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
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and m identifying compounds which may be agonists, antagonists and /or inhibitors which are potentially useful m 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-protems 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.
• G-proteins themselves, effector proteins, e.g., phosphohpase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g , protein kmase A and protein kmase 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-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 Hydrolysis of GTP to GDP, catalyzed by the G-protem itself, returns the G-protem 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-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 hydrophilic loops
• the G-protem family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders
• Other examples of members of this family include, but are not limited to, calciton , adrenergic, endothelin, cAMP, adenosme, musca ⁇ mc, acetylcholine, serotonin, mstamme, thrombin, kmm, follicle stimulating hormone, ops s, endothehal differentiation gene-1, rhodopsms, odorant, and cytomegalovirus receptors
• TM1, TM2, TM3, TM4, TM5, TM6, and TM7 TM3 has been implicated in signal transduction
• G-protem coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus
• G-protem 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 hydrophilic sockets formed by several G-protem coupled receptor transmembrane domains, said socket being surrounded by hydrophobic residues of the G-protem coupled receptors
• the hydrophilic side of each G-protem coupled receptor transmembrane helix is postulated to face mward and form polar ligand 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 m ligand binding G-protem coupled receptors can be mtracellularly coupled by heterot ⁇ me ⁇ c G-protems to va ⁇ ous mtracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc Rev., 1989, 10:317- 331)
• G-protem coupled receptors Phosphorylation of cytoplasmic residues of G-protem coupled receptors have been identified as an important mechanism for the regulation of G-protem coupling of some G- protem coupled receptors.
• G-protem coupled receptors are found m numerous sites withm a mammalian host. Over the past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane (7 TM) receptors have been successfully introduced onto the market.
• the present invention relates to AXOR17, m particular AXOR17 polypeptides and AXOR17 polynucleotides, recombmant mate ⁇ als and methods for their production.
• the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIN-l or HIN-2; pam; cancers; diabetes, obesity; anorexia, bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; u ⁇ nary retention; osteoporosis; angma 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 dyskinesias, such as Huntmgton's disease or Gilles de
• the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with AXOR17 imbalance with the identified compounds.
• the invention relates to diagnostic assays for detecting diseases associated with inapprop ⁇ ate AXOR17 activity or levels.
• the present invention relates to AXOR17 polypeptides.
• Such peptides include isolated polypeptides comprising an ammo acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID ⁇ O:2 over the entire length of SEQ ID NO:2.
• Such polypeptides include those comprising the ammo acid of SEQ ID NO:2.
• peptides of the present invention include isolated polypeptides in which the ammo acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the ammo acid sequence of SEQ ID NOJ over the entire length of SEQ ID NOJ.
• Such polypeptides include the polypeptide of SEQ ID NOJ
• peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: l. Polypeptides of the present invention are believed to be members of the G-Protem coupled,
• AXOR17 activity or "AXOR17 polypeptide activity” or "biological activity of AXOR17”.
• antigemc and immunogenic activities of said AXOR17 polypeptides in particular the antigemc and immunogenic activities of the polypeptide of SEQ ID NOJ.
• a polypeptide of the present invention exhibits at least one biological activity of AXOR17.
• 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 fusion protein. It is often advantageous to include an additional ammo acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid m purification such as multiple histidme residues, or an additional sequence for stability during recombinant production.
• the present invention also includes include va ⁇ ants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characte ⁇ stics. Typical such substitutions are among Ala,
• polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occumng polypeptides, recombmantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for prepa ⁇ ng such polypeptides are well understood in the art.
• the present invention relates to AXOR17 polynucleotides.
• Such polynucleotides include isolated polynucleotides comp ⁇ sing a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the ammo acid sequence of SEQ ID ⁇ OJ, over the entire length of SEQ ID NOJ.
• polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred.
• Such polynucleotides include a polynucleotide comp ⁇ smg the nucleotide sequence contained m SEQ ED NO- 1 encoding the polypeptide of SEQ ED NOJ
• polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ED NOJ, over the entire coding region.
• polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred
• polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ED NO: 1 over the entire length of SEQ ED NO: 1.
• polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identify are more highly preferred, and those with at least 99% identity are most highly preferred.
• Such polynucleotides include a polynucleotide comp ⁇ smg the polynucleotide of SEQ ED NO: 1 as well as the polynucleotide of SEQ ED NOJ .
• the invention also provides polynucleotides which are complementary to all the above described polynucleotides
• the nucleotide sequence of SEQ ED NO: 1 shows homology with Human Somatostatm SSTR4 [Y. Yammada et. al., Biochemical and Biophys Res. Com. 195, 844-852, 1993].
• the nucleotide sequence of SEQ ED NO: 1 is a cDNA sequence and comp ⁇ ses a polypeptide encoding sequence (nucleotide 1 to 1116) encoding a polypeptide of 372 ammo acids, the polypeptide of SEQ ED NOJ.
• the nucleotide sequence encoding the polypeptide of SEQ ED NOJ may be identical to the polypeptide encoding sequence contained in SEQ ED NO:l or it may be a sequence other than the one contained in SEQ ED NO: l, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NOJ.
• the polypeptide of the SEQ ED NOJ is structurally related to other proteins of the G-Protem coupled, 7TM family, having homology and/or structural similarity with Human P2Y9 Receptor [S.K. Bohm, et. al. Oct. 1997]
• 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 AXOR17 activity.
• Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library de ⁇ ved from mRNA in cells of human placenta genomic DNA, using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991)
• 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 which facilitates pu ⁇ fication of the fused polypeptide can be encoded.
• the marker sequence is a hexa-histidme peptide, as provided in the pQE vector (Qiagen, Inc.) and desc ⁇ bed in Gentz et al , Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
• the polynucleotide may also contain non-codmg 5 ' and 3 ' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that stabilize mRNA.
• polypeptide va ⁇ ants which comp ⁇ se the ammo acid sequence of SEQ ID NOJ and m which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, ammo acid residues are substituted, deleted or added, in any combination.
• Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 may be used as hyb ⁇ dization probes for cDNA and genomic DNA or as p ⁇ mers 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 human) that have a high sequence similarity to SEQ ED NO: 1
• these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to that of the referent.
• the probes or p ⁇ mers will generally comp ⁇ se at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides Particularly preferred probes will have between 30 and 50 nucleotides.
• a polynucleotide encoding a polypeptide of the present invention may be obtained by a process which comp ⁇ ses the steps of screening an approp ⁇ ate library under st ⁇ ngent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
• hyb ⁇ dization techniques are well known to the skilled artisan.
• Preferred st ⁇ ngent hyb ⁇ dization conditions include overnight incubation at 42°C in a solution comp ⁇ smg: 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in OJx SSC at about 65°C
• the present invention also includes polynucleotides obtainable by screening an approp ⁇ ate library under st ⁇ ngent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof.
• an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA 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 during the polymerization reaction), failing to complete a DNA copy of the mRNA template during 1st strand cDNA synthesis
• 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.
• Recombmant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comp ⁇ s g expression systems. Accordingly, in a further aspect, the present invention relates to expression systems which comp ⁇ se 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 recombmant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs de ⁇ ved from the DNA constructs of the present invention. For recombmant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
• polynucleotides into host cells can be effected by methods described m many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N.Y. (1989).
• Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, catiomc hpid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
• bacte ⁇ al cells such as streptococci, staphylococci, E. coli, 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
• a great va ⁇ ety of expression systems can be used, for instance, chromosomal, episomal and virus-de ⁇ ved systems, e.g., vectors de ⁇ ved from bacte ⁇ al plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors 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 which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
• the approp ⁇ ate nucleotide sequence may be inserted into an expression system by any of a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).
• 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 In this event, the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced mtracellularly, the cells must first be lysed before the polypeptide is recovered.
• Polypeptides of the present invention can be recovered and pu ⁇ fied from recombmant 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 lectm 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 du ⁇ ng isolation and or pu ⁇ fication.
• This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of the gene characterized by the polynucleotide of SEQ ED NO:l 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 expression of the gene. Individuals carrying mutations m the gene may be detected at the DNA level by a va ⁇ ety of techniques
• Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, u ⁇ ne, saliva, tissue biopsy or autopsy mate ⁇ al.
• the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques p ⁇ or 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 m compa ⁇ son to the normal genotype. Point mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled AXOR17 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments m gels, with or without denaturing agents, or by direct
• DNA sequencing (e.g., Myers et al , Science (1985) 230.1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton et al , Proc NatlAcad Set USA (1985) 85 4397-4401).
• nuclease protection assays such as RNase and S 1 protection or the chemical cleavage method (see Cotton et al , Proc NatlAcad Set USA (1985) 85 4397-4401).
• an array of ohgonucleotides probes comp ⁇ smg AXOR17 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g , genetic mutations.
• Array technology methods are well known and have general applicability and can be used to address a va ⁇ ety of questions in molecular genetics including gene expression, genetic linkage, and genetic va ⁇ abihty (see for example: M.Chee et al., Science, Nol 274, pp 610-613 (1996)).
• the diagnostic assays offer a process for diagnosing or determining a susceptibility to the Diseases through detection of mutation in the AXOR17 gene by the methods desc ⁇ bed.
• diseases may be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or mR ⁇ A. Decreased or increased expression can be measured at the R ⁇ A 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, R ⁇ ase 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 de ⁇ ved from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
• the present invention relates to a diagonostic kit which comprises:
• a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof ;
• b a nucleotide sequence complementary to that of (a);
• polypeptide of the present invention preferably the polypeptide of SEQ ID NOJ or a fragment thereof;
• kits an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NOJ.
• a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly infections such as bacte ⁇ al, fungal, protozoan and viral infections, particularly infections caused by HEV-1 or HIN-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, de ⁇ um, dementia, and severe mental retardation; and dyskinesias, such as Huntington's disease or Gilles dela Toure
• the nucleotide sequences of the present invention are also valuable for chromosome identification
• the sequence is specifically targeted to, and can hyb ⁇ dize with, a particular location on an individual human chromosome.
• the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, N. McKusick, Mendehan Inhe ⁇ tance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (comhe ⁇ tance of physically adjacent genes)
• the differences in the cD ⁇ A or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
• the gene of the present invention maps to human chromosome 12P 13.3
• polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as lmmunogens to produce antibodies lmmunospecific for polypeptides of the present invention.
• the term "lmmunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the p ⁇ or art.
• Antibodies generated against polypeptides of the present invention may be obtained by admmiste ⁇ ng the polypeptides or epitope-bea ⁇ ng fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols.
• an animal preferably a non-human animal
• any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hyb ⁇ doma technique (Kohler, G.
• Patent No. 4,946,778 can also be adapted to produce single chain antibodies to polypeptides of this invention.
• transgenic mice, or other organisms, including other mammals may be used to express humanized antibodies.
• the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to pu ⁇ fy the polypeptides by affinity chromatography.
• Antibodies against polypeptides of the present invention may also be employed to treat the Diseases, amongst others.
• the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and va ⁇ ous portions of the constant regions of heavy or light chains of lmmunoglobulms of various subclasses (IgG, IgM, IgA, IgE).
• lmmunoglobulm is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hmge region.
• the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa
• this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy.
• a further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found m International Patent Application Nos. W094/29458 and W094/22914.
• Another aspect of the invention relates to a method for inducing an immunological response m a mammal which comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases hereinbefore mentioned, amongst others.
• Yet another aspect of the invention relates to a method of inducing immunological response m a mammal which comprises, delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
• a further aspect of the invention relates to an lmmunological/vaccme formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a polypeptide of the present invention wherein the composition comprises a polypeptide or polynucleotide of the present invention.
• the vaccine 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, intramuscular, intravenous, or mtradermal injection).
• Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation mstomc with the blood of the recipient; and aqueous and non- aqueous sterile suspensions which 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-d ⁇ ed condition requiring only the addition of the sterile liquid carrier immediately prior to use.
• the vaccine formulation may also include adjuvant systems for enhancing the lmmunogemcity of the formulation, such as oil-m water systems and other systems known m the art.
• adjuvant systems for enhancing the lmmunogemcity of the formulation such as oil-m water systems and other systems known m the art.
• the dosage will depend on the specific activity of the vaccine and can be readily determined by routine expe ⁇ mentation.
• Polypeptides of the present invention are responsible for many biological functions, including many disease states, m particular the Diseases hereinbefore mentioned. It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide. Accordingly, m a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the polypeptide.
• agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned.
• Compounds may be identified from a va ⁇ ety of sources, for example, cells, cell-free preparations, chemical hbra ⁇ es, and natural product mixtures.
• Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology l(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.
• the screening method may involve competition with a labeled competitor.
• 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
• Constitutively active polypeptides may be employed m screening methods for inverse agonists or inhibitors, m the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide Further, 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 AXOR17 activity in the mixture, and comparing the screening methods.
• Fusion proteins such as those made from Fc portion and AXOR17 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 receptors 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 a 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 transfectmg a eukaryotic cell with the receptor of this invention to express the receptor on the cell surface The cell is then exposed to potential antagonists in the presence of the receptor of this invention. The amount of cAMP accumulation is then measured. If the potential antagonist binds the receptor, and thus inhibits receptor binding, the levels of receptor-mediated cAMP, or adenylate cyclase, activity will be reduced or increased.
• Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast based technology as desc ⁇ bed m 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 measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
• the polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art.
• ligand binding and crosslmkmg assays m which the polypeptide is labeled with a radioactive isotope (for instance, -*-*-"I), 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)
• a source of the putative receptor cells, cell membranes, cell supernatants, tissue extracts, bodily fluids
• Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art.
• polypeptide antagonists include antibodies or, in some cases, ohgonucleotides or proteins which are closely related to the hgands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the hgands, substrates, receptors, enzymes, etc.; or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented
• the present invention relates to a screening kit for identifying agonists, antagonists, hgands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which comprises'
• polypeptide of the present invention (c) a cell membrane expressing a polypeptide of the present invention; or (d) antibody to a polypeptide of the present invention, which polypeptide is preferably that of SEQ ED NOJ.
• kits may comprise a substantial component.
• 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:
• the present invention provides methods of treating abnormal conditions such as, for instance, infections such as bacte ⁇ al, fungal, protozoan and viral infections, particularly mfections caused by HIN-1 or HIN-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 dyskinesias, such as
• Huntingdon's disease or Gilles dela Tourett's syndrome related to either an excess of, or an under- expression of, AXOR17 polypeptide activity.
• One approach comp ⁇ ses admmiste ⁇ ng to a subject in need thereof an inhibitor compound (antagonist) as heremabove desc ⁇ bed, optionally m combination with a pharmaceutically acceptable earner, in an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of hgands, substrates, receptors, enzymes, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition.
• soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous polypeptide may be administered. Typical examples of such competitors include fragments of the
• expression of the gene encoding endogenous AXOR17 polypeptide can be inhibited using expression blocking techniques.
• Known such techniques involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56:560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
• ohgonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 3:173; Cooney et al , Science (1988) 241 :456; Dervan et al , Science (1991) 251: 1360). These o gomers can be administered per se or the relevant ohgomers can be expressed in vivo For treating abnormal conditions related to an under-expression of AXOR17 and its activity, several approaches are also available.
• a therapeutically effective amount of a compound which activates a polypeptide of the present invention i.e., an agonist as desc ⁇ bed above, in combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition.
• gene therapy may be employed to effect the endogenous production of AXOR17 by the relevant cells in the subject.
• a polynucleotide of the invention may be engineered for expression in a replication defective retro viral vector, as discussed above.
• the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing R ⁇ A encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
• These producer cells may be administered to a subject for engmee ⁇ ng cells in vivo and expression of the polypeptide in vivo.
• Another approach is to administer a therapeutic amount of a polypeptide of the present invention in combination with a suitable pharmaceutical earner.
• the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present invention, agonist/antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excrpient
• a pharmaceutically acceptable earner or excrpient include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
• the invention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
• Polypeptides and other compounds of the present invention may be employed alone or m conjunction with other compounds, such as therapeutic compounds.
• composition will be adapted to the route of administration, for instance by a systemic or an oral route.
• systemic administration include injection, typically by intravenous injection
• Other injection routes such as subcutaneous, intramuscular, or mtrapentoneal
• Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
• penetrants such as bile salts or fusidic acids or other detergents.
• oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like.
• the dosage range required depends on the choice of peptide or other compounds of the present invention, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of OJ-100 ⁇ g/kg of subject. Wide vanations m the needed dosage, however, are to be expected m view of the va ⁇ ety of compounds available and the diffe ⁇ ng efficiencies of va ⁇ ous routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection Vanations in these dosage levels can be adjusted using standard empi ⁇ cal routines for optimization, as is well understood in the art.
• Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as desc ⁇ bed above.
• cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector The cells are then introduced into the subject
• polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology. This is most easily facilitated by sto ⁇ ng the sequence in a computer readable medium and then using the stored data to search a sequence database using well known searching tools, such as GCC. Accordingly, in a further aspect, the present invention provides for a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ED NO:l and or a polypeptide sequence encoded thereby
• 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 Fab or other lmmunoglobulm expression library
• Isolated means altered “by the hand of man” from the natural state If an "isolated” composition or substance occurs m nature, it has been changed or removed from its original environment, or both
• a polynucleotide or a polypeptide naturally present in a living animal 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
• Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, 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 containing 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 mosme. A variety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabohcally 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 ohgonucleotides
• Polypeptide refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i.e , peptide isosteres. "Polypeptide” refers to both short chains, commonly referred to as peptides, ohgopeptides or 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 ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art Such modifications are well described m basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chams 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 in a given polypeptide. Also, a given polypeptide may contain 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- ⁇ bosylation, amidation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a hpid or hpid derivative, covalent attachment of phosphotidylmositol, cross-lmkmg, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation,
• 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 ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below.
• a typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, m many regions, identical.
• a variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions m any combination
• a substituted or inserted ammo 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.
• Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
• identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences
• Identity and similarity can be readily calculated by known methods, including but not limited to those described m (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin,
• Preferred computer program methods to determine identity and simila ⁇ ty between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol 215 403-410 (1990)
• the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al. , NCBI NLM NEH
• Preferred parameters for polypeptide sequence comparison include the following. 1) Algorithm: Needleman and Wunsch, J Mol Biol. 48: 443-453 (1970)
• Gap Penalty 12 Gap Length Penalty: 4
• Preferred parameters for polynucleotide comparison include the following- 1) Algorithm- Needleman and Wunsch, J. Mol Biol. 48 443-453 (1970)
• a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:l, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence.
• Such 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 withm the reference sequence.
• the number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ED NO: 1 by the numerical percent of the respective percent ⁇ dent ⁇ ty(d ⁇ v ⁇ ded by 100) and subtracting that product from said total number of nucleotides in SEQ ED NO: 1, or:
• n n is the number of nucleotide alterations
• x n is the total number of nucleotides in SEQ ID NO: l
• y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc, 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 .
• a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ED NOJ, that is be 100% identical, or it may include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the % identity is less than 100%.
• Such alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence.
• the number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids in SEQ ID NOJ by the numerical percent of the respective percent ⁇ dent ⁇ ty(d ⁇ v ⁇ ded by 100) and then subtracting that product from said total number of ammo acids in SEQ ED NOJ, or:
• n a is the number of ammo acid alterations
• x a is the total number of ammo acids m SEQ ED NOJ
• y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., 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
• 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 lmmunoglobulm molecules together with another human protein or part thereof.
• employing an lmmunoglobulm Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokmetic properties [see, e.g., EP-A 0232 262].
• Example 1 Mammalian Cell Expression
• 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 pnor to insertion into a pCDN or pCDNA3 vector.
• the cells are transfected with individual receptor cDNAs by hpofectm 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 m about 50% of the G418-res ⁇ stant clones analyzed
• a bank of over 200 putative receptor hgands has been assembled for screening.
• the bank compnses: transmitters, hormones and chemokmes known to act via a human seven transmembrane (7TM) receptor; naturally occumng 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 m nature, but which activate 7TM receptors with unknown natural hgands.
• This bank is used to initially screen the receptor for known hgands, using both functional (i.e., calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as binding assays.
• 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 radiolabehng 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 hgand. Where possible, more than one competing ligand is used to define residual nonspecific binding.
• Example 4 Functional Assay in Xenopus Oocytes
• RNA transc ⁇ pts from lmeanzed plasmid templates encoding the receptor cDNAs of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures. In vitro transc ⁇ pts are suspended in water at a final concentration of 0J mg/ml. Ova ⁇ an lobes are removed from adult female toads, Stage V defolhculated oocytes are obtained, and RNA transc ⁇ pts
• Activation of a wide vanety of secondary messenger systems results m extrusion of small amounts of acid from a cell.
• the acid formed is largely as a result of the increased metabolic activity required to fuel the mtracellular signaling process.
• the pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, CA).
• the CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing mtracellular signaling pathway such as the G-protem coupled receptor of the present invention.
• the 7TM receptor of the mvention is also functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify natural hgands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated identified.
• Example 8 Calcium and cAMP Functional Assays
• 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 in the normal, 100 nM to 200 nM, range.
• HEK 293 cells expressing recombmant receptors are loaded with fura 2 and m a single day > 150 selected hgands or tissue/cell extracts are evaluated for agonist induced calcium mobilization.
• HEK 293 cells expressing recombmant 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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