WO2001032833A2 - Monalisa, recepteur couple a la proteine g - Google Patents

Monalisa, recepteur couple a la proteine g Download PDF

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Publication number
WO2001032833A2
WO2001032833A2 PCT/US2000/030541 US0030541W WO0132833A2 WO 2001032833 A2 WO2001032833 A2 WO 2001032833A2 US 0030541 W US0030541 W US 0030541W WO 0132833 A2 WO0132833 A2 WO 0132833A2
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Prior art keywords
polypeptide
polynucleotide
sequence
seq
isolated
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PCT/US2000/030541
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English (en)
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WO2001032833A3 (fr
Inventor
Yuan Zhu
Xiaotong Li
Lisa Vawter
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Smithkline Beecham Corporation
Smithkline Beecham Plc.
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Publication of WO2001032833A2 publication Critical patent/WO2001032833A2/fr
Publication of WO2001032833A3 publication Critical patent/WO2001032833A3/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

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.
  • proteins participating in signal transduction pathways that involve G-protems 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 dopamme (Kobilka, B.K., et al , Proc Natl Acad. Sci , USA, 1987, 84 46-50; Kobilka, B.K, et al., Science, 1987,
  • G-protems themselves, effector proteins, e.g., phosphohpase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kmase C (Simon, M I , et al., Science, 1991, 252 802-8).
  • effector proteins e.g., phosphohpase C, adenyl cyclase, and phosphodiesterase
  • actuator proteins e.g., protein kinase A and protein kmase C (Simon, M I , et al., Science, 1991, 252 802-8).
  • GTP GTP also influences hormone binding.
  • a G-protem connects the hormone receptor to adenylate cyclase G-protem was shown to exchange GTP for bound GDP when activated by a hormone receptor.
  • the GTP-carrymg form then binds to activated adenylate cyclase.
  • the G-protem 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-protem coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
  • G-protem coupled receptors (otherwise known as 7TM receptors) have been characte ⁇ zed as including these seven conserved hydrophobic stretches of about 20 to 30 ammo acids, connecting at least eight divergent hydrophihc loops.
  • the G-protem family of coupled receptors includes dopamme receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders.
  • members of this family include, but are not limited to, calciton , adrenergic, endothelin, cAMP, adenosme, musca ⁇ nic, acetylcholme, serotonin, histamme, thrombm, kinm, follicle stimulating hormone, opsms, endothe al differentiation gene-1, rhodopsins, odorant, and cytomegalovirus receptors.
  • G-protem coupled receptors have single conserved cysteme residues m each of the first two extracellular loops which form disulf ⁇ de bonds that are believed to stabilize functional protein structure.
  • 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 Phosphorylation and hpidation (palmitylation or farnesylation) of cysteme residues can influence signal transduction of some G-protein coupled receptors.
  • Most G-protem coupled receptors contain potential phosphorylation sites withm the third cytoplasmic loop and/or the carboxy terminus.
  • G-protein coupled receptors such as the ⁇ -adrenoreceptor, phosphorylation by protein kmase A and/or specific receptor kmases mediates receptor desensitization.
  • the ligand binding sites of G-protem coupled receptors are believed to comprise hydrophihc sockets formed by several G-protem coupled receptor transmembrane domains, said socket being surrounded by hydrophobic residues of the G-protem coupled receptors.
  • the hydrophihc side of each G-protem coupled receptor transmembrane helix is postulated to face inward and form a polar hgand binding site TM3 has been implicated in several G-protem coupled receptors as having a ligand binding site, such as the TM3 aspartate residue.
  • TM5 se ⁇ nes, a TM6 asparagme and TM6 or TM7 phenylalanmes or tyrosmes are also implicated in ligand binding.
  • G-protein coupled receptors can be lntracellularly coupled by heterot ⁇ me ⁇ c G-protems to various mtracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331). Different G-protem ⁇ -subumts preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G- prote coupled receptors has been identified as an important mechanism for the regulation of G- protein coupling of some G-protem coupled receptors. G-protein coupled receptors are found m numerous sites withm a mammalian host.
  • the present invention relates to MonaLisa, m particular MonaLisa polypeptides and MonaLisa polynucleotides, recombinant mate ⁇ als 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; pam; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension, u ⁇ nary retention; 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, deh ⁇ um, dementia, and severe mental retardation; and dyskmesias, such as Huntmgton's disease or Gilles de
  • the invention relates to methods for identifying agonists and antagonists (e.g., inhibitors) using the materials provided by the invention, and treating conditions associated with MonaLisa imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with inappropriate MonaLisa activity or levels.
  • the present invention relates to MonaLisa polypeptides
  • Such polypeptides include (a) an isolated polypeptide encoded by a polynucleotide comp ⁇ smg the sequence of SEQ ID NO: 1 ;
  • Polypeptides of the present invention are believed to be members of the 7TM receptor family of polypeptides. They are therefore of interest because G-protem coupled receptors, more than other gene family, are targets of pharmaceutical intervention.
  • the biological properties of the MonaLisa are hereinafter referred to as "biological activity of MonaLisa” or “MonaLisa activity.”
  • a polypeptide of the present invention exhibits at least one biological activity of MonaLisa.
  • Polypeptides of the present invention also includes va ⁇ ants of the aforementioned polypeptides, including all allelic forms and splice va ⁇ ants. 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 va ⁇ ants are those m 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, m any combination.
  • Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an ammo acid sequence having at least 30, 50 or 100 contiguous amino acids from the ammo acid sequence of SEQ ID NO: 2, or an isolated polypeptide comprising an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO: 2.
  • Preferred fragments are biologically active fragments that mediate the biological activity of MonaLisa, including those with a similar activity or an improved activity, or with a decreased undesirable activity Also preferred are those fragments that are antigenic or lmmunogenic in an animal, especially in a human.
  • Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these va ⁇ ants 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 occurring sources, from genetically engineered host cells comprising expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesizers, or a combination of such methods. Means for prepa ⁇ ng such polypeptides are well understood m the art.
  • the present invention relates to MonaLisa polynucleotides.
  • Such polynucleotides include:
  • an isolated polynucleotide comprising a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2;
  • 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 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO: 1, or an isolated polynucleotide comprising an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO: 1.
  • Preferred va ⁇ ants of polynucleotides of the present invention include splice variants, allelic va ⁇ ants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • Polynucleotides of the present invention also include polynucleotides encoding polypeptide variants that comprise the ammo acid sequence of SEQ ID 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 2 to 1 or 1 ammo acid residues are substituted, deleted or added, in any combination.
  • the present invention provides polynucleotides that are RNA transc ⁇ pts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that is RNA transc ⁇ pts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that is RNA transc ⁇ pts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that
  • (a) comprises an RNA transc ⁇ pt of the DNA sequence encoding the polypeptide of SEQ ID NO.2,
  • (b) is the RNA transc ⁇ pt of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
  • (c) comprises an RNA transc ⁇ pt of the DNA sequence of SEQ ID NO: l; or
  • (d) is the RNA transc ⁇ pt of the DNA sequence of SEQ ID NO: 1 ,
  • RNA polynucleotides that are complementary thereto.
  • the polynucleotide sequence of SEQ ID NO: 1 shows homology with mouse G-protem coupled receptor, GPR27 (O'Dowd, B.F. et al. Genomics 47:310-313, 1998) .
  • the polynucleotide sequence of SEQ ID NO.1 is a cDNA sequence that encodes the polypeptide of SEQ ID NO:2.
  • the polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ ID NO: 1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO.2.
  • polypeptide of SEQ ID NO:2 is related to other proteins of the 7TM receptor family, having homology and/or structural similarity with mouse G-protem coupled receptor, GPR27 (O'Dowd, B.F., et al. Genomics 47:310-313, 1998).
  • Preferred polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one MonaLisa activity.
  • Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library de ⁇ ved from mRNA m cells of human fetal brain and placenta, (see for instance, Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Ed., Cold Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng 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-histidme peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et 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, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that stabilize mRNA.
  • Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO.1 may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification reaction (for instance, PCR). Such probes and p ⁇ mers 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 simila ⁇ ty to SEQ ED NO.1 , 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 p ⁇ mers 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 a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • Preferred st ⁇ ngent hyb ⁇ dization conditions include overnight incubation at 42oC in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium 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 m O.lx SSC at about 65oC.
  • 5xSSC 150mM NaCl, 15mM t ⁇ sodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5x Denhardt's solution 10 % dextran sulfate
  • 20 microgram/ml denatured, sheared salmon sperm DNA followed by washing the filters m O.lx SSC at about 65oC.
  • the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screening a library under st ⁇ ngent hybridization conditions with a labeled probe having the sequence of SEQ ID NO:l or a fragment thereof, preferably of at least 15 nucleotides.
  • an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide does not extend all the way through to the 5' terminus. This is a consequence of reverse transc ⁇ ptase, an enzyme with inherently low "processivity" (a measure of the ability of the enzyme to remain attached to the template du ⁇ ng the polymerization reaction), failing to complete a DNA copy of the mRNA template du ⁇ ng first strand cDNA synthesis.
  • cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the cDNA using a combination of gene specific and adaptor specific ohgonucleotide p ⁇ mers
  • PCR Nucleic acid amplification
  • the PCR reaction is then repeated using 'nested' primers, that is, p ⁇ mers designed to anneal within the amplified product (typically an adapter specific p ⁇ mer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' m the known gene sequence).
  • 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' p ⁇ mer.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems
  • 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
  • Polynucleotides may be introduced into host cells by methods desc ⁇ bed in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al.( ⁇ b ⁇ d)
  • Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micro-mjection, catiomc hpid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, or infection
  • bacterial cells such as Streptococci, Staphylococci, E coll, Streptomyces and Bacillus subtihs 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.
  • expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e g., vectors derived from bacterial plasmids, from bacte ⁇ ophage, from fransposons, 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 de ⁇ ved from combinations thereof, such as those de ⁇ ved 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.
  • 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 approp ⁇ ate polynucleotide 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., (ibid).
  • Approp ⁇ ate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the pe ⁇ plasmic space, or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • 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.
  • the cells may be harvested p ⁇ or to use m 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 pu ⁇ fied 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 pu ⁇ fication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during mtracellular synthesis, isolation, and/or purification.
  • Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations the associated gene Detection of a mutated form of the gene characterized by the polynucleotide of SEQ ID NO: 1 m 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 m 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 m 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 MonaLisa 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 m gels, with or without denatu ⁇ ng 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 nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401).
  • An array of ohgonucleotides probes comprising MonaLisa 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 va ⁇ abi ty, see, for example, M. Chee et al., Science, 274, 610-613 (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 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 from a host are well-known to those of skill in the art. Such assay methods include radio-immunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • the present invention relates to a diagnostic kit comprising: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment or an RNA transcript thereof;
  • polypeptide of the present invention preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or
  • kits may comprise a substantial component.
  • Such 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 localization 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 co ⁇ elated with genetic map data. Such data are found in, for example, V. McKusick, Mendehan Inhe ⁇ tance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the 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 m the art and include in situ hyb ⁇ dization techniques to clones arrayed on a grid, such as cDNA microarray hybridization (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR.
  • a preferred method uses the TAQMAN (Trade mark) technology available from Perkin Elmer. Results from these studies can provide an indication of the normal function of the polypeptide m the organism.
  • comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene can provide valuable insights into the role of the polypeptides of the present invention, or that of inapprop ⁇ ate expression thereof in disease.
  • Such inapprop ⁇ ate expression may be of a temporal, spatial or simply quantitative nature.
  • polypeptides of the present invention are expressed in at least human brain, placenta, testis, fetal brain, and fetal lung..
  • 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 lmmunogens to produce antibodies that are lmmunospecific 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 admmiste ⁇ ng the polypeptides or epitope-bea ⁇ ng fragments, or cells to an animal, preferably a non- human animal, using routine protocols.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hyb ⁇ doma technique (Kohler, G.
  • 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.
  • the present invention relates to a method for inducing an immunological response in a mammal that comp ⁇ ses inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine -producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established withm the individual or not.
  • An immunological response m a mammal may also be induced by a method comp ⁇ ses dehve ⁇ ng a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo m 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 nucleic acid, or a DNA/RNA hybrid
  • a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition).
  • the formulation may further comprise a suitable carrier Since a polypeptide may be broken down m the stomach, it is preferably administered parenterally (for instance, subcutaneous, intra-muscular, intravenous, or intra-dermal injection).
  • parenterally for instance, subcutaneous, intra-muscular, intravenous, or intra-dermal injection.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes that render the formulation mstonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions that may include suspending agents or thickening agents.
  • the formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze -dried condition requi ⁇ ng only the addition of the ste ⁇ le liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • 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 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, hgands, receptors, enzymes, etc., as the case may be, of the polypeptide; a structural or functional mimetic thereof (see Cohgan et al., Current Protocols in Immunology l(2):Chapter 5 (1991)) or a small molecule.
  • Such small molecules preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.
  • the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bea ⁇ ng 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 MonaLisa activity in the mixture, and comparing the MonaLisa 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 micotiter plates but also emerging methods such as the nanowell method desc ⁇ bed by Schullek et al, Anal Biochem., 246, 20-29, (1997) Fusion proteins, such as those made from Fc portion and MonaLisa 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 the 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.
  • 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 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 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.
  • 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 measu ⁇ ng 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 crosslmkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, 1251), chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods mclude biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art.
  • antagonists of polypeptides of the present invention include antibodies or, in some cases, oligonucleotides or proteins that are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, 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 MonaLisa gene.
  • transgenic animals are well established.
  • the MonaLisa 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-" animals in which an animal gene is replaced by the human equivalent withm the genome of that animal. Knock-m transgenic animals are useful m the drug discovery process, for target validation, where the compound is specific for the human target.
  • transgenic animals are so-called "knock-out" animals m which the expression of the 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 m certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells in the animal
  • Transgenic animal technology also offers a whole animal expression-clonmg system in which introduced genes are expressed to give large amounts of polypeptides of the present invention
  • Screening kits for use in the above described methods form a further aspect of the present invention
  • Such screening kits comprise
  • kits may comprise a substantial component.
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an
  • 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.
  • 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 comp ⁇ smg 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 ⁇ tylated bases and unusual bases such as inosine A variety of modifications may be made to DNA and RNA; thus, “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 characte ⁇ stic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides
  • Polypeptide refers to any polypeptide 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, ohgopeptides, or ohgomers, and to longer chains, generally referred to as proteins.
  • Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids
  • Polypeptides include amino 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 m more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere m a polypeptide, including the peptide backbone, the ammo acid side-chains and the ammo 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 m a given polypeptide. Also, a given polypeptide may contain many types of modifications.
  • Polypeptides may be branched as a result of ubiquitmation, 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, biotinylation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or hpid derivative, covalent attachment of phosphotidylmositol, cross-linking, cychzation, disulfide bond formation, demethylahon, formation of covalent cross-links, formation of cystme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as argmylation, and ubiquitmation
  • “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 ID NO.1.
  • “Va ⁇ ant” refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof. A typical va ⁇ ant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide.
  • Changes in the nucleotide sequence of the va ⁇ ant may or may not alter the amino 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 va ⁇ ant are closely similar overall and, m many regions, identical.
  • a va ⁇ ant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, insertions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe and Tyr.
  • a va ⁇ ant of a polynucleotide or polypeptide may be naturally occurring such as an allele, 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.
  • polypeptides having one or more post-translational modifications for instance glycosylation, phosphorylation, methylation, ADP ⁇ bosylation and the like.
  • Embodiments include methylation of the N-termmal ammo acid, phosphorylations of se ⁇ nes and threonmes and modification of C-termmal glycmes.
  • Allele refers to one of two or more alternative forms of a gene occurring at a given locus in the genome.
  • Polymorphism refers to a va ⁇ ation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome withm a population.
  • SNP Single Nucleotide Polymorphism
  • SNPs refers to the occurrence of nucleotide va ⁇ abihty at a single nucleotide position in the genome, withm a population.
  • An SNP may occur withm a gene or within mtergemc regions of the genome.
  • SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 p ⁇ mers are required.
  • a common p ⁇ mer 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) PCR reactions are then conducted on sample DNA, each using the common primer and one of the Allele Specific Primers.
  • "Splice Va ⁇ ant" as used herein refers to cDNA molecules produced from RNA molecules initially transc ⁇ bed from the same genomic DNA sequence but which have undergone alternative RNA splicing. Alternative RNA splicing occurs when a primary RNA transc ⁇ pt undergoes splicing, generally for the removal of mtrons, 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.
  • % Identity For sequences where there is not an exact co ⁇ espondence, 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" m 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.
  • Similar ⁇ ty is a further, more sophisticated measure of the relationship between two polypeptide sequences.
  • similar ⁇ ty means a comparison between the ammo acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences 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 correspondence, 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 uses the "local homology" algorithm of Smith and Waterman (J Mol Biol, 147,195-197, 1981, Advances in Applied Mathematics, 2, 482- 489, 1981) and finds the best single region of similarity between two sequences BESTFIT is more suited to compa ⁇ ng two polynucleotide or two polypeptide sequences that are dissimilar m 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 compa ⁇ ng 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 simila ⁇ ties are determined when the two sequences being compared are optimally aligned.
  • the BLOSUM62 ammo acid substitution mat ⁇ x (Hemkoff S and Hemkoff 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 ammo 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 desc ⁇ bed.
  • 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.
  • 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 m the reference sequence or in one or more contiguous groups withm the reference sequence.
  • the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore desc ⁇ bed.
  • na is the number of nucleotide or ammo acid differences
  • xa is the total number of nucleotides or ammo acids in SEQ ID NO.1 or SEQ ID NO:2, respectively,
  • 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 simila ⁇ ty between the two sequences as hereinbefore defined. Falling withm 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 polynucleotide or polypeptide that within the same species which is functionally similar.
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0 464 533-A 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].
  • 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 hpofectin 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-res ⁇ stant clones analyzed.
  • Example 2 Ligand
  • a bank of over 600 putative receptor ligands has been assembled for screening
  • the bank comp ⁇ ses transmitters, hormones and chemokmes known to act via a human seven transmembrane (7TM) receptor, naturally occur ⁇ ng 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.
  • Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format
  • the pu ⁇ fied 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 radiolabelmg does not dimmish 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 transc ⁇ pts from lmea ⁇ zed 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 defolhculated 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 Example 5.
  • Activation of a wide va ⁇ ety 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 su ⁇ oundmg 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 ligands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and 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 stimulation or inhibition.
  • Basal calcium levels in the HEK 293 cells in receptor-transfected or vector control cells were observed to be m 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.
  • 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 m vector control cells to determine if the response is unique to the transfected cells expressing receptor.
  • ATGGCCAACACTACCGGAGAGCCTGAGGAGGTGAGCGGCG 40 CTCTGTCCCCACCGTCCGCATCAGCTTATGTGAAGCTGGT 80 ACTGCTGGGACTGATTATGTGCGTGAGCCTGGCGGGTAAC 120 GCCATCTTGTCCCTGCTGGTGCTCAAGGAGCGTGCCCTGC 160 ACAAGGCTCCTTACTACTTCCTGCTGGACCTGTGCCTGGC 200 CGATGGCATACGCTCTCTGCCGTCTGCTTCCCCTTTGTGCTG 240 GCTTCTGTGCGCCACGGCTCTTCATGGACCTTCAGTGCAC 280 TCAGCTGCAAGATTGTGGCCTTTATGGCCGTGCTCTTTTG 320 CTTCCATGCGGCCTTCATGCTGTTCTGCATCAGCGTCACC 360 CGCTACATGGCCATCGCCCACCACCGCTTCTACGCCAAGC 400 GCATGACACTCTGGACATGCGCGGCTGTCATCTGCATGGC 440 CTGGACCCTGTCTGTGGCCATGGCCTTCCCACCTGTCT

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Abstract

L'invention concerne des polypeptides et polynucléotides de MonaLisa ainsi que des procédés de production de tels polypeptides à l'aide de techniques de recombinaison; elle concerne encore des procédés d'utilisation de polypeptides et polynucléotides de MonaLisa, dans des dosages diagnostiques.
PCT/US2000/030541 1999-11-04 2000-11-06 Monalisa, recepteur couple a la proteine g WO2001032833A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999046378A1 (fr) * 1998-03-12 1999-09-16 Yamanouchi Pharmaceutical Co., Ltd. Nouvelles proteines receptrices couplees aux proteines g
WO1999055733A1 (fr) * 1998-04-24 1999-11-04 Smithkline Beecham Corporation Clonage du recepteur axor-2 du 7tm

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999046378A1 (fr) * 1998-03-12 1999-09-16 Yamanouchi Pharmaceutical Co., Ltd. Nouvelles proteines receptrices couplees aux proteines g
WO1999055733A1 (fr) * 1998-04-24 1999-11-04 Smithkline Beecham Corporation Clonage du recepteur axor-2 du 7tm

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