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WO1996039440A1 - Recepteur d'amines humaines - Google Patents

Recepteur d'amines humaines Download PDF

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Publication number
WO1996039440A1
WO1996039440A1 PCT/US1995/007221 US9507221W WO9639440A1 WO 1996039440 A1 WO1996039440 A1 WO 1996039440A1 US 9507221 W US9507221 W US 9507221W WO 9639440 A1 WO9639440 A1 WO 9639440A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
receptor
compound
polynucleotide
dna
Prior art date
Application number
PCT/US1995/007221
Other languages
English (en)
Inventor
Yi Li
Steven M. Ruben
Original Assignee
Human Genome Sciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to PCT/US1995/007221 priority Critical patent/WO1996039440A1/fr
Priority to CA002224094A priority patent/CA2224094A1/fr
Priority to EP95922243A priority patent/EP0832124A4/fr
Priority to JP9500375A priority patent/JPH11507813A/ja
Priority to AU26996/95A priority patent/AU2699695A/en
Publication of WO1996039440A1 publication Critical patent/WO1996039440A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention are human 7- transmembrane receptors and has been putatively identified as a human amine receptor. The invention also relates to inhibiting the action of such polypeptides.
  • proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lef owitz, Nature, 351:353-354 (1991)).
  • cAMP Lef owitz, Nature, 351:353-354 (1991)
  • these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins.
  • GPC receptors such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., PNAS, 84:46-50 (1987); Kobilka, B.K., et al., Science, 238:650-656 (1987); Bunzow, J.R., et al., Nature, 336:783-787 (1988)), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 252:802-8 (1991)).
  • GPC receptors such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., PNAS, 84:46-50 (1987); Kobilka, B.K.,
  • the effect of hormone binding is activation of an enzyme, adenylate cyclase, inside the cell.
  • Enzyme activation by hormones is dependent on the presence of the nucleotide GTP, and GTP also influences hormone binding.
  • a G-protein connects the hormone receptors to adenylate cyclase. G- protein was shown to exchange GTP for bound GDP when activated by hormone receptors. The GTP-carrying form then binds to an activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G- protein to its basal, inactive form.
  • the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
  • G-protein coupled receptors The membrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane of-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
  • G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al . , Endoc, Rev., 10:317-331 (1989)). Different G-protein ⁇ - subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors have been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host. The Human Amine Receptor of the present invention is a G-protein coupled receptor. Neurosensory and neuromotor functions are carried out by neurotransmission.
  • Neurotransmission is the conductance of a nerve impulse from one neuron, called the presynaptic neuron, to another neuron, called the postsynaptic neuron, across the synaptic cleft. Transmission of the nerve impulse across the synaptic cleft involves the secretion of neurotransmitter substances. The neurotransmitter is packaged into vesicles in the presynaptic neuron and released into the synaptic cleft to find its receptor at the postsynaptic neuron. Transmission of the nerve impulse is normally transient.
  • synaptic transmission An essential property of synaptic transmission is the rapid termination of action following neurotransmitter release.
  • neurotransmitters including catecholamine, serotonin, and certain amino acids (e.g., gamma-aminobutyric acid (GABA) , glutamate and glycine)
  • GABA gamma-aminobutyric acid
  • glutamate and glycine gamma-aminobutyric acid
  • This rapid re-accumulation of a neurotransmitter is the result of re-uptake by the presynaptic terminals.
  • the various molecular structures for re-uptake are highly specific for such neurotransmitters as choline and the biogenic amines (low molecular weight neurotransmitter substances such as dopamine, norepinephrine, epinephrine, serotonin and histamine) .
  • These molecular apparatuses are receptors which are termed transporters. These transporters move neurotransmitter substances from the synaptic cleft back across the cell membrane of the presynaptic neuron into the cytoplasm of the presynaptic terminus and therefore terminate the function of these substances. Inhibition or stimulation of neurotransmitter uptake provides a means for modulating the effects of the endogenous neurotransmitters.
  • the neurotransmitter substances are implicated in numerous pathophysiologies and treatments including, movement disorders, schizophrenia, drug addiction, anxiety, migraine headaches, epilepsy, myoclonus, spastic paralysis, muscle spasm, schizophrenia, cognitive impairment, depression, Parkinson's Disease and Alzheimer's Disease, among others.
  • Re-uptake of neurotransmitter substances by the transporters may be sodium-dependent.
  • the GABA transporter is a member of the recently described sodium-dependent neurotransmitter transporter gene family.
  • These transporters are transmembrane receptor complexes having an extracellular portion, a transmembrane portion and an intracellular portion.
  • a significant degree of homology exists in the transmembrane domains of the entire family of sodium-dependent neurotransmitter transporter proteins, with considerable stretches of identical amino acids, while much less homology is apparent in the intracellular and extracellular loops connecting these domains.
  • the extracellular loop in particular seems to be unique for each transporter. This region may contribute to substrate and/or inhibitor specificities.
  • the polypeptide of the present invention has been putatively identified as an amine receptor. This identification has been made a ⁇ a result of amino acid sequence homology to the rat amine receptor.
  • novel mature receptor polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • the receptor polypeptides of the present invention are of human origin.
  • nucleic acid molecules encoding the receptor polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
  • processes for producing such receptor polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing nucleic acid sequences encoding the receptor polypeptides of the present invention, under conditions promoting expression of said polypeptides and subsequent recovery of said polypeptides.
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the polynucleotide sequences of the present invention.
  • diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences encoding such polypeptides and for detecting an altered level of the soluble form of the receptor polypeptides.
  • Figure 1 illustrates the cDNA sequence and corresponding deduced amino acid sequence of the human amine receptor of the present invention.
  • the standard one-letter abbreviations for amino acids are used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).
  • Figure 2 is an illustration of an amino acid homology alignment between the amine transporter or the present invention (top line) and murine ⁇ -1 Adrenoreceptor (bottom line) .
  • Figure 3 is an illustration of an amino acid homology alignment between the amine transporter or the present invention (top line) and human dopamine D2 receptor (bottom line) .
  • the amine receptor of the present invention may be responsible for re-uptake of one or any of the amine neurotransmitters present in mammalian cells.
  • amine transporters include, but are not limited to, dopamine, norepinephrine, epinephrine, serotonin and histamine, and other amino acid transmitters, including GABA, glycine and glutamate.
  • nucleic acid which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. on June 1, 1995.
  • a polynucleotide encoding a polypeptide of the present invention may be found in human monocytes.
  • the polynucleotide of this invention was discovered in a human genomic library. It is structurally related to the G protein-coupled receptor family. It contains an open reading frame encoding a protein of 337 amino acid residues.
  • the protein exhibits the highest degree of homology to a murine ⁇ -1 Adrenoreceptor with 32.099 % identity and 55.864 % similarity over a 330 amino acid stretch.
  • the protein also exhibits homology to a human dopamine D 2 receptor with 32 % identity and 58.333 % similarity over a 312 amino acid stretch.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID NO:l) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure l (SEQ ID NO:l) or the deposited cDNA.
  • the polynucleotide which encodes for the mature polypeptide of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non- naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone.
  • Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID NO:l) or of the coding sequence of the deposited clone.
  • an allelic variant is an alternate form of . polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • the polynucleotides may also encode for a soluble form of the amine receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved from the TM and intracellular domain of the full-length polypeptide of the present invention.
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • the marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
  • Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar biological activity.
  • Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases.
  • the probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns.
  • An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polypeptides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO:l) or the deposited cDNA(s) , i.e. function as a soluble amine receptor by retaining the ability to bind the ligands for the receptor even though the polypeptide does not function as a membrane bound amine receptor, for example, by eliciting a second messenger response.
  • the polynucleotides may have at least 20 bases, preferably 30 bases and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which have an identity thereto, as hereinabove described, and which may or may not retain activity.
  • Such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO: 1, or for variants thereof, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
  • the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.
  • the present invention further relates to a human amine receptor polypeptide which has the deduced amino acid sequence of Figure 1 (SEQ ID No. 2) or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
  • fragment when referring to the polypeptide of Figure 1 (SEQ ID No. 2) or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide, i.e. functions as an amine receptor, or retains the ability to bind the ligand for the receptor even though the polypeptide does not function as a G-protein coupled receptor, for example, a soluble form of the receptor.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID No. 2) or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) or (iv) one in which the additional amino acids are fused to the mature polypeptide which are employed for purification of the mature polypeptide or (v) one in which a fragment of the polypeptide is soluble, i.e. not membrane bound, yet still binds ligands to the membrane bound receptor.
  • Such fragments, derivatives and analogs
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and to portions of such polypeptide with such portion of the polypeptide generally contains at least 30 amino acids and more preferably at least 50 amino acids.
  • similarity between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide.
  • Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region “leader and trailer” as well as intervening sequences (introns) between individual coding segments (exons) .
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
  • similarity between two polypeptides is determined by comparing the amino acid sequence ar ⁇ its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques.
  • the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmid ⁇ ; vectors derived from combinations of plasmid ⁇ and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be u ⁇ ed as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site( ⁇ ) by procedures known in the art. Such procedures and others are deemed to be within the ⁇ cope of tho ⁇ e ⁇ killed in the art.
  • the DNA ⁇ equence in the expression vector is operatively linked to an appropriate expres ⁇ ion control sequence(s) (promoter) to direct mRNA synthesis.
  • promoter an appropriate expres ⁇ ion control sequence(s) (promoter) to direct mRNA synthesis.
  • promoters there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or ⁇ uch as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • bacterial cell ⁇ ⁇ uch as E. coli, Streptomyces, Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, HEK, COS or Bowes melanoma
  • adenoviruses plant cells, etc.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the construct ⁇ compri ⁇ e a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the con ⁇ truct further comprises regulatory sequence ⁇ , including, for example, a promoter, operably linked to the ⁇ equence.
  • suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen), pBS, pDIO, phagescript, psiX174, pblue ⁇ cript SK, pb ⁇ k ⁇ , pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia) .
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) .
  • any other plasmid or vector may be used as long a ⁇ they are replicable and viable in the ho ⁇ t.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are PKK232-8 and PCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to host cells containing the above-described construct ⁇ .
  • the ho ⁇ t cell can be a higher eukaryotic cell, ⁇ uch a ⁇ a mammalian cell, or a lower eukaryotic cell, ⁇ uch a ⁇ a yea ⁇ t cell, or the ho ⁇ t cell can be a prokaryotic cell, ⁇ uch a ⁇ a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated tran ⁇ fection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)) .
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be ⁇ ynthetically produced by conventional peptide ⁇ ynthe ⁇ izers.
  • Mature proteins can be expre ⁇ ed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.
  • Cell-free translation sy ⁇ tem ⁇ can al ⁇ o be employed to produce ⁇ uch protein ⁇ u ⁇ ing RNA ⁇ derived from the DNA con ⁇ truct ⁇ of the pre ⁇ ent invention.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic host ⁇ are de ⁇ cribed by Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the di ⁇ closure of which is hereby incorporated by reference.
  • Enhancers are cis-acting element ⁇ of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • recombinant expres ⁇ ion vector ⁇ will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resi ⁇ tance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes ⁇ uch a ⁇ 3-pho ⁇ phoglycerate kina ⁇ e (PGK) , ⁇ -factor, acid phosphatase, or heat ⁇ hock protein ⁇ , among other ⁇ .
  • heterologous structural sequence is as ⁇ embled in appropriate pha ⁇ e with tran ⁇ lation initiation and termination ⁇ equences.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristic ⁇ , e.g., ⁇ tabilization or ⁇ implified purification of expre ⁇ sed recombinant product.
  • Useful expression vectors for bacterial use are constructed by in ⁇ erting a ⁇ tructural DNA ⁇ equence encoding a de ⁇ ired protein together with ⁇ uitable translation initiation and termination signal ⁇ in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillu ⁇ ⁇ ubtilis. Salmonella typhimurium and various specie ⁇ within the genera P ⁇ eudomona ⁇ , Streptomyces, and Staphylococcu ⁇ , although other ⁇ may al ⁇ o be employed a ⁇ a matter of choice.
  • useful expre ⁇ ion vector ⁇ for bacterial u ⁇ e can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) .
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemical ⁇ , Upp ⁇ ala, Sweden) and GEMl (Promega Biotec, Madi ⁇ on, WI, USA) .
  • the ⁇ e pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the ⁇ elected promoter i ⁇ induced by appropriate means e.g., temperature shift or chemical induction
  • cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expres ⁇ ion of protein ⁇ can be di ⁇ rupted by any convenient method, including freeze-thaw cycling, ⁇ onication, mechanical di ⁇ ruption, or u ⁇ e of cell ly ⁇ ing agents, such methods are well know to those skilled in the art.
  • mammalian cell culture systems can al ⁇ o be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expres ⁇ ing a compatible vector, for example, the C127, 3T3, CHO, HEK, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and al ⁇ o any nece ⁇ ary ribo ⁇ ome binding ⁇ ite ⁇ , polyadenylation ⁇ ite, splice donor and acceptor ⁇ ite ⁇ , tran ⁇ criptional termination ⁇ equence ⁇ , and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • the human amine receptor polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as neces ⁇ ary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification ⁇ teps.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
  • a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may also include an initial methionine amino acid residue.
  • Fragments of the full length human amine transporter gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other gene ⁇ which have a high ⁇ equence similarity to the gene or similar biological activity.
  • Probes of thi ⁇ type are at lea ⁇ t 20 ba ⁇ es, preferably at lea ⁇ t 30 bases and most preferably at least 50 base ⁇ or more.
  • the probe may al ⁇ o be u ⁇ ed to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete human amine transporter gene including regulatory and promotor regions, exons, and introns.
  • a screen comprises isolating the coding region of the human amint transporter gene by using the known DNA sequence to synthe ⁇ ize an oligonucleotide probe.
  • Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • This invention provides a method for determining amine neurotran ⁇ mitter ⁇ which are transported by the human amine receptor of the present invention.
  • An example of an assay which will identify these neurotransmitter ⁇ comprises infecting mammalian cell ⁇ with recombinant vaccinia virus strain VTF-7 encoding a T7 RNA polymerase and following ⁇ uch infection with lipo ⁇ ome-mediated tran ⁇ fection with the amine receptor gene of the pre ⁇ ent invention through the u ⁇ e of a vector, for example, pBSSKII(-). Controlled tran ⁇ fection ⁇ are also done with equivalent amounts of vector alone. As ⁇ ay ⁇ are performed eight hours following transfection in modified Kreb ⁇ -Rxnger-HEPES buffer.
  • This invention also provides a method of detecting expression of the amine receptor of the present invention on the surface of a cell by detecting the presence of mRNA coding for the amine receptor.
  • This method comprises obtaining total mRNA from the cell using methods well-known in the art and contacting the mRNA so obtained with a nucleic acid probe of at lea ⁇ t 10 nucleotides and which is capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human amine receptor of the present invention under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expres ⁇ ion of the amine receptor by the cell.
  • Hybridization of probe ⁇ to target nucleic acid molecules employs techniques well known in the art.
  • nucleic acids are extracted by precipitation from lysed cells and the mRNA is isolated from the extract using a column which binds the poly-A tails of the mRNA molecules.
  • the mRNA is then exposed to radioactively labelled probe on a nitrocellulose membrane, and the probe hybridizes to and thereby label ⁇ complementary mRNA ⁇ equence ⁇ . Binding may be detected by autoradiography or ⁇ cintillation counting.
  • other method ⁇ for performing the ⁇ e ⁇ tep ⁇ are well known to tho ⁇ e of ⁇ kill in the art.
  • an antibody directed to the human amine receptor may be employed under condition ⁇ permitting binding of the antibody to the tran ⁇ porter, and detecting the pre ⁇ ence of the receptor on the ⁇ urface of the cell.
  • Such a method may be employed for determining whether a given cell i ⁇ defective in expre ⁇ sion of the amine receptor. Detection methods include fluorescent markers bound to the antibodies.
  • the invention also provides a method for determining whether a compound not known to be capable of specifically binding to a human amine receptor can specifically bind to the human amine receptor, which comprise ⁇ contacting a mammalian cell compri ⁇ ing a pla ⁇ mid adapted for expre ⁇ ion in a mammalian cell which pla ⁇ mid further compri ⁇ e ⁇ a DNA which expresses the amine receptor on the cell surface with the compound under conditions permitting binding of ligands known to bind to the amine receptor, detecting the presence of any compound bound to the amine receptor, the presence of bound compound indicating that the compound is capable of specifically binding to the human amine receptor.
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human di ⁇ ea ⁇ e.
  • the amine receptor of the pre ⁇ ent invention may be employed in a proce ⁇ for ⁇ creening for compounds which activate (agonists) or inhibit activation (antagonists) of the receptor polypeptide of the present invention .
  • such screening procedures involve providing appropriate cells which expres ⁇ the receptor polypeptide of the present invention on the surface thereof.
  • Such cells include cells from mammals, yeast, drosophila or E. Coli .
  • a polynucleotide encoding the receptor of the pre ⁇ ent invention is employed to transfect cells to thereby express the amine receptor.
  • the expres ⁇ ed receptor i ⁇ then contacted with a te ⁇ t compound to ob ⁇ erve binding, ⁇ timulation or inhibition of a functional response.
  • such as ⁇ ay may be employed for ⁇ creening for a compound which inhibit ⁇ activation of the receptor polypeptide of the pre ⁇ ent invention by contacting the melanophore cells which encode the receptor with both the receptor ligand and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor.
  • the screen may be employed for determining a compound which activates the receptor by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the receptor.
  • amine receptor for example, transfected CHO cells
  • compound ⁇ may be contacted with a cell which expresse ⁇ the receptor polypeptide of the pre ⁇ ent invention and a second mes ⁇ enger response, e.g. signal transduction or pH changes, may be mea ⁇ ured to determine whether the potential compound activates or inhibits the receptor.
  • a second mes ⁇ enger response e.g. signal transduction or pH changes
  • Another such screening technique involves introducing RNA encoding the amine receptor into Xenopus oocytes to transiently expres ⁇ the receptor.
  • the receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for compounds which are thought to inhibit activation of the receptor.
  • Another screening technique involves expressing the amine receptor in which the receptor is linked to a phospholipa ⁇ e C or D.
  • a ⁇ representative examples of such cells there may be mentioned endothelial cells, smooth mu ⁇ cle cell ⁇ , embryonic kidney cell ⁇ , etc.
  • the screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal.
  • Another method involve ⁇ ⁇ creening for compound ⁇ which inhibit activation of the receptor polypeptide of the pre ⁇ ent invention antagoni ⁇ t ⁇ by determining inhibition of binding of labeled ligand to cell ⁇ which have the receptor on the surface thereof.
  • Such a method involves transfecting a eukaryotic cell with DNA encoding the amine receptor such that the cell expres ⁇ e ⁇ the receptor on it ⁇ ⁇ urface and contacting the cell with a compound in the pre ⁇ ence of a labeled form of a known ligand.
  • the ligand can be labeled, e.g., by radioactivity.
  • the amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the compound binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.
  • Amine receptors are ubiquitou ⁇ in the mammalian ho ⁇ t and are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate the amine receptor on the one hand and which can inhibit the function of a amine receptor on the other hand.
  • Examples of compounds which bind to and inhibit the amine receptor of the present invention includes antibodies, or in some cases an oligopeptides, which bind to the amine receptor but do not elicit a ⁇ econd messenger response such that the activity of the amine receptor is prevented.
  • Antibodies include anti-idiotypic antibodies which recognize unique determinants generally as ⁇ ociated with the antigen- binding ⁇ ite of an antibody.
  • Another example includes proteins which are closely related to the ligand of the amine receptors, i.e. a fragment of the ligand, which has lost biological function and when binding to the amine receptor, elicits no respon ⁇ e.
  • An anti ⁇ en ⁇ e construct prepared through the use of antisen ⁇ e technology may be u ⁇ ed to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of mRNA molecules into amine receptor (antisense - Okano, J. Neurochem. , 56:560 (1991) ; 01igodeoxynucleotides a ⁇ Anti ⁇ en ⁇ e Inhibitor ⁇ of Gene Expression, CRC Press, Boca Raton, FL (1988)) .
  • the oligonucleotides de ⁇ cribed above can al ⁇ o be delivered to cell ⁇ ⁇ uch that the anti ⁇ en ⁇ e RNA or DNA may be expressed in vivo to inhibit production of the amine receptor.
  • a small molecule which binds to the amine receptor, making it inaccessible to ligands such that normal biological activity is prevented for example small peptides or peptide- like molecule ⁇ , may al ⁇ o be u ⁇ ed to inhibit activation of the receptor polypeptide of the pre ⁇ ent invention.
  • a ⁇ oluble form of the amine receptor e.g. a fragment of the receptor, may be u ⁇ ed to inhibit activation of the receptor by binding to the ligand to the receptor polypeptide of the pre ⁇ ent invention and preventing the ligand from interacting with membrane bound amine receptor ⁇ .
  • This invention additionally provides a method of treating an abnormal condition related to expression of the amine receptor of the present invention which comprises administering to a subject an inhibitory compound as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to block bind to a human amine receptor can specifically bind to the human amine receptor, which comprises contacting a mammalian cell comprising a plasmid adapted for expression in a mammalian cell which plasmid further comprises a DNA which expresses the amine receptor on the cell surface with the compound under condition ⁇ permitting binding of ligands known to bind to the amine receptor, detecting the presence of any compound bound to the amine receptor, the presence of bound compound indicating that the compound is capable of specifically binding to the human amine receptor.
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostic ⁇ to human disease.
  • the amine receptor of the present invention may be employed in a process for screening for compounds which activate (agonists) or inhibit activation (antagonist ⁇ ) of the receptor polypeptide of the present invention .
  • Such screening procedures involve providing appropriate cell ⁇ which express the receptor polypeptide of the present invention on the surface thereof.
  • Such cells include cells from mammals, yeast, drosophila or E. Coli .
  • the invention al ⁇ o provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition ⁇ of the invention.
  • a ⁇ ociated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, u ⁇ e or ⁇ ale for human admini ⁇ tration.
  • the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.
  • the pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramu ⁇ cular, subcutaneous, intranasal or intradermal route ⁇ .
  • the pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 g/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
  • human amine receptor and agonist and antagonist compounds which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy. "
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • cells may be engineered by procedures known in the art by u ⁇ e of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cell ⁇ in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosi ⁇ virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the retroviral pla ⁇ mid vector i ⁇ derived from Moloney Murine Leukemia Virus.
  • the vector includes one or more promoters.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and jS-actin promoter ⁇ ) .
  • CMV human cytomegalovirus
  • viral promoters which may be employed include, but are not limited to, adenovirus promoter ⁇ , thymidine kina ⁇ e (TK) promoters, and B19 parvovirus promoter ⁇ .
  • TK thymidine kina ⁇ e
  • B19 parvovirus promoter ⁇ The ⁇ election of a ⁇ uitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, ⁇ uch a ⁇ the adenoviral major late promoter; or hetorologou ⁇ promoter ⁇ , ⁇ uch a ⁇ the cytomegalovirus (CMV) promoter; the respiratory ⁇ yncytial viru ⁇ (RSV) promoter; inducible promoter ⁇ , ⁇ uch a ⁇ the MMT promoter, the metallothionein promoter; heat ⁇ hock promoter ⁇ ; the albumin promoter,- the ApoAI promoter; human globin promoter ⁇ ; viral thymidine kina ⁇ e promoter ⁇ , ⁇ uch a ⁇ the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described) ; the 0-actin promoter; and human
  • the promoter al ⁇ o may be the native promoter which control ⁇ the gene ⁇ encoding the polypeptide ⁇ .
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ⁇ -2 , ⁇ -AM, PA12, T19-14X, VT-19-17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety.
  • the vector may transduce the packaging cells through any means known in the art.
  • retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
  • the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides.
  • retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vi tro or in vivo.
  • the transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • This invention is also related to the use of the human amine receptor gene as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the human amine receptor genes. Such diseases are related to under-expression of the human amine receptor.
  • Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al . , Nature, 324:163-166 (1986)) prior to analysis.
  • RNA or cDNA may also be used for the same purpose.
  • PCR primers complementary to the nucleic acid encoding the human amine receptor protein can be used to identify and analyze human amine receptor mutations.
  • deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled human amine receptor RNA or alternatively, radiolabeled human amine receptor antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al . , Science, 230:1242 (1985) ) .
  • Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)) .
  • nuclease protection assays such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)) .
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP) ) and Southern blotting of genomic DNA.
  • restriction enzymes e.g., Restriction Fragment Length Polymorphisms (RFLP)
  • RFLP Restriction Fragment Length Polymorphisms
  • mutations can also be detected by in si tu analysis.
  • the present invention also relates to a diagnostic assay for detecting altered levels of soluble forms of the amine receptor polypeptides of the present invention in various tissues which may be employed to diagnose diseases related to under-expression of the amine receptor.
  • Assays used to detect levels of the soluble receptor polypeptides in a sample derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western blot analysis and preferably as ELISA assay.
  • An ELISA assay initially comprises preparing an antibody specific to antigens of the amine receptor polypeptides, preferably a monoclonal antibody.
  • a reporter antibody is prepared against the monoclonal antibody.
  • a detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme.
  • a sample is now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non ⁇ specific protein such as bovine serum albumin.
  • the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any amine receptor proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer.
  • the reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to amine receptor proteins. Unattached reporter antibody is then washed out.
  • Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of amine receptor proteins present in a given volume of patient sample when compared against a standard curve.
  • the sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in si tu hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in si tu hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • FISH Fluorescence in si tu hybridization
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb) .
  • polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments .
  • Antibodies generated against the polypeptides corresponding to a sequence of the oresent invention can be obtained by direct injection of whoge polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this ' manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) , and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) .
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • For analytical purposes typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • For the purpose of isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
  • Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980) .
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al. , Id., p. 146) . Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • Example 1 Bacterial Expression and Purification of Human Amine Receptor
  • the DNA sequence encoding human amine receptor, ATCC # is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the processed amine receptor nucleic acid sequence (minus the signal peptide sequence) . Additional nucleotides corresponding to amine receptor gene are added to the 5' and 3' sequences respectively.
  • the 5' oligonucleotide primer has the sequence 5' CGGAATTCCTUATGAGAGCTGTCTTCATC 3' (SEQ ID No. 3) contains an EcoRI restriction enzyme site followed by 18 nucleotides of human amine receptor coding sequence starting from the presumed terminal amino acid of the processed protein.
  • the 3' sequence 5' CGGAAGCTTCGTCATTCTTGGTACAAATCAAC 3' contains complementary sequences to an Hindlll site and is followed by 18 nucleotides of the human amine receptor gene.
  • the restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, CA) .
  • pQE-9 encodes antibiotic resistance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a ribosome binding site (RBS) , a 6-His tag and restriction enzyme sites.
  • pQE-9 is then digested with Hindlll and EcoRI.
  • the amplified sequences are ligated into pQE-9 and are inserted in frame with the sequence encoding for the histidine tag and the RBS .
  • the ligation mixture is then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al . , Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989) .
  • M15/rep4 contains ' multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kana ycin resistance (Kan r ) .
  • Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected.
  • Plasmid DNA is isolated and confirmed by restriction analysis .
  • Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) .
  • the O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250.
  • the cells are grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG "Isopropyl-B-D- thiogalacto pyranoside" is then added to a final concentration of 1 mM. IPTG induces by inactivating the lad repressor, clearing the P/O leading to increased gene expression.
  • Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCI. After clarification, solubilized human amine receptor is purified from this solution by chromatography on a Nickel- Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)) .
  • Human amine receptor protein is eluted from the column in 6 molar guanidine HCI pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCI, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.
  • Example 2 Cloning and expression of human amine receptor using the baculovirus expression system
  • the DNA sequence encoding the full length human amine receptor protein, ATCC # is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
  • the 5' primer has the sequence 5' 5' CGGGATCCCTCCATGAGA GCTGTCTTCATC 3' (SEQ ID No. 5) and contains a BamHI restriction enzyme site followed by 4 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (Kozak, M. , J. Mol . Biol., 196:947-950 (1987) which is just behind the first 18 nucleotides of the human amine receptor gene.
  • the 3' primer has the sequence 5' CGGGATCCCGCTCATTCTTGG TACAAATC 3' (SEQ ID No. 6) and contains the cleavage site for the restriction endonuclease BamHI and 18 nucleotides complementary to the 3' non-translated sequence of the human amine receptor gene.
  • the amplified sequences are isolated from a 1% agarose gel using a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.) .
  • the fragment is then digested with the endonucleases BamHI and then purified again on a 1% agarose gel. This fragment is designated F2.
  • the vector pRGl (modification of pVL941 vector, discussed below) is used for the expression of the human amine receptor protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) .
  • This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction endonucleases BamHI .
  • the polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation.
  • the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene.
  • the polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-transfected wild-type viral DNA.
  • Many other baculovirus vectors could be used in place of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31-39) .
  • the plasmid is digested with the restriction enzymes BamHI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art .
  • the DNA is then isolated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) . This vector DNA is designated V2.
  • Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase.
  • E.coli HB101 cells are then transformed and bacteria identified that contained the plasmid (pBac-Human amine receptor) with the human amine receptor gene using the enzyme BamHI .
  • the sequence of the cloned fragment is confirmed by DNA sequencing.
  • 5 ⁇ g of the plasmid pBac-Human amine receptor is co- transfected with 1.0 ⁇ g of a commercially available linearized baculovirus ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987) ) .
  • the plate is then incubated for 5 hours at 27°C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation continued at 27°C for four days.
  • plaque assay After four days the supernatant is collected and a plaque assay performed similar as described by Summers and Smith (supra) . As a modification an agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue stained plaques. (A detailed description of a "plaque assay” can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9- 10) .
  • the viruses are added to the cells and blue stained plaques are picked with the tip of an Eppendorf pipette.
  • the agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 ⁇ l of Grace's medium.
  • the agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
  • the supernatants of these culture dishes are harvested and then stored at 4°C.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS.
  • the cells are infected with the recombinant baculovirus V-Human amine receptor at a multiplicity of infection (MOD of 2.
  • MOD multiplicity of infection
  • the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg) .
  • the cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS- PAGE and autoradiography.
  • Example 3 Expression of Recombinant Human Amine Receptor in COS cells
  • plasmid, Human amine receptor HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire Human amine receptor precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter.
  • the HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, et al., Cell, 37:767, (1984)) .
  • the infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the plasmid construction strategy is described as follows :
  • the DNA sequence encoding Human amine receptor, ATCC # is constructed by PCR using two primers: the 5' primer 5' GTCCAAGCTTGCCACCATGAGAGCTGTCTTCATC 3' (SEQ ID No. 7) contains a Hindlll site followed by 18 nucleotides of Human amine receptor coding sequence starting from the initiation codon; the 3' sequence 5' CTAGCTCGAGTCAAGCGTA GTCTGGGACGTCGTATGGGTAGCATTCTTGGTACAAATCAAC 3' (SEQ ID No. 8) contains complementary sequences to an Xhol site, translation stop codon, HA tag and the last 18 nucleotides of the Human amine receptor coding sequence (not including the stop codon) .
  • the PCR product contains a Hindlll site, human amine receptor coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Hindlll site.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp are digested with Hindlll and Xhol restriction enzymes and ligated.
  • the ligation mixture is transformed into E. coli strain SURE (Stratagene Cloning Systems, La Jolla, CA) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
  • COS cells are transfected with the expression vector by DEAE- DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989) ) .
  • the expression of the Human amine receptor HA protein is detected by radiolabelling and im unoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)) .
  • Cells are labelled for 8 hours with 35 S-cysteine two days post transfection.
  • Culture media is then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I. et al. , Id. 37:767 (1984)) . Both cell lysate and culture media are precipitated with a HA specific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE gels.
  • RIPA buffer 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5
  • RNAzolTM B system Biotecx Laboratories, Inc. Houston, TX
  • About lO ⁇ g of total RNA isolated from each human tissue specified is separated on 1% agarose gel and blotted onto a nylon filter (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press, (1989)) .
  • the labeling reaction is done according to the Stratagene Prime-It kit with 50ng DNA fragment.
  • the labeled DNA is purified with a Select-G-50 column (5 Prime - 3 Prime, Inc. Boulder, CO) .
  • the filter is then hybridized with radioactive labeled full length Human amine receptor gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 , pH 7.4 and 7% SDS overnight at 65 °C. After wash twice at room temperature and twice at 60 'C with 0.5 x SSC, 0.1% SDS, the filter is then exposed at -70 °C overnight with an intensifying screen.
  • Fibroblasts are obtained from a subject by skin biopsy.
  • the resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask.
  • the flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added. This is then incubated at 37°C for approximately one week. At this time, fresh media is added and subsequently changed every several days.
  • fresh media e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin
  • pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and Hindlll and subsequently treated with calf intestinal phosphatase.
  • the linear vector is fractionated on agarose gel and purified, using glass beads.
  • the cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequences respectively.
  • the 5' primer contains an EcoRI site and the 3' primer contains a Hindlll site.
  • Equal quantities of the Moloney murine sarcoma virus linear backbone and the EcoRI and Hindlll fragment are added together, in the presence of T4 DNA ligase.
  • the resulting mixture is maintained under conditions appropriate for ligation of the two fragments .
  • the ligation mixture is used to transform bacteria HB101, which are then plated onto agar- containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
  • the amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS) , penicillin and streptomycin.
  • DMEM Dulbecco's Modified Eagles Medium
  • CS calf serum
  • penicillin and streptomycin The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector.
  • the packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells) .
  • Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells.
  • the spent media containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells.
  • Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.
  • the engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
  • the fibroblasts now produce the protein product.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,

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Abstract

La présente invention concerne un polypeptide récepteur d'amines humaines et l'ADN (ARN) codant ce polypeptide, ainsi qu'un procédé de production dudit polypeptide par techniques de recombinaison. Elle concerne également des procédés de détection de composés liant et activant ou liant et inhibant ce polypeptide et l'utilisation de ces composés dans le traitement de maladies liées à la sous-expression ou à la surexpression du récepteur d'amines humaines décrit. Linvention a en outre pour objet des procédés permettant de détecter des mutations dans la séquence d'acide nucléique codant le polypeptide, ou encore des modifications de la concentration de la forme soluble du polypeptide.
PCT/US1995/007221 1995-06-06 1995-06-06 Recepteur d'amines humaines WO1996039440A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
PCT/US1995/007221 WO1996039440A1 (fr) 1995-06-06 1995-06-06 Recepteur d'amines humaines
CA002224094A CA2224094A1 (fr) 1995-06-06 1995-06-06 Recepteur d'amines humaines
EP95922243A EP0832124A4 (fr) 1995-06-06 1995-06-06 Recepteur d'amines humaines
JP9500375A JPH11507813A (ja) 1995-06-06 1995-06-06 ヒトアミン受容体
AU26996/95A AU2699695A (en) 1995-06-06 1995-06-06 Human amine receptor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/US1995/007221 WO1996039440A1 (fr) 1995-06-06 1995-06-06 Recepteur d'amines humaines
CA002224094A CA2224094A1 (fr) 1995-06-06 1995-06-06 Recepteur d'amines humaines

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0859055A1 (fr) * 1997-01-24 1998-08-19 Smithkline Beecham Corporation Clonage d'un nouveau récepteur à 7 passages transmembranaires couplé à la protéine-G
WO2001036473A2 (fr) * 1999-11-16 2001-05-25 Pharmacia & Upjohn Company Recepteurs couples a une proteine g
WO2004008153A2 (fr) * 2002-07-16 2004-01-22 Bayer Healthcare Ag Diagnostique et therapie des maladies associees au recepteur presume de neurotransmetteurs chez l'humain (pnr)

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
FEBS LETTERS, Volume 271, Numbers 1-2, issued October 1990, C. EVA et al., "Molecular Cloning of a Novel G Protein-Coupled Receptor that May Belong to the Neuropeptide Receptor Family", pages 81-84. *
FEBS LETTERS, Volume 284, Number 2, issued June 1991, W. MEYERHOF et al., "Molecular Cloning of a Novel Putative G-Protein Coupled Receptor Expressed During Rat Spermiogenesis", pages 155-160. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 87, issued April 1990, P.C. ROSS et al., "RTA, a Candidate G Protein-Coupled Receptor: Cloning, Sequencing and Tissue Distribution", pages 3052-3056. *
SCIENCE, Volume 244, issued 05 May 1989, F. LIBERT et al., "Selective Amplification and Cloning of Four New Members of the G Protein-Coupled Receptor Family", pages 569-572. *
See also references of EP0832124A4 *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 265, Number 16, issued 05 June 1990, T. HLA et al., "An Abundant Transcript Induced in Differentiating Human Endothelial Cells Encodes a Polypeptide with Structural Similarities to G-Protein-Coupled Receptors", pages 9308-9313. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0859055A1 (fr) * 1997-01-24 1998-08-19 Smithkline Beecham Corporation Clonage d'un nouveau récepteur à 7 passages transmembranaires couplé à la protéine-G
US5871967A (en) * 1997-01-24 1999-02-16 Smithkline Beecham Corporation Cloning of a novel G-Protein coupled 7TM receptor
WO2001036473A2 (fr) * 1999-11-16 2001-05-25 Pharmacia & Upjohn Company Recepteurs couples a une proteine g
WO2001036473A3 (fr) * 1999-11-16 2002-07-11 Upjohn Co Recepteurs couples a une proteine g
AU784543B2 (en) * 1999-11-16 2006-04-27 Pharmacia & Upjohn Company Novel G protein-coupled receptors
WO2004008153A2 (fr) * 2002-07-16 2004-01-22 Bayer Healthcare Ag Diagnostique et therapie des maladies associees au recepteur presume de neurotransmetteurs chez l'humain (pnr)
WO2004008153A3 (fr) * 2002-07-16 2004-03-04 Bayer Healthcare Ag Diagnostique et therapie des maladies associees au recepteur presume de neurotransmetteurs chez l'humain (pnr)

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CA2224094A1 (fr) 1996-12-12
EP0832124A4 (fr) 1999-12-22
EP0832124A1 (fr) 1998-04-01
AU2699695A (en) 1996-12-24

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