinjection, virus infection, ligand-DNA conjugation, virus-ligand-DNA conjugation, or liposomes.
- mice obtained by a method of producing recombinant mice as defined above, among others.
- APJ in vitro APJ "knock-outs" i.e., tissue cultures.
- Animals of any species including, but not limited to: mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, chimpanzees, may be used to generate in vitro APJ "knock-outs".
- Methods for "knocking out" genes in vitro are described in Galli-Taliadoros, et al, Journal of Immunological Methods 181: 1-15 (1995).
- Transgenic, "knock-in”, and “knock-out” animals are a particularly advantageous model, from a physiological point of view, for studying 7TM receptor. Such animals will be valuable tools to study the functions of a APJ gene. Moreover, such animal models are expected to provide information about potential toxicological effects in humans of any compounds discovered by an aforementioned screening method, among others.
- Rattus norvegicus APJ gene functions in these animal models is expected to provide an insight into treating and preventing human diseases including, but not limited to: infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HEV-1 or HEV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; 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 or Gilles dela Tourett's syndrome.
- infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HEV-1 or HEV-2
- pain cancers
- diabetes obesity
- anorexia bulimia
- asthma
- Polypeptides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases mentioned herein. It is, therefore, an aspect of the invention to devise screening methods to identify compounds that stimulate
- the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function of the polypeptide.
- agonists or antagonists may be employed for therapeutic and prophylactic purposes for the Diseases mentioned herein mentioned.
- Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
- Such agonists and antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case maybe, of the polypeptide; or may be structural or functional mimetics thereof (see Coligan, et al, CURRENT PROTOCOLS IN IMMUNOLOGY 1(2): Chapter 5 (1991)).
- 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.
- a screening method may involve measuring or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the polypeptide with a labeled competitor (e.g., agonist or antagonist).
- screening methods may test whether the candidate compound results in a signal generated by an agonist or antagonist of the polypeptide, using detection systems appropriate to cells bearing the polypeptide.
- Antagonists are generally assayed in the presence of a known agonist and an effect on activation by the agonist by the presence of the candidate compound is observed.
- screening methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide of the present invention, to form a mixture, measuring Rattus norvegicus APJ activity in the mixture, and comparing a Rattus norvegicus APJ activity of the mixture to a control mixture which contains no candidate compound.
- Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats.
- HTS formats include not only the well-established use of 96- and, more recently, 384-well microtiter 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 Rattus norvegicus APJ polypeptide, as herein described, can also be used for high-throughput screening assays to identify antagonists of antagonists of the polypeptide of the present invention (see D.
- a screening technique includes the use of cells which express the receptor of this invention (for example, transfected CHO cells) in a system which measures extracellular pH or intracellular 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. Such a method involves transfecting 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 c AMP 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 method for detecting agonists or antagonists for the receptor of the present invention is the yeast based technology as described in U.S. Patent No. 5,482,835
- potential polypeptide antagonists include antibodies or, in some cases, oligopeptides or proteins that are closely related to ligands, substrates, receptors, enzymes, etc., as the case may be, of a APJ polypeptide, e.g., a fragment of a ligand, substrate, receptor, enzyme, etc.; or small molecules which bind to a APJ polypeptide but do not elicit a response, so that an activity of a APJ polypeptide is prevented.
- the present invention relates to a screening kit for identifying agonists, antagonists, inhibitors, ligands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which compounds comprise a member selected from the group consisting of:
- (c) a cell membrane expressing a polypeptide of the present invention; which polypeptide is preferably that of SEQ ID NO:2. It will be appreciated that in any such kit, (a), (b) or (c) may comprise a substantial component.
- a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by: (a) determining in the first instance the three-dimensional structure of the polypeptide;
- the present invention relates to the use of Rattus norvegicus APJ polypeptides, polynucleotides, and recombinant materials thereof in selection screens to identify compounds which are neither agonists nor antagonist/inhibitors of Rattus norvegicus APJ.
- the data from such a selection screen is expected to provide in vitro and in vivo comparisons and to predict oral absorption, pharmacokinetics in humans.
- Allele refers to one or more alternative forms of a gene occurring at a given locus in the genome.
- “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 polynucleotide sequence that is shorter than the reference sequence of SEQ ED NO:l.
- Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
- EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
- employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e.g., EP-A 0232 262].
- “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 polynucleotides/genes or polypeptide that are homologs via speciation, that is closely related and assumed to have commend descent based on structural and functional considerations. "Paralog” refers to polynucleotides/genes or polypeptide that are homologs via gene duplication, for instance, duplicated variants within a genome.
- Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences.
- identity refers to an exact nucleotide to nucleotide or amino acid to amino acid co ⁇ espondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared.
- a "% identity” may be determined.
- the two sequences to be compared are aligned to give a maximum co ⁇ elation 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 lengths (so-called local alignment), that is more suitable for sequences of unequal length.
- Similarity is a further, more sophisticated measure of the relationship between two polypeptide sequences.
- similarity means a comparison between the amino acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact co ⁇ espondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact co ⁇ espondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated 'score' from which the "% similarity" of the two sequences can then be determined.
- BESTFIT Garnier et al, Nucleic Acids Res, 12, 387-395, 1984, available from Genetics Computer Group, Madison, Wisconsin, USA
- BESTFIT uses the "local homology” algorithm of Smith and Waterman (J. Mol.
- BESTFIT is more suited to comparing two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer.
- 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” and “Length Weight” used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively.
- % identities and similarities are determined when the two sequences being compared are optimally aligned.
- the BLOSUM62 amino 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 amino 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 polynucleotide or a polypeptide sequence of the present invention, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore described.
- a polynucleotide sequence having, for example, at least 95% identity to a reference polynucleotide sequence is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference sequence.
- Such point mutations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion.
- point mutations 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.
- a polynucleotide sequence having at least 95 %> identity to a reference polynucleotide sequence up to 5%> of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described.
- % identities such as 96%, 97%, 98%, 99% and
- a polypeptide sequence having, for example, at least 95%) identity to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include up to five point mutations per each 100 amino acids of the reference sequence.
- Such point mutations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non- conservative substitution, or insertion. These point mutations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
- Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100%> identity to the reference sequence of SEQ ID NO:l, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ED NO:l or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ED NO:l by the integer defining the percent identity divided by 100 and
- n n is the number of nucleotide alterations
- x n is the total number of nucleotides in
- SEQ ED NO: 1 y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
- Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ED NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
- Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of
- polypeptide sequence may be identical to the reference sequence of SEQ ED NO:2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ED NO:2, or: n a ⁇ x a - (x a • y),
- n a is the number of amino acid alterations
- x a is the total number of amino acids in
- y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
- Isolated means altered “by the hand of man” from its natural state, i.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.
- “Knock-in” refers to the fusion of a portion of a wild-type gene to the cDNA of a heterologous gene
- “Knock-out” refers to partial or complete suppression of the expression of a protein encoded by an endogenous DNA sequence in a cell.
- the “knock-out” can be affected by targeted deletion of the whole or part of a gene encoding a protein, in an embryonic stem cell. As a result, the deletion may prevent or reduce the expression of the protein in any cell in the whole animal in which it is normally expressed.
- RNA Variant refers to cDNA molecules produced from RNA molecules initially transcribed from the same genomic DNA sequence but which have undergone alternative RNA splicing.
- Alternative 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 amino acid sequences.
- the term splice variant also refers to the proteins encoded by the above cDNA molecules.
- Transgenic animal refers to an animal to which exogenous DNA has been introduced while the animal is still in its embryonic stage. In most cases, the transgenic approach aims at specific modifications of the genome, e.g., by introducing whole transcriptional units into the genome, or by up- or down-regulating pre-existing cellular genes. The targeted character of certain of these procedures sets transgenic technologies apart from experimental methods in which random mutations are confe ⁇ ed to the germline, such as administration of chemical mutagens or treatment with ionizing solution.
- Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA 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 comprising one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
- Modified bases include, for example, tritylated bases and unusual bases such as inosine.
- polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
- Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
- Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
- Polypeptide refers to both short chains, commonly refe ⁇ ed to as peptides, oligopeptides or oligomers, and to longer chains, generally refe ⁇ ed to as proteins. Polypeptides may comprise amino acids other than the 20 gene-encoded amino acids.
- Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art.
- Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may comprise many types of modifications. 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- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyro glutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, PROTEINS -
- Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties.
- a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino 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 amino acid sequence from another, reference polypeptide.
- a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
- a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
- a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
- Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
- the receptors of the present invention are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells.
- HEK293 human embryonic kidney 293
- adherent dhfr CHO cells 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 lipofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis.
- HEK293 or CHO cells transfected with the vector alone serve as negative controls.
- To isolate cell lines stably expressing the individual receptors about 24 clones are typically selected and analyzed by Northern blot analysis. Receptor mRNAs are generally detectable in about 50%> of the G418-resistant clones analyzed.
- a bank of over 200 putative receptor ligands 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 ligands.
- This bank is used to initially screen the receptor for known ligands, using both functional (i.e. calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as binding assays.
- Example 3 Ligand Binding Assays
- 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
- RNA transcripts (10 ng/oocyte) are injected in a 50 nl bolus using a micro injection apparatus.
- Two electrode voltage clamps are used to measure the cu ⁇ ents 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.
- 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 ligands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.
- Example 8 Calcium and cAMP Functional Assays 7TM receptors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimulation 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 ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization. Similarly, 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 fluctuation 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 APJ de Rattus norvegicus et un procédé pour produire ces polypeptides par des techniques recombinantes. Elle concerne aussi des procédés de criblage à la recherche de composés agonistes ou antagonistes des APJ de Rattus norvegicus. On s'attend à ce que ces composés soient utiles dans le traitement des maladies de l'homme, y compris de façon non limitative ce qui suit: les infections bactériennes, fongiques, causées par des protozoaires ou par des virus et autres infections, notamment celles causées par VIH-1 ou VIH-2; la douleur; les cancers; le diabète; l'obésité; l'anorexie; la boulimie; l'asthme; la maladie de Parkinson; l'insuffisance cardiaque aiguë; l'hypotension; la rétention d'urine; l'ostéoporose; l'angine de poitrine; l'infarctus du myocarde; l'ictus; les ulcères; l'asthme; les allergies; l'hypertrophie bénigne de la prostate; la migraine; le vomissement; les troubles psychotiques et neurologiques, y compris l'anxiété, la schizophrénie, le trouble bipolaire, la dépression, le délire, la démence et le retard mental grave, ainsi que des dyskinésies telles que le syndrome de Gilles de la Tourette et la chorée de Huntington.
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US13375499P | 1999-05-12 | 1999-05-12 | |
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DATABASE GENBANK [online] 1 November 1999 (1999-11-01), RIZZOTI K.: "The G protein-coupled receptor msr/APJ is expressed in endothelial precursers", XP002931468, Database accession no. Q9WV08 * |
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