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WO2006005473A2 - Recepteurs associes a une amine de trace de rat - Google Patents

Recepteurs associes a une amine de trace de rat Download PDF

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Publication number
WO2006005473A2
WO2006005473A2 PCT/EP2005/007187 EP2005007187W WO2006005473A2 WO 2006005473 A2 WO2006005473 A2 WO 2006005473A2 EP 2005007187 W EP2005007187 W EP 2005007187W WO 2006005473 A2 WO2006005473 A2 WO 2006005473A2
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seq
polypeptide
isolated
recombinant
sequence
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WO2006005473A3 (fr
Inventor
Martin Ebeling
Marius Hoener
Lothar Lindemann
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F Hoffmann La Roche AG
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F Hoffmann La Roche AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Definitions

  • the present invention provides a fingerprint sequence which is specific and selective for trace amine associated receptors (TAAR) in rat, forming a subfamily of G protein coupled receptors.
  • the invention also provides the rat polypeptides identified as members of this family, nucleic acids encoding said polypeptides, and vectors, host cells and non-human animals comprising the novel family members.
  • the invention provides methods of identifying rat TAARs.
  • Trace amines are endogenous compounds structurally related to biogenic amines and are found in the mammalian nervous system in trace amounts. TAs are stored in nerve terminals and released together with classical biogenic amines. To date there is no evidence for the existence of synapses using TAs as their exclusive transmitter. Recently, receptors specifically binding trace amines were reported by Borowski et al (Trace amines: identification of a family of mammalian G protein-coupled receptors.
  • TARs TAA Receptors
  • TAARs do not respond to TAs at all, hence the term "associated". It covers all receptors of this GPCR family and is compatible with the different number of receptor genes in different species.
  • the novel nomenclature is strictly based on the sequential order of the receptor genes on the respective human chromosome as well as a detailed phylogenetic analysis (Fig. 3) of the receptor genes across different species. The nomenclature adheres to the following rules:
  • the present invention provides novel polypeptides identified as TAAR family members, the use of said polypeptides as drug target, the polynucleotide sequences encoding said polypeptides, and vectors, host cells and non-human animals comprising said polynucleotides. Furthermore, the present invention pertains the fingerprint motif specific and selective for the TAAR family and the use of it.
  • the present invention provides fingerprint motif comprising the sequence NSXXNPXXZXXXYXWF (SEQ. ID NO: 1), wherein X is any natural occurring amino acid and Z may be a tyrosine or histidine.
  • the term "fingerprint”, "fingerprint motif or “fingerprint sequence” as used herein relates to an amino acid sequence which is specific and selective for the rat GPCR subfamily TAAR.
  • the fingerprint motif is specific for functional TAARs.
  • the tryptophan residue in the context of the fingerprint motif is found exclusively in TAARs and not in any other known GPCR; rather, the corresponding sequence position is almost invariably occupied by polar or even charged amino acids in other GPCRs.
  • the fingerprint motif may be used for identifying TAARs, preferably for identifying functional TAARs.
  • the present invention also provides a method of identifying rat TAARs using the fingerprint sequence (SEQ. ID NO: 1).
  • SEQ. ID NO: 1 the fingerprint sequence
  • a polypeptide may have 100% identity.
  • the present invention pertains a method of identifying TAARs of other species, preferably of mammalians, using the fingerprint sequence (SEQ. ID NO: 1).
  • a polypeptide may have at least 75 % identity with the fingerprint sequence, preferably more than 87% identity, more preferably 100% identity.
  • the fingerprint sequence may be used as a "query sequence" to perform a search against sequence databases to, for example to identify TAAR family members.
  • Such searches can be performed using a pattern recognition program as for example fuzzpro of EMBOSS (Rice et al, EMBOSS: the European Molecular Biology Open Software Suite. Trends in Genetics, 2000, 16(6):276-277).
  • the rat TAAR family contains nineteen members (rTAARl to rTAAR 9) wherein two of them are pseudogenes: rTAAR7f ⁇ and rTAAR7i ⁇ . All genes encoding the rat
  • TAAR family members are located in the same region of chromosome 1 (Ipl2). With the exception of rTAAR 2, which is encoded by two exons, the coding sequences of all rat
  • TAAR genes are located in a single exon.
  • gene refers to any segment of DNA associated with a biological function.
  • a gene is an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a specific functional product.
  • pseudogene as used herein relates to an inactive gene which may have evolved by mutation events from an active ancestor gene. Inactive means that the gene is not translated to functional polypeptide.
  • the present invention provides an isolated or recombinant polypeptide comprising sequence SEQ. ID NO: 5.
  • Said polypeptide was identified as trace amine associated receptor (TAAR) and was named rTAAR2.
  • polypeptide sequence e.g., a protein, polypeptide, peptide, etc.
  • polypeptide sequence relates to a polymer of amino acids comprising naturally occurring amino acids.
  • isolated means altered “ by the hand of man” form the natural state. If an "isolated” composition or substance occures in nature, it has been changed or removed from its orignial environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated”, but the same polynucleotide or polypeptide separated form the coexisting materials of its natural state is "isolated” as the term is employed herein.
  • polynucleotide or polypeptide when used with reference, e.g. to a polynucleotide or polypeptide typically indicates that the polynucleotide or polypeptide has been modified by the introduction of a heterologuous (or foreign) nucleic acid of the alteration of a native nucleic acid, or that the protein or polypeptide has been mdified by the introduction of a hteerologous amino acid.
  • the present invention further pertains an isolated or recombinant polynucleotide sequence comprising SEQ. ID NO: 4 that encodes the polypeptide rTAAR2.
  • polynucleotide sequence e.g., a nucleic acid, polynucleotide, oligonucleotide, etc.
  • polynucleotide sequence e.g., a nucleic acid, polynucleotide, oligonucleotide, etc.
  • the present invention provides a polypeptide comprising sequence SEQ. ID NO: 7.
  • Said polypeptide belongs to the family of trace amine associated receptors and was named as rTAAR3.
  • the present invention further pertains an isolated or recombinant polynucleotide sequence comprising SEQ. ID NO: 6 that encodes the polypeptide rTAAR3.
  • the present invention provides a polypeptide comprising sequence SEQ. ID NO: 11. Said polypeptide belongs to the family of trace amine associated receptors and was named as rTAAR5.
  • the present invention further pertains an isolated or recombinant polynucleotide sequence comprising SEQ. ID NO: 10 that encodes the polypeptide rTAAR5.
  • the present invention provides an isolated or recombinant polypeptide comprising sequence SEQ. ID NO: 19.
  • Said polypeptide belongs to the family of trace amine associated receptors and was named as rTAAR7c.
  • the present invention further pertains an isolated or recombinant polynucleotide sequence comprising SEQ. ID NO: 18 that encodes the polypeptide rTAAR7c.
  • the present invention provides an isolated or recombinant polypeptide comprising sequence SEQ. ID NO: 35 Said polypeptide belongs to the family of trace amine associated receptors and was named as rTAAR8a.
  • the present invention further pertains an isolated or recombinant polynucleotide sequence comprising SEQ. ID NO: 34 that encodes the polypeptide rTAAR ⁇ a.
  • the present invention provides an isolated or recombinant polypeptide comprising sequence SEQ. ID NO: 37.
  • Said polypeptide belongs to the family of trace amine associated receptors and was named as rTAAR ⁇ b.
  • the present invention further pertains an isolated or recombinant polynucleotide sequence comprising SEQ. ID NO: 36 that encodes the polypeptide rTAAR ⁇ b.
  • the present invention provides an isolated or recombinant polypeptide comprising sequence SEQ. ID NO: 39.
  • Said polypeptide belongs to the family of trace amine associated receptors and was named as rTAAR ⁇ c.
  • the present invention further pertains an isolated or recombinant polynucleotide sequence comprising SEQ. ID NO: 38 that encodes the polypeptide rTAAR8c.
  • the present invention further pertains an isolated or recombinant polynucleotide sequence comprising SEQ. ID NO: 31.
  • This polynucleotide sequence was identified as pseudogene rTAAR7i ⁇ .
  • the other members of the rat TAARs family (rTAARl, rTAAR4, rTAAR6, rTAAR7a, rTAAR7b, rTAAR7d to 7h and rTAAR9) were previously described (Borowski et al Trace amines: identification of a family of mammalian G protein-coupled receptors. Proc Natl Acad Sci USA (2001) 98(16):8966-71.), however some of the published sequences have minor errors (see Table 1).
  • the present invention also provides the correct polynucleotide sequences of rTAARl (SEQ. ID NO: 2), rTAAR7b (SEQ. ID NO: 16), rTAAR7d (SEQ. ID NO: 20), rTAAR7e (SEQ. ID NO: 22), rTAAR7f ⁇ (SEQ. ID NO: 24).
  • This invention pertains further the correct amino acid sequences of rTAARl (SEQ. ID NO: 3), rTAAR7b (SEQ. ID NO: 17), rTAAR7d (SEQ. ID NO: 21), rTAAR7e (SEQ. ID NO: 23).
  • the present invention also provides the repaired nucleotide sequences of rTAAR7f ⁇ (SEQ. ID NO: 25) and rTAAR7i ⁇ (SEQ. ID NO: 32).
  • This invention further provides the polynucleotide sequences encoded by the fixed nucleotide sequence hTAAR7f (SEQ. ID NO: 26) and rTAAR7i (SEQ. ID NO: 33).
  • the term "repaired gene” or "fixed gene” as used herein relates to a pseudogene whose sequence was altered in a way that the gene becomes active.
  • the polynucleotide sequence of i.e. hTAAR3 is inactive due to a mutation that caused an early stop codon.
  • the sequence was repaired by insertion of two nucleotides at position 133-134 (see Figure 4).
  • the present invention also provides isolated or recombinant polpeptides comprising an amino acid sequence which comprise the fingerprint sequence and differs from the sequences SEQ. ID NOs: 3, 9, 13, 15, 17, 21, 23, 26, 28, 30, 33, 41.
  • the invention relates to the use of the polypeptides of the invention as drug target.
  • the polypeptides of the invention are used as a drug target for identifying compounds useful in depression therapy, schizophrenia therapy, migraine therapy, or in therapy of attention- deficit hyperactivity disorder or eating disorder as anorexia or obesity.
  • the drug target may be suitable for the design, screening and development of pharmaceutically active compounds
  • polypeptide having a polypeptide sequence SEQ. ID NO: 5, 7, 11, 19, 35, 37 or 39 as drug target Preferably, the polypeptides of the invention are used as a drug target for identifying compounds in depression therapy, schizophrenia therapy, migraine therapy, or in therapy of attention- deficit hyperactivity disorder or eating disorder as anorexia or obesity.
  • Another embodiment of the invention relates to the receptors having an amino acid sequence which comprise the fingerprint sequence and differs from the sequences SEQ. ID NOs: 3, 9, 13, 15, 17, 21, 23, 26, 28, 30, 33, 41 as drug target.
  • these receptors are drug target for identifying compounds in depression therapy, schizophrenia therapy, migraine therapy, or in therapy of attention-deficit hyperactivity disorder or eating disorder as anorexia or obesity.
  • polypeptide of the invention relates to the novel polypetides provided in the present invention, i.e. rTAAR2, rTAAR3 and rTAAR7c.
  • the present invention also pertain vectors comprising polynucleotides of the invention, host cells which are transduced with said vectors and the production of the polypeptides of the invention by recombinant techniques.
  • the vector comprises the polynucleotide comprising SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 10, SEQ. ID NO: 18, SEQ. ID NO: 34, SEQ. ID NO: 36 or SEQ. ID NO: 38 and preferably, the host cells are transduced with said vector.
  • Host cells can be genetically engineered (i.e., transduced, transformed or transfected) to incorporate expression systems or portion thereof for polynucleotides of the present invention.
  • Introduction of the vector into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic methods in molecular biology Elsevier, New York (1986); Davis JM (ed.): Basic cell culture: a practical approach, sec. edition. Oxford University Press (2002); R. Ian Freshney: Culture of Animal Cells: A Manual of Basic Technique, fourth edition. John Wiley & Sons (Sd) 1999; and Sambrook et al., Molecular cloning: a laboratory manual, 2.
  • a host cell maybe mammalian cell as HEK 293, CHO, COS, HeLa, neuronal, neuroendicrinal, neuroblastoma! or glial cell lines like SH-SY5Y, PC12, HN-IO (Lee HJ, Hammond DN, Large TH, Roback JD, Sim JA, Brown DA, Otten UH, Wainer BH.: Neuronal properties and trophic activities of immortalized hippocampal cells from embryonic and young adult mice. J Neurosci.
  • IMR-32 IMR-32, NB41A3, Neuro-2a, TE 671, primary neuronal or glia cells from mammals like rat or mouse, Xenopus oocytes, bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells such as Saccharomyces cerevisiae and Aspergillus cell; insect cells such as Drosophila S2 and Spodoptera Sf9 cells and plant cells.
  • mammals like rat or mouse
  • Xenopus oocytes bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as Saccharomyces cerevisiae and Aspergillus cell
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells and plant cells.
  • Such a system include, among others, chromosomal, episomal and virus -derived systems, i.e. vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episoms, from yeast chromosomal elements, from viruses such as baculovirus, papova viruses, such as SV40, vaccinia viruses, adenovirus, fowl pox virus, pseudorabies, retroviruses and vectors derived from combinations thereof, such as those derived from plasmids and bacteriophage 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 suitable to maintain, progagate or express polynucleotides to produce a polypeptide in a host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth on Sambrook et al., Molecular cloning: a laboratory manual, 2. Ed., Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y. (1989) or Borowski et al Trace amines: identification of a family of mammalian G protein-coupled receptors. Proc Natl Acad Sci USA (2001) 98(16):8966-71).
  • the present invention further provides a transgenic non-human animal comprising a polynucleotide encoding a polypeptide of the invention.
  • the transgenic non- human animal comprises a polynucleotide comprising SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 10, SEQ. ID NO: 18, SEQ. ID NO: 34, SEQ. ID NO: 36 or SEQ. ID NO: 38.
  • the transgenic non-human animal may be any non-human animal known in the art.
  • the transgenic non-human animal is a mammal, more preferably the transgenic non-human animal is a rodent.
  • the transgenic animal of the invention is a mouse.
  • transgenic non-human animal Methods of producing a transgenic non-human animal are well known in the art such as for example those set forth on Hogan, B.C., F; Lacy, E, Manipulating the Mouse Embryo: A Laboratory Manual. 1986, New York: Cold Spring Harbor Laboratory Press Hogan, B., et al., Manipulating the mouse embryo. 1994, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor and Joyner, A. (ed.): Gene Targeting - A Practical Approach Second Edition. Practical Approach Series, Oxford University Press 1999.
  • polypeptides of the invention may be employed in a screening process for compounds which bind the receptor and which activate (agonists) or inhibit (antagonists) activation of the receptor polypeptide of the present invention or compounds which modulate receptor function e.g. by acting on receptor trafficking.
  • the present invention provides a method for identifying compounds which bind to a polypeptide of the invention comprising: a) contacting the polypeptide of the invention with a candidate compound and b) determing whether compound binds to polypeptide of the invention.
  • the invention also provides a method for identifying compounds which have a stimulatory or inhibitory effect on the biological activity of a polypeptide of the invention or its expression comprising a) contacting the polypeptide of the invention with a candidate compound and b) determining if said compound has modulated the function or activity of the polypeptide of the invention.
  • such screening procedures as described above involve producing appropriate cells which express the receptor polypeptide of the present invention on the surface thereof, however, the receptor expressed in appropiate cells may also be localized intracellularly.
  • Such cells include i.e. cells from mammals, Xenopus, yeast, Drosophila or E. coli. Cells expressing the receptor (or cell membrane containing the expressed receptor) are then contacted with a test compound to measure binding, or stimulation or inhibition of a functional response.
  • One screening technique includes the use of cells which express receptor of this invention (for example, transfected CHO cells or HEK293) in a system which measures extracellular pH or intracellular calcium changes caused by receptor activation.
  • compounds may be contacted with cells expressing the receptor polypeptide of the present invention.
  • a second messenger response e.g., signal transduction, pH changes, PI hydrolyse, GTP - ⁇ - [ 35 S] release 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.
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the receptor is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the receptor, using detection systems appropriate to the cells bearing the receptor at their surfaces. 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. Standard methods for conducting such screening assays are well understood in the art.
  • polypeptide of the invention antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligand of the polypeptide of the invention, e.g., a fragment of the ligand, or small molecular weight molecules such as e.g. metabolites of neurotransmitters or of amino acids, which bind to the receptor but do not elicit a response, so that the activity of the receptor is prevented.
  • Figure 1 shows the chromosomal localization of TAARs genes in rat.
  • the relative position of TAAR genes on the chromosomes is indicated by boxes, representing the single coding exons of the respective genes.
  • rTAAR 2 which is encoded by two exons
  • the box indicates the position of the second coding exon harboring more than 95% of the open reading frame.
  • the width of the boxes is not to scale with regard to the length of the respective coding sequences, and the colors indicate a distinction in receptor subgroups as discussed in the text.
  • Figure 2 shows an alignment of all functional TAARs. Amino acid residues conserved in all human, rat and mouse TAARs are highlighted by black shading. The characteristic TAAR fingerprint motif is located in TM VII.
  • TM II 26-46, TM II: 60-80, TM III: 99-109, TM IV: 137-157, TM V: 189-209, TM VI: 253-273, TM VII: 288-308); hTAAR 2 (TM I: 49-71, TM II: 80-102, TM III: 129-151, TM IV: 163- 185, TM V: 208-230, TM VI: 263-285, TM VII: 300-322); hTAAR 5 (TM I: 36-58, TM II: 71-93, TM III: 108-130, TM IV: 150-172, TM V: 203-225, TM VI: 253-270, TM VII: 285- 307); hTAAR 6 (TM I: 33-53, TM II: 69-89, TM III: 108-128, TM IV: 148-168, TM V: 203-223, TM VI: 260-276, TM VII: 283-302);
  • Figure 3 shows the phylogenetic relationship of TAAR genes in human, rat and mouse based on their DNA sequence.
  • Figure 4 shows the ,,repair" of the pseudogene hTAAR 3 (old name GPR57 ⁇ ): The two bps CC in position 133-134 of the fixed ORF were added, which repairs the otherwise early stop codon TCA.
  • Figure 5 shows the relationship of TAARs based on the properties of the ligand pocket vectors (amino acid whose residues probably take part in the ligand binding, determined by calculation model).
  • LUVs The Ligand Pocket Vectors (LPVs) of human, rat and mouse TAAR proteins, as predicted according to Kratochwil et al. (2004). Individual TAARs are ordered such that proteins with closest similar ligand binding pockets are placed next to each other.
  • the physicochemical properties of the amino acid residues are indicated by colored shading (blue: hydrophobic/aromatic residues; green: aliphatic polar residues; red/plum: positively/negatively charged residues; orange/yellow: glycine/proline residues, potentially inducing kinks in the TM domains; pink: histidine; light blue: tyrosine).
  • the position of the respective amino acids in the transmembrane domains (TM 1-7) as well as the extracellular loop II (EC II) is indicated by boxes in the lower part of the figure.
  • TM 1--7 The position of the respective amino acids in the transmembrane domains
  • EC II extracellular loop II
  • B Hierarchical tree representation of the pharmacophore similarity of the ligand binding pockets of TAAR proteins.
  • the following proteins were included into the analysis as "repaired pseudogenes", modification in parenthesis: hTAAR 3 ⁇ (insertions: C135-, A136-), hTAAR 4 ⁇ (pointmutation: T41 IA, deletions: -605G, -750A), mTAAR 7c ⁇ (insertion: C513-), rTAAR 7f ⁇ (pointmutation: G515A), rTAAR 7i ⁇ (insertion: A90-).
  • Scale bar Pharmacophore diversity units.
  • the TAAR genes were identified by comparison of the previously published TAAR receptor genes with the human, rat and mouse genomic sequence information available from Genbank (see also Table 1) employing standard algorithms like BLAST.
  • TAAR genes were amplified by means of PCR from either genomic DNA or cDNA of the respective species. Were not further specified, all procedures were carried out basically described in: Sambrook, J., Fritsch, E.F., und Maniatis, T. (1989). Molecular Cloning: A laboratory manual (New York: Cold Spring Harbor Laboratory Press). All reagents were of highest available purity, and all solutions and reagents were sterilized prior to use unless otherwise stated.
  • oligonucleotide primers (table 2) were designed based on the
  • TAAR coding sequences which had been derived from the genomic sequence information available at Genbank.
  • the primers were designed such that the amplicons contain the entire open reading frames.
  • the primer were designed using the VectorNTI software (Vector NTI version 9.0.0, Informax) following standard rules for PCR primers, mainly: a) Primers should have a length of 18-25 nt b) a G/C content of about 50% c) should not contain inverted repeats longer than 3 nt d) should carry an G or C at least at the 5' end e) should have no simple repetitions of the same nts more than 3, and f) the annealing temperatures (TM) of primers used in the same PCR reaction should differ as little as possible (for details see for example: McPherson M.J., Hames B.D., Taylor G.R.
  • the annealing temperatures of the primer oligonucleotides were calculated based on rules described in: Breslauer KJ, Frank R, Blocker H, Marky LA.: Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A. 1986 Jun;83(ll):3746-50 (These rules assume that the sequences are not symmetric and contain at least one G or C and have a length of minimally 8 nts). Oligonucleotides were ordered from Microsynth (Microsynth AG, Sch ⁇ tzenstrasse 15, 9436 Balgach, Switzerland) in HPLC purified quality.
  • Table. 2 Primer for cloning of rTAARs.
  • rTAAR2 The main reason of cloning the rTAAR 2 from cDNA was to assure whether the transcript of the corresponding genes would in fact contain the two exons. Independent from these experiments, the two coding exons have been cloned separately from genomic DNA.
  • Tm melting temperature in 0 C calculated according to Breslauer KJ, Frank R, Blocker H, Marky LA.: Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A. 1986 Jun;83(ll):3746-50.; "5" in the primer name indicates 5' primer, "3" in the primer name indicates a 3' primer).
  • the actual PCR reactions had a total volume of 50 ⁇ l and the following composition: Template equaling 5-100 ng genomic DNA or cDNA equivalent to 100-200 ng of total RNA (sources specified below), 200 nM oligonucleotide primer, 1.5 mM MgCl 2 (Invitrogen), 200 mM dNTPs each (Invitrogen), Ix concentrated PCR reaction buffer (Invitrogen) and 5 U/reaction recombinant Taq DNA polymerase (Invitrogen).
  • the PCR reactions were assembled under a sterile working bench with UV irradiated equipment, and a) preparation of template material, b) assembly of PCR reactions, c) running the PCR reactions and agarose gel electrophoresis, and d) plasmid preparation were each performed in separated rooms in order to avoid any possible cross contamination of reagents with PCR amplified material or plasmid DNA.
  • the PCR reactions were assembled in 200 ⁇ l PCR cups (Eppendorf) including Taq DNA polymerase at room temperature (RT) and transferred to the thermocycler (Geneamp 9700 with gold block, Applied Biosystems) preheated to 95°C. The temperature profile was adjusted as follows:
  • annealing temperature the Tm (melting temperature) of the oligonucleotide primer with the lower Tm value for the PCR reaction (calculated as specified above) -1°C was used as annealing temperature.
  • Tm primer 1 55 0 C
  • Tm primer 2 57°C
  • annealing temperature 54°C.
  • extension time was calculated by length of amplicon (in kb) x 1 min.
  • cycle number For each gene several PCR reactions were run in parallel with cycle numbers between 25 - 40. All PCR reactions were subsequently analyzed by agarose gel electrophoresis, and the PCR reaction with the lowest cycle number still producing a clearly visible PCR product of the correct size was used for subsequent cloning.
  • PCR products were analyzed by agarose gel electrophoresis using 1% ultraPure agarose (GibcoBRL) made up in TAE buffer (Invitrogen). Agarose gels were run in PerfectBlue Horizontal Mini Electrophoresis Systems (Pequlab Biotechnologie GmbH, Er Weg, Germany) according to standard protocols (Sambrook et al., 1989). Agarose gels were stained with ethidium bromide, PCR products were visualized on a UV transilluminator (Syngene, Cambridge, UK), and the size of the PCR products was analyzed in comparison with the molecular weight standard 1 kb DNA ladder (Invitrogen).
  • PCR products of the expected sizes were cut out from the gels with sterile scalpels (Bayha, Tuttlingen, Germany) and extracted from the agarose gel slices using the QIAquick Gel Extraction Kit (QIAGEN AG, Basel, Switzerland) following the instructions of the manufacturer.
  • the extracted PCR products were precipitated with a cold ethanol/sodium acetate precipitation (Sambrook et al., 1989), and DNA pellets were dissolved in a volume of 10 ⁇ l of 10 mM Tris/HCl pH 7.5.
  • the PCR products in this solutions were subsequently cloned using the TOPO cloning kit for sequencing (Invitrogen; kits containing the vectors pCR4-TOPO and pCR2.1-TOPO were used) following the instructions of the manufacturer.
  • Ligations were transformed into TOPlO chemically competent bacteria (included in the TOPO cloning kit for sequencing) following the instructions of the manufacturer and subsequently plated on LB agar plates containing 100 ⁇ g/ml ampicillin (Sigma, Division of FLUBCA Chemie GmbH, Buchs, Switzerland) and incubated at 37°C over night.
  • the bacterial colonies were analyzed by a method called "miniprep PCR": Colonies were picked with a sterile inoculation loop (Copan Diagnostic S.P.A., Brescia, Italy) and transferred to new LB agar plates containing 100 ⁇ g/ml ampicillin.
  • PCR products were analyzed by agarose gel electrophoresis as described above. Bacterial colonies for which cloned DNA fragments of the expected sizes could be detected by the method outlined above were used for subsequent plasmid preparation.
  • bacterial colonies identified to carry inserts of the expected sizes by "miniprep PCR” were used to inoculate bacterial liquid cultures in LB medium containing 100 ⁇ g/ml ampicillin, and cultures were incubated on a horizontal shaker at 37°C over night (Sambrook et al., 1989). Plasmids were isolated from the bacterial liquid cultures using the HiSpeed Plasmid Maxi Kit (QIAGEN AG, Basel, Switzerland) following the instructions of the manufacturer.
  • Plasmid concentrations were determined by measuring the OD at 260 nm using an UV/VIS spectrophotometer ultrospec 3300 pro (Amersham Biosciences Europe GmbH, Otelfingen, Switzerland).
  • the size of inserts in the isolated plasmids was analyzed by means of restriction digests using an restriction endonuclease i.e. EcoRI (New England Biolabs, products distributed in Switzerland by Bioconcept, Allschwil, Switzerland); EcoRI restriction sites are flanking the multiple cloning sites in both vectors pCR4-TOPO and pCR2.1-TOPO, therefore the cloned inserts can be released from the plasmids by EcoRI restriction digests.
  • EcoRI New England Biolabs, products distributed in Switzerland by Bioconcept, Allschwil, Switzerland
  • Plasmids which turned out to carry inserts of the expected sizes as revealed by restriction analysis were subjected to DNA sequence analysis, carried out by an external company (Microsynth AG, Sch ⁇ tzenstrasse 15, 9436 Balgach, Switzerland).
  • DNA sequences of the cloned TAAR gene open reading frames, as revealed by DNA sequence analysis, were compared to either previously published sequences and/or to the sequence of the TAAR genes retrieved from the genome sequence information. Potential errors in the DNA sequence information which could have been introduced by the PCR reaction were eliminated by comparing the sequences of the two to three DNA plasmids which were cloned each from independent PCR reactions per TAAR gene: Since the probability of the same PCR error to occur at the very same position in independent PCR reactions is basically zero, the alignment of the independent sequences for each gene revealed the very correct DNA sequence.
  • Template material We paid particular attention to use standardized template material derived, were applicable, from the same inbred strains from which the genome sequence information available at Genbank was derived. Mice of the inbred strain C57BL/6, or genomic DNA derived from this mouse strain, was purchased from Jackson Laboratory (600 Main Street, Bar Harbor, Maine 04609 USA), rats of the inbred strain Crl:Wi was purchased from Charles River Laboratories France (Les Oncins, France; belongs to Charles River Laboratories, Inc.,251 Ballardvale Street,Wilmington, MA 01887-1000,USA).
  • Genomic DNA of tail biopsies taken from adult CrhWi rats was prepared basically according to: Laird PW, Zijderveld A, Linders K, Rudnicki MA, Jaenisch R, Berns A.: Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 1991 Aug 11;19(15):4293. The DNA concentration was determined by measuring the OD at 260 nm as described above and the DNA concentration was adjusted to 100 ng/ ⁇ l. The DNA was stored in aliquots at 4°C. Human genomic DNA was purchased from Stratagene.
  • cDNAs were either commercially purchased (human: Human brain, cerebellum Marathon-Ready cDNA, BD Biosciences Clontech), or made from total RNA.
  • RNA was either commercially purchased (human: human adult total brain, male, total RNA, Stratagene, Stratagene Europe, P.O. Box 12085 1100 AB Amsterdam, The Netherlands) or extracted from postnatal day 3 C57BL/6 mouse or CrhWi rat pups as described below.
  • Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction.
  • RNA pellet derived from each half brain was dissolved on 200 ⁇ l 10 mM Tris/HCl pH 7.0. Traces of genomic DNA were removed by DNAseI digest (addition of 10 ⁇ l 100 ⁇ l MgCl 2 and 5 ⁇ l RNAse free DNAse 1, 10 3 U/ ⁇ l, Roche; incubation at 37°C for 1 hr).
  • RNAse I was removed from the solution by phenol/chloroform extraction (H 2 O saturated phenol pH 7.0, Sigma) according to Sambrook et al. (1989).
  • the RNA was precipitated by cold ethanol/sodium acetate precipitate (Sambrook et al., 1989), dissolved in 10 mM Tris/HCl pH 7.0, and the RNA concentration was determined by measuring the OD at 260 nm as described above.
  • This RNA preparation was used for the synthesis of cDNA.
  • cDNA synthesis In an 1.5 ml Eppendorf cup was mixed: 2 ⁇ g total RNA in a total volume of ll ⁇ l, 1 ⁇ l 0.5 ⁇ g/ ⁇ l oligo dT12-18 (Invitrogen). The mixture was heated to 70 0 C for 10 min, placed on ice, and the following reagents were added:
  • Example 2 Cell culture & generating stable cell lines
  • DNA fragments carrying the entire TAAR open reading frames were removed from the respective TOPO vectors by restriction digest with i.e. EcoRI and subsequent purification of the ⁇ lkb fragments by agarose gel electrophoresis and gel extraction as described above.
  • the pIRES-NEO2 vector was linearized with EcoRI and dephosphorylated with shrimp alkaline phosphatase (Roche) following the instructions of the manufacturer.
  • the DNA fragments carrying the entire TAAR open reading frames were ligated into the linearized pIRES-NEO2 vector using T4 DNA ligase (New England Biolabs) by mixing 30 ng of linearized pIRES-NEO2 vector, 100-300 ng of DNA fragments carrying the respective TAAR coding sequence, Ix ligation buffer (final concentration; provided with the enzyme as 10x concentrate) and 1 ⁇ l of T4 DNA ligase in a total volume of 20 ⁇ l. The reaction was proceeded at room temperature for 2-3 hrs, and the ligation was transformed into chemically competent TOPlO cells as described for the TOPO ligations.
  • T4 DNA ligase New England Biolabs
  • Plating of transformed bacteria and picking of bacterial clones was performed as described earlier for the PCR cloning of TAAR genes from genomic and cDNA.
  • the picked bacterial clones were used to inoculate 5 ml liquid cultures in LB containing 100 ⁇ g/ml ampidllin. These liquid cultures were used for mini plasmid preparations using the QIAprep Miniprep Kit (QIAGEN), following the instructions of the manufacturer.
  • QIAprep Miniprep Kit QIAGEN
  • the purified pIRES-NEO2 derived plasmid constructs were analyzed for the presence of inserts of the expected sizes by restriction analysis using EcoRI and agarose gel electrophoresis.
  • Plasmids which were identified to carry inserts of the expected sizes were subjected to DNA sequence analysis carried out by Microsynth in order to make sure for the correct orientation and sequence of the inserts. Plasmids carrying the correct inserts in the correct orientation were used for expression of the respective TAARs in mammalian cell lines.
  • Stable cell lines For the generation of stably transfected cell lines HEK cells were transfected with the pIRES-NEO2 expression vectors of the respective TAARs by using Lipofectamin 2000 transfection reagent and Optimem 1 reduced serum medium with Glutamax (Invitrogen Cat. # 51985-026) following the recommendations given in the Lipofectamine 2000 datasheet. After 24 hr post transfection cells were trypsinated and transferred into 90 mm tissue culture dishes (Nunc) at dilutions between 1:10 - 1:300 in culture medium supplemented with 1 mg/ml G418 which was renewed on a daily basis.
  • Rat TAAR 1 Rat TAAR 1
  • the amount of cAMP produced by HEK293 cells stably expressing the indicated receptor in response to stimulation by each of the listed compounds was measured using the cAMP Biotrak EIA System (Amersham). Shown are the EC 50 values in ⁇ M (means of at least three independent experiments; EC 50 values are calculated from experiments carried out with 12 concentrations each in duplicates evenly distributed in the range of 30 ⁇ M - 0.1 nM). The maximal response is represented as percentage of the maximal cAMP levels induced by p-TYR. In the experiments with dopamine and serotonin (5-HT) the EC 5 Q values were calculated based on a maximal cAMP level of 50% compared to the level reached by p-Tyramine.
  • cAMP assay cAMP was assayed using the cAMP Biotrak Enzymeimmunoassay (EIA) System from Amersham as described in the product manual, following the so- called Non-acetylation EIA procdure. The general procedure is also described i.e. in Notley-McRobb L et al.
  • chromosome 6q23.1 The genes mapped to very compact regions spanning a total of about 109 kb in human (chromosome 6q23.1), 192 kb in mouse (chromosome 10A4) and 216 kb in rat (chromosome Ipl2; all chromosomal regions as assigned by LocusLink), with no other annotated or predicted genes in the same chromosomal regions.
  • TAAR 2 which is encoded by two exons
  • the coding sequences of all TAAR genes are located in a single exon and are very similar in their length of about 1 kb throughout all three species (range: 999 - 1089 bp). It is interesting to note that the region 6q23.1 on human chromosome no.
  • TAAR 1-5 As well as TAAR 6, 8 and 9 were found to be arranged in two blocks of genes with an inverse orientation of the open reading frames.
  • the most striking difference between human and rodents is the complete absence of any functional TAAR 7 paralogues from the human genome, which represent almost half of the entire TAAR family in rat and for which we detected only a degenerate gene fragment in human with closest similarity to rTAAR 7h.
  • TAAR 3 and 4 are functional receptors in rodents, but pseudogenes in human. The pseudogenization of hTAAR 3 ⁇ and 4 ⁇ likely is a recent event, since these genes are overall very well preserved with only two and three bp changes in the coding sequences, respectively, rendering them non-functional.
  • the different total number of nine TAAR 7 paralogues in rat (including two pseudogenes, rTAAR 7f ⁇ and rTAAR 7i ⁇ ) and six TAAR 7 paralogues in mouse (including one pseudogene, mTAAR 7c ⁇ ) represents a remarkable inter-rodent difference within a family of such closely related genes, which is reflected in strong predicted ligand binding differences between individual mouse and rat TAARs determined by the GPCR pharmacophore relationship of the ligand pocket vector analysis (see below).
  • TAAR genes Triggered by the compact chromosomal arrangement of TAAR genes in human and rodents and the high number of very similar genes within each species, the phylogenetic relationship of TAAR genes was analyzed across all three species.
  • the phylogenetic analysis shows that the TAAR genes originate in a putative common ancestor from which they evolved through a total of eight gene duplication events, leading to a set of nine genes before the primate and rodent lineage split (Fig. 2; gene duplication events from the common ancestor, speciations leading to human and primate lineage, and gene duplications within the rodent lineage are each collectively indicated by numbers 1, 2 and 3, respectively).
  • hTAAR 3 ⁇ became a pseudogene most likely due to a deletion of two bps (CA) at position 135 causing an early termination.
  • a pointmutation (A411T) turning a Cys into a stop codon and two insertions (G605, A750) might have caused the pseudogenisation of hTAAR 4 ⁇ .
  • rTAAR 7f ⁇ has two obvious differences to all the other TAAR 7 members of 12 additional bps at position 726, probably caused by an insertion event and of a pointmutation (A515G) turning a Trp into a stop codon.
  • the second rat pseudogene rTAAR 7i ⁇ misses 10 bps at position 90 causing an early termination
  • mTAAR 7c ⁇ misses 15 bps at position 895 (intracellular loop 3) and 7 bps at position 513 causing an early termination.
  • pseudogenisation seems to be caused by all possible mutational events like pointmutations, deletion and insertions. The events seem to have occurred fairly recently due to the highly conserved overall sequence. The pharmacology of such repaired pseudogenes might give insights into the importance of TAARs in the context of adaptation processes during evolution.

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Abstract

La présente invention concerne une séquence d'empreinte qui est spécifique de récepteurs associés à une amine de trace de rat (TAAR) et qui est sélective vis-à-vis de ces récepteurs formant une sous-famille de récepteurs couplés à la protéine G. Cette invention a aussi pour objet les nouveaux polypeptides de rat identifiés en tant que membres de cette famille, des acides nucléiques codant ces polypeptides, et des vecteurs et des cellules hôtes renfermant les nouveaux membres de la famille. Ladite invention a aussi trait à des méthodes d'identification desdits récepteurs TAAR de rat.
PCT/EP2005/007187 2004-07-08 2005-07-04 Recepteurs associes a une amine de trace de rat Ceased WO2006005473A2 (fr)

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US7381542B2 (en) 2001-07-09 2008-06-03 Oregon Health & Science University Nucleic acids encoding a biogenic amine receptor
CN118308457A (zh) * 2024-06-06 2024-07-09 汉王科技股份有限公司 嗅觉受体在识别三甲胺中的用途和检测三甲胺的方法

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US20020099200A1 (en) * 1999-12-29 2002-07-25 Borowsky Beth E. Dna encoding orphan snorf66 receptor
GB2373501A (en) * 2000-11-10 2002-09-25 Smithkline Beecham Corp GPR58a
US20040197766A1 (en) * 2001-07-05 2004-10-07 Millennium Pharmaceuticals, Inc. Methods and compositions for the treatment and diagnosis of body weight disorders
AU2003300776A1 (en) * 2002-09-09 2004-05-25 Omeros Corporation G protein coupled receptors and uses thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7381542B2 (en) 2001-07-09 2008-06-03 Oregon Health & Science University Nucleic acids encoding a biogenic amine receptor
CN118308457A (zh) * 2024-06-06 2024-07-09 汉王科技股份有限公司 嗅觉受体在识别三甲胺中的用途和检测三甲胺的方法

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