EP0578784A1 - Recepteurs de substances odorantes et leurs utilisations - Google Patents

Recepteurs de substances odorantes et leurs utilisations

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Publication number
EP0578784A1
EP0578784A1 EP92917380A EP92917380A EP0578784A1 EP 0578784 A1 EP0578784 A1 EP 0578784A1 EP 92917380 A EP92917380 A EP 92917380A EP 92917380 A EP92917380 A EP 92917380A EP 0578784 A1 EP0578784 A1 EP 0578784A1
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EP
European Patent Office
Prior art keywords
odorant
leu
sequence
ser
val
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
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EP92917380A
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German (de)
English (en)
Other versions
EP0578784A4 (fr
Inventor
Linda B. Buck
Richard Axel
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Columbia University in the City of New York
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Columbia University in the City of New York
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Publication of EP0578784A1 publication Critical patent/EP0578784A1/fr
Publication of EP0578784A4 publication Critical patent/EP0578784A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L9/00—Disinfection, sterilisation or deodorisation of air
    • A61L9/01—Deodorant compositions
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • peripheral neurons respond to environmental stimuli and transmit these signals to higher sensory centers in the brain where they are processed to allow the discrimination of complex sensory information.
  • the delineation of the peripheral mechanisms by which environmental stimuli are transduced into neural information can provide insight into the logic underlying sensory processing.
  • the basic logic underlying olfactory sensory perception has remained elusive. Mammals possess an olfactory system of enormous discriminatory power (5, 6). Humans, for example, are thought to be capable of distinguishing among thousands of distinct odors.
  • the specificity of odor recognition is emphasized by the observation that subtle alterations in the molecular structure of an odorant can lead to profound changes in perceived odor.
  • the primary events in odor detection occur in a specialized olfactory neuroepithelium located in the posterior recesses of the nasal cavity.
  • the olfactory sensory neuron is bipolar: a dendritic process extends to the mucosal surface where it gives rise to a number of specialized cilia which provide an extensive, receptive surface for the interaction of odors with olfactory sensory neurons.
  • the olfactory neuron also gives rise to an axon which projects to the olfactory bulb of the brain, the first relay in the olfactory system.
  • the axons of the olfactory bulb neurons project to subcortical and cortical regions where higher level processing of olfactory information allows the discrimination of odors by the brain.
  • the initial events in odor discrimination are thought to involve the association of odors with specific receptors on the cilia of olfactory neurons. Selective removal of the cilia results in the loss of olfactory response (9).
  • Elevations in cyclic AMP are thought to elicit depolarization of olfactory neurons by direct activation of a cyclic nucleotide-gated, cation permeable channel (17, 18). This channel is opened upon binding of cyclic nucleotides to its cytoplasmic domain, and can therefore transduce changes in intracellular levels of cyclic AMP into alterations in the membrane potential.
  • This invention address the problem of olfactory perception at a molecular level. Eighteen different members of an extremely large multigene family have been cloned and characterized which encodes seven transmembrane domain proteins whose expression is restricted to the olfactory epithelium. The members of this novel gene family encode the individual odorant receptors.
  • the invention provides an isolated nucleic acid, e.g. a DNA and cDNA molecule, encoding an odorant receptor.
  • the invention further provides expression vectors containing such nucleic acid.
  • a purified protein encoding an odorant receptor.
  • the invention further provides a method of transforming cells which comprises transfecting a suitable host cell with a suitable expression vector containing the nucleic acid encoding the odorant receptor.
  • the invention also provides methods of identifying odorant ligands and of identifying odorant receptors.
  • the invention further provides methods of developing fragrances, of identifying appetite suppressant compounds, of controlling appetite.
  • the invention also provides methods of controlling insect and other animal populations.
  • the invention additionally provides a method of detecting odors such as the vapors emanating from Cocaine, Marijuana, Heroin, Hashish, Angel Dust, gasoline, decayed human flesh, alcohol, gun powder explosives, plastic explosives, firearms, poisonous or harmful smoke, or natural gas.
  • FIG. 1 The Olfactory Neuroepithelium and a Pathway for Olfactory Signal Transduction.
  • A. The Olfactory Neuroepithelium. The initial event in odor perception occurs in the nasal cavity in a specialized neuroepithelium which is diagrammed here. Odors are believed to interact with specific receptors on the cilia of olfactory sensory neurons. The signal generated by these initial binding events are propagated by olfactory neuron axons to the olfactory bulb.
  • B A Pathway of Olfactory Signal Transduction. In this scheme, the binding of an odorant molecule to an odor-specific transmembrane receptor leads to the interaction of the receptor with a GTP-binding protein (G s[olf] ).
  • GTP-binding protein G s[olf]
  • FIG. 1 A PCR Amplification Product Containing Multiple Species of DNA.
  • cDNA prepared from olfactory epithelium RNA was subjected to PCR amplification with a series of different primer oligonucleotides and the DNA products of appropriate size were isolated, further amplified by PCR, and size fractionated on agarose gels (A) (For details, see text).
  • Each of these semipurified PCR products was digested with the restriction enzyme, Hinf I, and analyzed by agarose gel electrophoresis.
  • Lanes marked "M” contain size markers of 23.1, 9.4, 5.6, 4.4, 2.3, 2.0, 1.35, 1.08, 0.87, 0.60, 0.31, 0.28, 0.23, 0.19, 0.12 and 0.07kb.
  • PCR 13 Twenty-two of the 64 PCR products that were isolated and digested with Hinf I are shown here. Digestion of one of these, PCR 13, yielded a large number of fragments whose sizes summed to a value much greater than that of the undigested PCR 13 DNA, indicating that PCR 13 might contain multiple species of DNA which are representatives of a multigene family.
  • FIG. 3 Northern Blot Analysis with a Mixture of Twenty Probes.
  • One ⁇ g of polyA+ RNA isolated from rat olfactory epithelium, brain, or spleen was size-fractionated in formaldehyde agarose, blotted onto a nylon membrane, and hybridized with a 32 P-labeled mixture of segments of 20 cDNA clones.
  • the DNA segments were obtained by PCR using primers homologous to transmembrane domains 2 and 7.
  • FIG. 4 The Protein Sequences Encoded by Ten Divergent cDNA Clones. Ten divergent cDNA clones were subjected to DNA sequence analyses and the protein sequence encoded by each was determined. Amino acid residues which are conserved in 60% or more of the proteins are shaded. The presence of seven hydrophobic domains (I-VII), as well as short conserved motifs shared with other members of the superfamily, demonstrate that these proteins belong to the seven transmembrane domain protein superfamily. Motifs conserved among members of the superfamily and the family of olfactory proteins include the GN in TM1 (transmembrane domain 1), the central W of TM4 , the Y near the C-terminal end of TM5, and the NP in TM7.
  • DRY motif C-terminal to TM3 is common to many members of the G-protein-coupled superfamily. However, all of the proteins shown here share sequence motifs not found in other members of this superfamily and are clearly members of a novel family of proteins.
  • FIG. 1 Positions of Greatest Variability in the Olfactory Protein Family.
  • the protein encoded by cDNA clone 115 is shown traversing the plasma membrane seven times with its N-terminus located extracellularly, and its C-terminus intracellularly.
  • the vertical cylinders delineate the seven putative ⁇ -helices spanning the membrane.
  • Positions at which 60% or more of the 10 clones shown in Figure 4 share the same residue as 115 are shown as white balls. More variable residues are shown as black balls.
  • the high degree of variability encountered in transmembrane domains III, IV, and V is evident in this schematic.
  • FIG. 6 The Presence of Subfamilies in a Divergent Multigene Family. Partial nucleotide sequences and deduced protein sequences were obtained for 18 different cDNA clones. Transmembrane domain V along with the flanking loop sequences, including the entire cytoplasmic loop between transmembrane domains V and VI, are shown here for each protein. Amino acid residues found in 60% or more of the clones in a given position are shaded (A). This region of the olfactory proteins (particularly transmembrane domain V) appears to be highly variable (see Figure 4). These proteins, however, can be grouped into subfamilies (B,C,D) in which the individual subfamily members share considerable homology in this divergent region of the protein.
  • FIG. 7 Southern Blot Analyses with Non-crosshvbridizing Fragments of Divergent cDNAs.
  • Five ⁇ g of rat liver DNA was digested with Eco RI (A) or Hind III (B), electrophoresed in 0.75% agarose, blotted onto a nylon membrane, and hybridized to the 32 P-labeled probes indicated.
  • the probes used were PCR-generated fragments of: 1, clone F9 (identical to F12 in Figure 4); 2, F5; 3, F6; 4, I3; 5, I7; 6, I14; or 7, I15.
  • the lane labeled "1-7" was hybridized to a mixture of the seven probes.
  • the probes used showed either no crosshybridization or only trace crosshybridization with one another.
  • the size markers on the left correspond to the four blots on the left (1-4) whereas the marker positions noted on the right correspond to the four blots on the right ( 5-7 , " 1-7" ) .
  • Figure 8 Northern Blot Analysis with a Mix of Seven Divergent Clones.
  • One ⁇ g of polyA+ RNA from each of the tissues shown was size-fractionated, blotted onto a nylon membrane, and hybridized with a 32 P-labeled mixture of segments of seven divergent cDNA clones (see Legend to Figure 7).
  • Figure 9 The amino acid and nucleic acid sequence of clone
  • Figure 10 The amino acid and nucleic acid sequence of clone F5.
  • Figure 11 The amino acid and nucleic acid sequence of clone F6.
  • Figure 13 The amino acid and nucleic acid sequence of clone 13.
  • Figure 14 The amino acid and nucleic acid sequence of clone 17.
  • Figure 15 The amino acid and nucleic acid sequence of clone 18.
  • Figure 17. The amino acid and nucleic acid sequence of clone 114.
  • Figure 18. The amino acid and nucleic acid sequence of clone 115.
  • Figure 19 The amino acid and nucleic acid sequence of human clone H5.
  • Figure 20 The amino acid and nucleic acid sequence of clone J1, where the reading frame starts at nucleotide position 2.
  • Figure 21 The amino acid and nucleic acid sequence of clone J2.
  • Figure 22 The amino acid and nucleic acid sequence of clone J4, where the reading frame starts at nucleotide position 2.
  • Figure 23 The amino acid and nucleic acid sequence of clone J7, where the reading frame starts at nucleotide position 2.
  • Figure 24 The amino acid and nucleic acid sequence of clone J8, where the reading frame starts at nucleotide positon 2.
  • Figure 25 The amino acid and nucleic acid sequence of clone J11.
  • Figure 26 The amino acid and nucleic acid sequence of clone J14, where the reading frame starts at nucleotide position 2.
  • Figure 27 The amino acid and nucleic acid sequence of clone J15, where the reading frame starts at nucleotide psition 2.
  • Figure 28 The amino acid and nucleic acid sequence of clone J16, where the reading frame starts at nucleotide position 2.
  • Figure 29 The amino acid and nucleic acid sequence of clone J17, where the reading frame starts at nucleotide position 2.
  • Figure 30 The amino acid and nucleic acid sequence of clone J19, where the reading frame starts at nucleotide position 2.
  • Figure 31 The amino acid and nucleic acid sequence of clone J20, where the reading frame starts at nucleotide position 2.
  • FIG. 32 SOUTHERN BLOT: Five micrograms of DNA isolated from 1. Human placenta, 2. NCI-H-1011 neuroblastoma cells, or 3. CHP 134 neuroblastoma cells were treated with the restriction enzyme A. Eco RI, B. Hind III, C. Bam HI, or D. Pst I, and then electrophoresed on an agarose gel and blotted onto a nylon membrane. The blotted DNA was hybridized to the 32P-labeled H3/H5 sequence. An autoradiograph of the hybridized blot is shown with the sizes of co-electrophoresed size markers noted in kilobases.
  • the invention provides an isolated nucleic acid, e.g. a DNA or cDNA molecule, encoding an odorant receptor.
  • a receptor is a receptor which binds an odorant ligand and include but not limited to pheromone receptors.
  • An odorant ligand may include, but is not limited to, molecules which interact with the olfactory sensory neuron, molecules which interact with the olfactory cilia, pheromones, and molecules which interact with structures within the vomeronasal organ.
  • the invention specifically provides the isolated cDNAs encoding odorant receptors the sequences of which are shown in Figures 9-31.
  • the nucleic acid is most typically a cDNA and encodes an insect, a vertebrate, a fish or a mammalian odorant receptor.
  • the mammalian odorant receptor is preferably a human, rat, mouse or dog receptor.
  • human odorant receptor cDNA sequence and the correspondent protein is isolated ( Figure 19).
  • phermone receptors are isolated and shown as clones J1, J2, J4, J7, J8, J11, J14, J15, J16, J17, J19 and J20 ( Figures 20-31).
  • the invention further provides expression vectors containing cDNA which encodes odorant receptors.
  • Such expression vectors are well known in the art and include in addition to the nucleic acid the elements necessary for replication and expression in a suitable hosts. Suitable hosts are well known in the art and include without limitation bacterial hosts such as E. coli, animal hosts such as CHO cells, insect cells, yeast cells and like.
  • the invention also provides purified proteins encoding odorant receptors.
  • proteins may be prepared by expression of the forementioned expression vectors in suitable host cells and recovery and purification of the receptors using methods well known in the art. Examples of such proteins include those having the amino acid sequences shown in figures 9-31.
  • the purified protein typically encodes an insect, vertebrate, fish or mammalian odorant receptor.
  • the mammalian odorant receptor may be a human, rat, mouse or dog.
  • the invention provides a novel purified protein which belong to a class of proteins which have 7 transmembrane regions and a third cytoplasmic loop from the N-terminus which is approximately 17 amino acid long and to nucleic acid molecules encoding such proteins.
  • the invention provides methods of transforming cells which comprises transfecting a suitable host cell with a suitable expression vector containing nucleic acid encoding of the odorant receptor. Techniques for carrying out such transformations on cells are well known to those skilled in the art. (41,42) Additionally, the resulting transformed cells are also provided by the invention. These transformed cells may be either olfactory cells or non-olfactory cells. One advantage of using transformed non-olfactory cells is that the desired odorant receptor will be the only odorant receptor expressed on the cell's surface. In order to obtain cell lines that express a single receptor type, standard procedures may be used to clone individual cDNAs or genes into expression vectors and then transfect the cloned sequences into mammalian cell lines.
  • the invention also provides a method of identifying a desired odorant ligand which comprises contacting transformed non-olfactory cells expressing a known odorant receptor with a series of odorant ligands to determining which ligands bind to the receptors present on the non- olfactory cells.
  • the invention provides a method of identifying a desired odorant receptor comprising contacting a series of transformed non-olfactory cells with a known odorant ligand and determining which odorant receptor binds with the odorant ligand.
  • the invention provides a method of detecting an odor which comprises: a) identifying a odorant receptor which binds the desired odorant ligand and; b) imbedding the receptor in a membrane such that when the odorant ligand binds to the receptor so identified a detectable signal is produced.
  • the membrane used in this method is cellular, including a membrane of an olfactory cell or a synthetic membrane.
  • the ligand tested for may be the vapors emanating from Cocaine, Marijuana, Heroin, Hashish, Angel Dust, gasoline, decayed human flesh, alcohol, gun powder explosives, plastic explosives or firearms.
  • the ligand tested for may be natural gas, a pheromone, toxic fumes, noxious fumes or dangerous fumes.
  • the detectable signal is a lightbulb lighting up, a buzzer buzzing, a bell ringing, a color change, phosphorescence, or radioactivity.
  • the invention further provides a method of quantifying the amount of an odorant ligand present in a sample which comprises utilizing the above-mentioned method for odor detection and then quantifying the amount of signal produced.
  • the invention further provides a method of developing fragrances which comprises identifying a desired odorant receptor by the above method, then contacting non-olfactory cells, which have been transfected with an expression vector containing nucleic acid encoding the desired odorant receptor such that the receptor is expressed upon the surface of the non-olfactory cell, with a series of compounds to determine which compound or compounds bind the receptor.
  • the invention provides to a method of identifying an "odorant fingerprint" which comprises contacting a series of cells, which have been transformed such that each express a known odorant receptor, with a desired sample and determining the type and quantity of the odorant ligands present in the sample.
  • the invention provides a method of identifying odorant ligands which inhibit the activity of a desired odorant receptor which comprises contacting the desired odorant receptor with a series of compounds and determining which compounds inhibit the odorant ligand - odorant receptor interaction.
  • the invention also provides for a method of identifying appetite suppressant compounds which comprises identifying odorant ligands by the method mentioned in the preceding paragraph wherein the desired odorant receptor is that which is associated with the perception of food.
  • the invention provides a method of controlling appetite in a subject which comprises contacting the olfactory epithelium of the subject with these odorant ligands. Further the invention provides a nasal spray, to control appetite comprising the compounds identified by the above method in a suitable carrier.
  • the invention provides a method of trapping odors which comprises contacting a membrane which contains multiples of the desired odorant receptor, with a sample such that the desired odorant ligand is absorbed by the binding of the odorant ligand to the odorant receptor.
  • the invention also provides an odor trap employing this method.
  • the invention also provides a method of controlling pest populations which comprises identifying odorant ligands by the method mentioned above which are alarm odorant ligands and spraying the desired area with the identified odorant ligands.
  • a method of controlling a pest population which comprises identifying odorant ligands by the above mentioned method, which interfere with the interaction between the odorant ligands and the odorant receptors which are associated with fertility.
  • the pest population is a population of insects or rodents, including mice and rats.
  • the invention also provides a method of promoting fertility which comprises identifying odorant ligands which interact with the odorant receptors associated with fertility by the above mentioned method. Further, the invention provides a method of inhibiting fertility which comprises employing the above mentioned method to identifying odorant ligands which inhibit the interaction between the odorant ligands and the odorant receptors associated with fertility.
  • RNA was prepared from the olfactory epithelia of Sprague Dawley rats according to Chirgwin et al. (40) or using RNAzol B (Cinna/Biotecx) and then treated with DNase I (0.1 unit/ ⁇ g RNA) (Promega).
  • this RNA was incubated at 0.1 ⁇ g/ ⁇ l with 5 ⁇ M random hexamers (Pharmacia) 1 mM each of dATP, dCTP, dGTP, TTP, and 2 units/ ⁇ l RNase inhibitor (Promega) in 10 mM TrisCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2 , and 0.001% gelatin for 10 min. at 22°C, and then for a further 45 min. at 37°C following the addition of 20 u./ ⁇ l of Moloney murine leukemia virus reverse transcriptase (BRL).
  • Pharmacia random hexamers
  • dATP 1 mM each of dATP, dCTP, dGTP, TTP, and 2 units/ ⁇ l RNase inhibitor (Promega) in 10 mM TrisCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2 , and 0.001%
  • cDNA prepared from 0.2 ⁇ g of RNA was used in each of a series of polymerase chain reactions (PCR) containing 10 mM TrisCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl 2 , 0.001% gelatin, 200 ⁇ M each of dATP, dCTP, dGTP, and TTP, 2.5 u. Taq polymerase (Perkin Elmer Cetus), and 2 ⁇ M of each PCR primer.
  • PCR reactions were performed according to the following schedule: 96°C for 45 sec., 55°C for 4 min. (or 45°C for 2 min.), 72°C for 3 min. with 6 sec. extension per cycle for 48 cycles.
  • the primers used for PCR were a series of degenerate oligonucleotides made according to the amino acid sequences found in transmembrane domain 2 and 7 of a variety of different members of the 7 transmembrane domain protein superfamily (19). The regions used correspond to amino acids number 60-70 and 286-295 of clone 115 ( Figure 4). Each of five different 5' primers were used in PCR reactions with each of six different 3' primers. The 5' primers had the sequences:
  • the 3' primers were:
  • CDNA libraries were prepared according to standard procedures (41, 42) in the cloning vector, ⁇ ZAP II (Stratagene) using poly A + RNA prepared from Sprague Dawley rat epithelia (see above) or from an enriched population of olfactory neurons which had been obtained by a 'panning' procedure, using an antibody against the H blood group antigen (Chembiomed) found on a large percentage of rat olfactory neurons.
  • phage clones were analyzed by PCR using primers A4 and B6 and those which showed the appropriate size species were purified. In later screens, all position clones were purified, but only those that could be amplified with the B6 primer and a primer specific for vector sequence were analyzed further.
  • phagemid rescue was performed according to the instructions of the manufacturer of ⁇ ZAP II (Stratagene). DNA sequence analysis was performed on plasmid DNAs using the Sequenase system (USB), initially with the A4 and B6 primers and later with oligonucleotide primers made according to sequences already obtained.
  • RNAs from various tissues were prepared as described above or purchased from Clontech. One ⁇ g of each RNA was size fractionated on formaldehyde agarose gels and blotted onto nylon membranes (41, 42).
  • genomic DNA prepared from Sprague Dawley rat liver was digested with the restriction enzymes Eco RI or Hind III, size fractionated on agarose gels and blotted onto nylon membranes (41, 42). The membranes were dried at 80°C, and then prehybridized in 0.5 M sodium phosphate buffer (pH 7.3) containing 1% bovine serum albumin and 4% sodium dodecyl sulfate.
  • Hybridization was carried out in the same buffer at 65°-70°C for 14-20 hrs. with DNAs labeled with 32 P.
  • the 'P1' probe (see above under cDNA clone isolation) was used.
  • Rat Sequences used to obtain similar sequences expressed in Humans There are genes similar to the rat genes discussed above present in humans, these genes may be readily isolated by screening human gene libraries with the cloned rate sequences or by performing PCR experiments on human genomic DNA with primers homologous to the rat sequences. First, PCR experiments were performed with genomic DNA from rat, human, mouse, and several other species. When primers homologous to transmembrane domains 2 and 6 (the A4/B6 primer set used to isolate the original rat sequences) were used, DNA of the appropriate size was amplified from rat, human and mouse DNAs.
  • Hybridization under high or low stringency conditions reveals the presence of a large number of cloned human DNA segments that are homologous to the rat sequences.
  • RNA from a human olfactory tumor (neuroesthesioma, NCI-H-1011) cell line has been examined for sequences homologous to those cloned in the rat.
  • cDNA prepared from this RNA was subjected to PCR with the A4/B6 primer set and a DNA species of the appropriate size was seen. This DNA was subcloned and partially sequenced and clearly encodes a member of the olfactory protein family identified in the rat.
  • the inserted sequence in human clones H3/H5 was amplified by PCR with the A4/B6 primers, gel purified, and then labeled with 32P.
  • the labeled DNA was then hybridized to restriction enzyme human placenta. Multiple hybridizing species were observed with each DNA (See Figure 32). This observation is consistent with the presence of a family of odorant receptor genes in the human genome.
  • degenerated primer oligonucleotides homologous to conserved regions within the rat odorant receptor family may be used in PCR reactions with genomic DNA or with cDNA prepared from olfactory tissue RNA from those species.
  • PCR products within the size range expected for this family of receptors were subsequently picked and amplified further with the appropriate primer pair in order to isolate individual PCR bands.
  • Sixty-four PCR bands isolated in this fashion revealed only one or a small number of bands upon agarose gel electrophoresis. Representatives of these isolated PCR products are shown in Figure 2A.
  • the isolated PCR products were digested with the ndonuclease, Hae III or Hinf I, which recognize four base restriction sites and cut DNA at frequent intervals. In most instances, digestion of the PCR product with Hinf I generated a set of fragments whose molecular weights sum to the size of the original DNA ( Figure 2B). These PCR bands are therefore likely to each contain a single DNA species. In some cases, however, restriction digestion yielded a series of fragments whose molecular weights sum to a value greater than that of the original PCR product.
  • PCR product 13 consists of a number of different species of DNA, each of which could be amplified with the same pair of primer oligonucleotides.
  • PCR experiments similar to those described were performed using cDNA library DNAs as templates, a 710 bp PCR product was obtained with the PCR13 primer pair (A4/B6) with DNA from olfactory cDNA libraries, but not a glioma cDNA library.
  • digestion of one of this 710 bp product also revealed the presence of multiple DNA species.
  • digestion yielded a series of restriction fragments whose molecular weights also sum to a size greater than the starting material. Further analysis, however, revealed that the original PCR product consisted of multiple bands of similar but different sizes.
  • PCR 13 DNA was cloned into the plasmid vector Bluescript and five individual clones were subjected to DNA sequence analysis. Each of the five clones exhibited a different DNA sequence, but each encoded a protein which displayed conserved features of the superfamily of seven transmembrane domain receptor proteins. In addition, the proteins encoded by all five clones shared distinctive sequence motifs not found in other superfamily members indicating they were all members of a new family of receptors.
  • cDNA libraries prepared from olfactory epithelium RNA or from RNA of an enriched population of olfactory sensory neurons were screened.
  • the probe used in these initial screens was a mixture of PCR 13
  • DNA or DNA from two olfactory cDNA libraries with the same primers used to generate PCR 13 (A4 and B6 primers).
  • Hybridizing plaques were subjected to PCR amplification with the A4/B6 primer set and only those giving a PCR product of the appropriate size (approximately 710 bp) were purified.
  • the frequency of such positive clones in the enriched olfactory neuron cDNA library was approximately five times greater than the frequency in the olfactory epithelium cDNA library.
  • the increased frequency of positive clones observed in the olfactory neuron library is comparable to the enrichment in olfactory neurons generally obtained in the purification procedure.
  • the original pair of primers used to amplify PCR 13 DNA were then used to amplify coding segments of 20 different cDNA clones.
  • a mix of these PCR products were labeled and used as probe for further cDNA library screens.
  • This mixed probe was also used in a Northern blot ( Figure 3) to determine whether the expression of the gene family is restricted to the olfactory epithelium.
  • the mixed probe detects two diffuse bands centered at 2 and 5 kb in RNA from olfactory epithelium; no hybridization can be detected in brain or spleen.
  • Each of the ten sequences contains seven hydrophobic stretches (19-26 amino acids) that represent potential transmembrane domains. These domains constitute the regions of maximal sequence similarity to other members of the seven transmembrane domain superfamily (see legend to Figure 4).
  • the amino termini of the olfactory proteins are located on the extracellular side of the plasma membrane and the carboxyl termini are located in the cytoplasm.
  • three extracellular loops alternate with three intracellular loops to link the seven transmembrane domains (see Figure 5).
  • Receptors which belong to the superfamily of seven transmembrane domain proteins interact with G-proteins to generate intracellular signals.
  • In vitro mutagenesis experiments indicate that one site of association between receptor and G-protein resides within the third cytoplasmic loop (22, 23).
  • the sequence of this cytoplasmic loop in 18 different clones we have characterized is shown in Figure 6A. This loop which is often quite long and of variable length in the receptor superfamily is relatively short (only 17 amino acids) and of fixed length in the 18 clones examined. Eleven of the 18 different clones exhibit the sequence motif K/R I V S S I (or a close relative) at the N- terminus of this loop.
  • Two of the cDNA clones reveal a different H I T C/W A V motif at this site. If this short loop is a site of contact with G-proteins, it is possible that the conserved motifs may reflect sites of interaction with different G-proteins that activate different intracellular signalling systems in response to odors.
  • the receptors cloned reveal several serine or threonine residues within the third cytoplasmic loop. By analogy with other G-protein coupled receptors, these residues may represent sites of phosphorylation for specific receptor kinases involved in desensitization. (24) Subfamilies within the Multigene Family
  • Figure 6A displays the sequences of the fifth transmembrane domain and the adjacent cytoplasmic loop encoded by L8 of the cDNA clones we have analyzed. As a group, the 18 sequences exhibit considerable divergence within this region.
  • the multigene family can be divided into subfamilies such that the members of a given subfamily share significant sequence conservation.
  • subfamily B clones F12 and F13, for example, differ from one another at only four of 44 positions (91% identify), and clearly define a subfamily.
  • Clones F5 and 111 (subfamily D) differ from F12 and F13 at 34-36 positions within this region and clearly define a separate subfamily.
  • this olfactory-specific multigene family consists of highly divergent subfamilies.
  • Genomic Southern blotting experiments were preformed and genomic libraries were screened to obtain an estimate of the sizes of the multigene family and the member subfamilies encoding the putative odor receptors.
  • DNAs extending from the 3' end of transmembrane domain 3 to the middle of transmembrane domain 6 were synthesized by PCR from DNA of seven of the divergent cDNA clones ( Figure 4). In initial experiments, these DNAs were labeled and hybridized to each other to define conditions under which minimal crosshybridization would be observed among the individual clones. At 70°C, the seven DNAs showed no crosshybridization, or crosshybridized only very slightly.
  • a single band is obtained with clone 17 (probe 5).
  • PCR experiments using nested primers (TM2/TM7 primers followed by primers to internal seguences) and genomic DNA as template indicate that the coding regions of the members of this multigene family, like those of many members of the G-protein coupled superfamily, may not be interrupted by introns.
  • This observation, together with the fact that most of the probes only encompasses 400 nucleotides suggests that each band observed in these experiments is likely to represent a different gene.
  • the individual probes chosen are representatives of subfamilies which range in size from a single member to as many as 17 members. A total of about 70 individual bands were detected in this analysis which could represent the presence of at least 70 different genes.
  • cDNA probes isolated may not be representative of the full complement of subfamilies within the larger family of olfactory proteins.
  • the isolation of cDNAs relies heavily on PCR with primers from transmembrane domains 2 and 7 and biases our clones for homology within these regions. Thus, estimates of gene number as well as subsequent estimates of RNA abundance should be considered as minimal.
  • the frequency of positive clones in cDNA libraries made from olfactory epithelium RNA suggests that the abundance of the RNAs in the epithelium is about one in 20,000.
  • the frequency of positive clones is approximately five-fold higher in a cDNA library prepared from RNA from purified olfactory neurons (in which 75% of the cells are olfactory neurons).
  • the increased frequency of positive clones obtained in the olfactory neuron cDNA library is comparable to the enrichment we obtain upon purification of olfactory neurons.
  • the vomeronasal organ (vomeronasal gland) is an accessory olfactory structure that is located near the nasal cavity. Like the olfactory epithelium of the nasal cavity, the olfactory epithelium of the vomeronasal organ contains olfactory sensory neurons.
  • the vomeronasal organ is believed to play an important role in the sensing of pheromones in numerous species. Pheromones are believed to have profound effects on both physiological and behavioral aspects of reproduction. the identification of pheromone receptors would permit the identification of the pheromones themselves.
  • RNA from the vomeronasal organs of female rats was isolated from this RNA.
  • the cDNA was subjected to PCR with several different pairs of degenerate oligonucleotide primers that match sequences present in the rat odorant receptor family.
  • the PCR products were subcloned and the nucleotide sequences of the subcloned DNAs were determined.
  • Each of the subcloned DNAs encodes a protein that belongs to the odorant receptor family.
  • the sequences of the following vomeronasal subclones are shown: J1, J2, J4, J7, J8, J11, J14, J15, J16, J17, J19, J20.
  • J2, J4 the same sequence was amplified with two different primer pairs and the sequence shown is a composite of the two sequences. It is possible that one or more of these molecules, or closely related molecules, serve as pheromone receptors in the rat.
  • the mammalian olfactory system can recognize and discriminate a large number of odorous molecules. Perception in this system, as in other sensory systems, initially involves the recognition of external stimuli by primary sensory neurons. This sensory information is then transmitted to the brain where it is decoded to permit the discrimination of different odors. Elucidation of the logic underlying olfactory perception is likely to require the identification of the specific odorant receptors, the analysis of the extent of receptor diversity and receptor specificity, as well as an understanding of the pattern of receptor expression in the olfactory epithelium. The odorant receptors are thought to transduce intracellular signals by interacting with G-proteins which activate second messenger systems (12, 13, 14, 15).
  • the odorant receptors should be expressed specifically in the tissue in which odorant are recognized.
  • the family of olfactory proteins cloned is expressed in the olfactory epithelium. Hybridizing RNA is not detected in brain or retina, or in a host of nonneural tissues. Moreover, expression of this gene family the epithelium may be restricted to olfactory neurons.
  • the family of odorant receptors must be capable of interacting with extremely diverse molecular structures.
  • the genes cloned are members of any extremely large multigene family which exhibit variability in regions thought to be important in ligand binding.
  • the size of the receptor repertoire is likely to reflect the range of detectable odors and the degree of structural specificity exhibited by the individual receptors. It has been estimated that humans can identify over 10,000 structurally-distinct odorous ligands. However, this does not necessarily imply that humans possess an equally large repertoire of odorant receptors. For example, binding studies in lower vertebrates suggest that structurally-related odorant may activate the same receptor molecules. In fish which smell amino acids, the binding of alanine to isolated cilia can be competed by other small polar residues (threonine and serine), but not by the basic amino acids, lysine or arginine (11).
  • the characterization of a large multigene family encoding putative odorant receptors suggests that the olfactory system utilizes a far greater number of receptors than the visual system.
  • Color vision allows the discrimination of several hundred hues, but is accomplished by only three different photoreceptors (1, 2, 3 and 4).
  • the photoreceptors each have different, but overlapping absorption spectra which cover the entire spectrum of visible wavelengths. Discrimination of color results from comparative processing of the information from these three classes of photoreceptors in the brain.
  • the olfactory proteins identified in this application are clearly members of the superfamily of receptors which traverse the membrane seven time. Analysis of the proteins encoded by the 18 distinct cDNAs we have cloned reveals structural features which may render this family particularly well suited for the detection of a diverse array of structurally distinct odorant. Experiments with other members of this class of receptors suggest that the ligand binds to its receptor within the plane of the membrane such that the ligand contacts many, if not all of the transmembrane helices. The family of olfactory proteins can be divided into several different subfamilies which exhibit significant sequence divergence within the transmembrane domains.
  • Nonconservative changes are commonly observed within blocks of residues in transmembrane regions 3, 4, and 5 ( Figures 4, 5, 6); these blocks could reflect the sites of direct contact with odorous ligands.
  • Some members for example, have acidic residues in transmembrane domain 3, which in other families are thought to be essential for binding aminergic ligands (20) while other members maintain hydrophobic residues at these positions. This divergence within transmembrane domains may reflect the fact that the members of the family of odorant receptors must associate with odorant of widely different molecular structures.
  • each of the individual subfamilies encode receptors which bind distinct structural classes of odorant.
  • sequence differences are far less dramatic and are often restricted to a small number of residues.
  • the members of a subfamily may recognize more subtle variations among odor molecules of a given structural class.
  • individual subfamilies may recognize grossly different structures such that one subfamily may associate, for example, with the aromatic compound, benzene and its derivatives, whereas a second subfamily may recognize odorous, short chain, aliphatic molecules.
  • Subtle variations in the structure of the receptors within, for example, the hypothetical benzene subfamily could facilitate the recognition and discrimination of various substituted derivatives such as toluene, xylene or phenol. It should be noted that such a model, unlike previous stereochemical models, does not necessarily predict that molecules with similar structures will have similar odors. The activation of distinct receptors with similar structures could elicit different odors, since perceived odor will depend upon higher order processing of primary sensory information.
  • the multigene family encoding the olfactory proteins is large: all of the member genes clearly have a common ancestral origin, but have undergone considerable divergence such that individual genes encode proteins that share from 40-80% amino acid identity. Subfamilies are apparent with groups of genes sharing greater homology among themselves than with members of other subfamilies. Examination of the sequences of even the most divergent subfamilies, however, reveals a pattern in which several blocks of conserved residues are interspersed with variable regions. This segmental homology is conceptually similar to the organization of framework and hypervariable domains within the families of immunoglobulin and T cell receptor variable region sequences (27, 28).
  • each family consists of a large number of genes which have diversified over evolutionary time to accommodate the binding of a highly diverse array of ligands.
  • the evolutionary mechanisms responsible for the diversification and maintenance of these large gene families may also be similar. It has been suggested that gene conversion has played a major role in the evolution of immunoglobulin and T cell receptor variable domains (29, 30 and 31). Analysis of the sequence of the putative olfactory receptors reveals at least one instance where a motif from a variable region of one subfamily is found imbedded in the otherwise divergent sequence of a second subfamily, suggesting that conversion has occurred.
  • the gene conversion event could be stochastic, such that a given neuron could randomly express any one of several hundred receptor genes, or regulated (perhaps by positional information), such that a given neuron could only express one or a small number of predetermined receptor types.
  • positional information in the olfactory epithelium controls the expression of the family of olfactory receptors by more classical mechanisms that do not involve DNA rearrangement. What ever mechanisms will regulate the expression of receptor genes within this large, multigene family, these mechanisms must accommodate the requirement that olfactory neurons are regenerated every 30-60 days (8) and therefore the expression of the entire repertoire of receptors must be accomplished many times during the life of an organism.
  • a second model argues that sensory neurons expressing distinct odorant receptors are randomly distributed in the epithelium but that neurons responsive to a given odorant project to restricted regions within the olfactory bulb. In this instance, the discrimination of odors would be a consequence of the position of second order neurons in the olfactory bulb, but would be independent of the site of origin of the afferent signals within the epithelium.
  • Mapping of the topographic projections of olfactory neurons has been performed by extracellular recordings from different regions of the bulb (37, 38) and by 2-deoxyglucose autoradiography to map regional activity after exposure to different odorant (39). These studies suggest that spatially-localized groups of bulbar neurons preferentially respond to different odorant. The existence of specific odorant receptors, randomly distributed through the olfactory epithelium, which converge on a common target within the olfactory bulb, would raise additional questions about the recognition mechanisms used to guide these distinct axonal subsets to their central targets. Other sensory systems also spatially segregate afferent input from primary sensory neurons.
  • the spatial segregation of information employed, for example, by the visual and somatosensory systems, is used to define the location of the stimulus within the external environment as well as to indicate the quality of the stimulus.
  • olfactory processing does not extract spatial features of the odorant stimulus. Relieved of the necessity to encode information about the spatial localization of the sensory stimulus, it is possible that the olfactory system of mammals uses the spatial segregation of sensory input solely to encode the identity of the stimulus itself.
  • the molecular identification of the genes likely to encode a large family of olfactory receptors should provide initial insights into the underlying logic of olfactory processing in the mammalian nervous system.
  • Odorant-sensitive adenylate cyclase may mediate olfactory reception. Nature 316. 255-258. 13. Sklar, P.B., Anholt, R.R.H., and Snyder, S.H. (1986).
  • odorant-sensitive adenylate cyclase of olfactory receptor cells Differential stimulation by distinct classes of odorant. J. Biol. Chem. 261, 15538-15543. 14. Breer, H., Boekhoff, I., and Tareilus, E. (1990).
  • G olf an olfactory neuron specific-G-protein involved in odorant signal transduction. Science 244, 790-795.
  • ORGANISM rat olfactory epithelium
  • AAGATAGTAT CCTCCATACA TTCTATCTCC ACAGTTCAGG GGAAGTACAA GGCATTTTCT 720
  • ORGANISM rat olfactory epithelium
  • AAAGACCTAC AACCCCTTAT TTATGGTCTT TTTCTCTCTA TGTACCTGGT TACTGTCATT 120
  • ORGANISM rat olfactory epithelium
  • TTCTTCCTCA GTAACCTGTC CTTTGTGGAT GTCTGCTTCT CCTCTACCAC TGTCCCTAAA 240
  • ORGANISM rat olfactory epithelium
  • ORGANISM rat olfactory epithelium
  • CTGTACTTCT TTATGGTTTT TGGAGATATG GAGAGCTTCC TTCTTGTGGT CATGGCCTAT 360
  • ORGANISM rat olfactory epithelium
  • ORGANISM rat olfactory epithelium
  • ORGANISM rat olfactory epithelium
  • ORGANISM rat olfactory epithelium
  • ORGANISM rat olfactory epithelium
  • ORGANISM horaosapien
  • ORGANISM rat olfactory epithelium
  • GGT CAT CAT GAA CCN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN 238
  • ORGANISM rat olfactory epithelium
  • ORGANISM rat olfactory epithelium
  • ORGANISM rat olfactory epithelium
  • STRAIN Srpague-Dawley rat
  • ORGANISM rat olfactory epithelium
  • ORGANISM rat olfactory epithelium

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Abstract

La présente invention se rapporte à un acide nucléique isolé, par exemple de l'ADNc codant un récepteur de substances odorantes, ainsi qu'à des vecteurs d'expression contenant un tel acide nucléique, et à une protéine purifiée codant un récepteur de substances odorantes, avec les vecteurs d'expression susmentionnés, et à la cellule transformée résultante. On décrit également des procédés d'identification de ligands de substances odorantes et d'identification de récepteurs de ces substances, ainsi que des procédés de développement d'arômes, d'identification de composés supprimant l'appétit et de régulation de l'appétit, de même que des procédés permettant de maîtriser des colonies de parasites, de stimuler et d'inhiber la fertilité et de détecter les odeurs.
EP9292917380A 1991-04-05 1992-04-06 Recepteurs de substances odorantes et leurs utilisations. Withdrawn EP0578784A4 (fr)

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WO2001025431A1 (fr) * 1999-10-01 2001-04-12 The Rockefeller University Recepteur de type vomerien pour de primate, en particulier pour l'homme
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