WO1999000422A1 - Nouvelle famille de recepteurs de pheromones - Google Patents

Nouvelle famille de recepteurs de pheromones Download PDF

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WO1999000422A1
WO1999000422A1 PCT/US1998/013680 US9813680W WO9900422A1 WO 1999000422 A1 WO1999000422 A1 WO 1999000422A1 US 9813680 W US9813680 W US 9813680W WO 9900422 A1 WO9900422 A1 WO 9900422A1
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ser
phe
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PCT/US1998/013680
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WO1999000422A9 (fr
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Linda Buck
Catherine Dulac
Gilles Herrada
Hiroaki Matsunami
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President And Fellows Of Harvard College
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Priority to EP98933045A priority Critical patent/EP0996635A4/fr
Priority to JP50590499A priority patent/JP2002511871A/ja
Priority to CA002294473A priority patent/CA2294473A1/fr
Publication of WO1999000422A1 publication Critical patent/WO1999000422A1/fr
Publication of WO1999000422A9 publication Critical patent/WO1999000422A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to nucleic acids and encoded polypeptides which are part of a multigene family encoding a collection of novel mammalian pheromone receptors.
  • the invention further provides representative nucleic acids and encoded polypeptides in this multigene family.
  • the representative polypeptides are expressed in the murine and rat vomeronasal organ (VNO).
  • Agents which bind the nucleic acids or polypeptides also are provided.
  • the invention further relates to methods of using such nucleic acids and polypeptides in the diagnosis and/or treatment of disease, including the use of these molecules in controlling fertility and behavior in vertebrates and invertebrates.
  • Pheromones are intraspecific chemical signals found throughout the animal kingdom. They regulate populations of animals by inducing innate behaviors and stereotyped changes in physiology (Karlson and Luscher, Nature, 1959, 183:55-56; Wilson, Sci. Am., 1963, 208:100- 114; Sorensen, Chem. Sens., 1996, 21:245-256). Pheromones can serve as cues for overcrowding, impending danger, reproductive status, gender, or dominance. In rodents, a variety of pheromone effects have been reported. These include effects on estrus and the onset of puberty as well as the induction of mating and aggressive behaviors (Singer, A.G., J Steroid. Biochem. Molec.
  • pheromones The detection of pheromones is mediated by the olfactory system.
  • sensory neurons that detect pheromones are typically segregated from those that detect volatile odorants (Keverne, E.B., Trends Neurosci., 1983, 6:381-384; Halpern, M., Ann. Rev. Neurosci., 1987, 10:325-362; Wysocki, C.J., et al, In the Neurobiology of Taste and Smell, 1987, 125-150; Hildebrand, J.G., et al., Brain Res., 1997, 677:157-161).
  • VNO vomeronasal organ
  • Volatile odorants are detected in the OE by as many as 1000 different types of odorant receptors (ORs), which are differentially expressed by olfactory sensory neurons (Buck and Axel, Cell, 1991, 65:175-187; Levy, N.S., et al, J Steroid Biochem. Mol. Biol, 1991, 39:633-637, 1991; Nef, P., et al, Proc. Natl. Acad.
  • ORs odorant receptors
  • VNO VNO neurons
  • VNs also lack a number of other olfactory sensory transduction molecules, including the G protein a subunit,G ⁇ 01f (Reed, R., Neuron, 1992, 8:205- 209), which is highly expressed in olfactory neurons (Dulac and Axel, Cell, 1995, 83:195-206; Berghard, A., et al, Proc. Natl. Acad. Sci. USA, 1996, 93:2365-2369; Wu, Y., et al, Biochem. Biopys. Res. Com., 1996, 220:900-904).
  • VNs express high levels of two other G protein a subunits,G ⁇ 0 and G ⁇ i 2 (Dulac and Axel, Cell, 1995, 83:195-206; Halpern, M., Brain Res., 41995, 677:157-161; Berghard, A., et al, Proc. Natl. Acad. Sci. USA, 1996, 93:2365-2369)
  • G ao and G ⁇ i 2 are expressed in spatially-segregated subsets of VNs that form longitudinal zones in the VNO neuroepithelium.
  • VNRs pheromones receptors
  • This invention differs from the state of the art in providing a novel family of mammalian pheromone receptors. Accordingly, the objects of the invention relate to providing compositions containing these novel receptors and their binding partners and methods for using such compositions to modulate pheromone receptor activity.
  • the invention involves the discovery of a multigene family of mammalian pheromone receptors.
  • the invention involves the cDNA cloning of multiple pheromone receptors from a murine VNO cDNA library and from a rat VNO cDNA library. Partial sequences of human homologs of these pheromone receptors also are provided.
  • the invention provides isolated nucleic acid molecules encoding the novel pheromone receptors, unique fragments of the isolated nucleic acid molecules, expression vectors containing the foregoing, and host cells transfected with the foregoing.
  • the invention also provides isolated pheromone receptor polypeptides and agents which bind such polypeptides, including antibodies.
  • the foregoing can be used in the diagnosis or treatment of conditions, including the control of fertility, that are characterized by the expression ofa pheromone receptor polypeptide. Methods for identifying pharmacological agents useful in the diagnosis or treatment of such conditions and methods for identifying additional members of this multigene family also are provided.
  • VNO vomeronasal organ
  • G ⁇ 0 protein expressing neurons This is in contrast to the prior art VNO pheromone receptors which are expressed in neurons which express different G-coupled proteins (G ⁇ i 2 -expressing neurons).
  • G ⁇ i 2 -expressing neurons G-coupled proteins
  • a family of pheromone receptor polypeptides is provided.
  • Each polypeptide of the family shares amino acid sequence homology and structural organization with a pheromone receptor polypeptide selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52.
  • Each polypeptide member of the receptor family contains, from amino terminus to carboxyl terminus, the following domains: (a) an amino-terminal extracellular domain containing from 30 to 600 amino acids; (b) a transmembrane region comprising: (i) seven non-contiguous transmembrane domains designated TM1, TM2, TM3, TM4, TM5, TM6 and TM7, (ii) three noncontiguous extracellular domains designated EC2, EC3 and EC4, and (iii) three non-contiguous intracellular domains designated ICl, IC2, and IC3, wherein the transmembrane domains, the extracellular domains and the intracellular domains are attached to one another from amino terminus to carboxyl terminus in the order TM1 -IC 1 -TM2-EC2-TM3- IC2-TM4-EC3-TM5-IC3- TM6-EC4-TM7, and wherein the transmembrane region has at least about 35% homology and
  • Each polypeptide member of the family is expressed in a G ⁇ 0 protein-expressing vomeronasal organ neuron or are expressed in another olfactory organ neuron in an animal which does not possess a vomeronasal organ.
  • One skilled in the art can readily identify olfactory organs in animals which do not possess a vomeronasal organ.
  • the amino-terminal extracellular domains (NTDs) of the receptor family members share sequence homology to a pheromone receptor polypeptide selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50 to a lesser extent than that observed for the transmembrane region.
  • the length of the extracellular domain can vary among members of the family. Accordingly, certain embodiments of the invention have extracellular domains that contain at least 50, 100, 200, 300, 400 or 500 amino acids.
  • the transmembrane region has greater than 40% homology with the corresponding region of a pheromone receptor polypeptide selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 34, 36, 38, 40, 42, 44, 46, 48, and 50, and more preferably, have even greater sequence homology (e.g., more than 50%), 60%, 70%, 80% or 90% homology).
  • the length of the carboxyl-terminal intracellular domain can vary among members of the family. Accordingly, certain embodiments of the invention have carboxyl-terminal intracellular domains that contain at least between 5 and 50 amino acids. More preferably, carboxyl-terminal intracellular domains contain between 15 and 25 amino acids.
  • a method for identifying a nucleic acid encoding a pheromone receptor involves contacting a mixture of nucleic acid molecules (genomic library, cDNA library, genomic DNA, RNA, etc.) with at least one nucleic acid probe ofa nucleic acid selected from the group consisting of: (a) a nucleic acid molecule selected from the group consisting of SEQ ID NO.
  • the nucleic acid probe further includes a detectable label to facilitate identification of the sequence in the library which hybridizes to the probe.
  • the probe is represented by a pair of degenerate polymerase chain reaction ("PCR") primers that amplify a unique fragment of a nucleic acid molecule selected from the group consisting of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 54, and 55.
  • PCR polymerase chain reaction
  • degenerate PCR primers that amplify a unique fragment degenerate primers which result in the amplification of a unique fragment following a polymerase chain reaction.
  • the method for identifying a nucleic acid encoding a pheromone receptor polypeptide further involves subjecting a mixture of nucleic acids and the degenerate PCR primers to amplification conditions prior to identifying the sequences of the mixture that hybridize to the probe and that form part of the amplification reaction products.
  • the pair of degenerate polymerase chain reaction primers is selected from a conserved sequence motif of a pheromone receptor polypeptide.
  • a "conserved sequence motif” can be determined using the side-by-side comparison of the amino acid sequences of the different pheromone receptor polypeptides of the invention.
  • Exemplary conserved sequence motifs include regions selected from the group consisting of amino acids 191-397, amino acids 565-825, amino acids 637-825, amino acids 637-804, amino acids 619-784, of the polypeptide of, for example, SEQ ID NO. 2 (VR1).
  • the pair of degenerate polymerase chain reaction primers is selected from the group consisting of SEQ ID NOs. 60 and 61 , SEQ ID
  • an isolated nucleic acid molecule hybridizes under high or low stringency conditions to a molecule consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 54, and 55.
  • the invention further embraces nucleic acid molecules that differ from the foregoing isolated nucleic acid molecules in codon sequence due to the degeneracy of the genetic code.
  • the invention also embraces complements of the foregoing nucleic acids.
  • the pheromone receptors of the invention are expressed in the vomeronasal organ or, in an animal which lacks such an organ, are expressed in another olfactory organ. More particularly, the receptors of the invention are expressed in a G ⁇ 0 protein-expressing vomeronasal organ neuron. Although not intending to be bound to a particular mechanism, it is believed that the receptors of the invention are G-protein coupled receptors. This is supported by Applicants' discovery that the receptors of the invention are expressed in G ⁇ 0 protein-expressing vomeronasal organ neurons.
  • the pheromone receptors of the invention bind to ligands (pheromones) which induce certain changes in receptor conformation.
  • Methods for identifying ligands which bind to the pheromone receptors of the invention are provided below, e.g., by forming an affinity matrix containing immobilized receptor and using the matrix to isolate a cognate ligand from a complex mixture.
  • the particular ligand bound by a particular receptor is dictated by the primary and secondary structure of the receptor.
  • the immobilized pheromone receptor polypeptide is a pheromone receptor polypeptide selected from the group consisting of SEQ ID NO.
  • an isolated nucleic acid molecule that is a unique fragment of any of the foregoing isolated nucleic acid molecules is provided.
  • the isolated nucleic acid molecule consists of a unique fragment between 12 and 4000 nucleotides in length, and complements thereof, of any cDNA (SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 54, and 55) encoding a pheromone receptor polypeptide selected from the group consisting of SEQ ID NO.
  • the unique fragment can be between 12 and 2000, 1000, 500, 250, 100, 50 or 25 nucleotides in length.
  • the isolated nucleic acid molecule consists of between 12 and 35 contiguous nucleotides of the foregoing cDNAs encoding the pheromone receptor polypeptides, or complements of such nucleic acid molecules.
  • the unique fragment is at least 14, 15, 16, 17, 18, 20 or 22 contiguous nucleotides of the nucleic acid sequence of the foregoing cDNAs encoding the pheromone receptor polypeptides, or complements thereof.
  • Particularly preferred isolated nucleic acid molecules are isolated fragments of the foregoing cDNAs which encode one or more of the following pheromone receptor polypeptide domains, alone or in combination (e.g., as fusion proteins): an amino-terminal extracellular domain, a transmembrane region, and a carboxy- terminal intracellular domain.
  • the unique fragments are a pheromone receptor extracellular domain or a pheromone receptor intracellular domain coupled to at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) transmembrane domain.
  • an isolated nucleic acid molecule comprising a molecule having a sequence selected from the group consisting of SEQ ID NO. 51, 53, 54, 55, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, and 92, that encodes a pheromone receptor polypeptide are provided.
  • This aspect of the invention further embraces nucleic acid molecules that differ from these nucleic acid molecules in codon sequence due to the degeneracy of the genetic code, and diversity among pheromone receptors and complements of foregoing.
  • an expression vector comprising any of the foregoing isolated nucleic acid molecules operably linked to a promoter and host cells transformed or transfected with the same also are provided.
  • an isolated polypeptide encoded by any of the above-described isolated nucleic acid molecules is provided.
  • the isolated polypeptide is a pheromone receptor polypeptide that has a pheromone receptor activity or an antigenic fragment thereof.
  • a pheromone receptor activity refers to the ability of the pheromone receptor to selectively bind to its cognate ligand (pheromone) and, optionally, upon binding, to induce signal transduction in a cell that expresses the pheromone receptor.
  • the isolated polypeptide comprises a pheromone receptor polypeptide having a sequence selected from the group consisting of SEQ ID NO.
  • the isolated polypeptide comprises a polypeptide encoded by a nucleic acid which hybridizes under high or low stringency conditions to the extracellular domain, transmembrane region and/or intracellular domain of a cDNA sequence selected from the group consisting of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 54, and 55 that encodes a pheromone receptor polypeptide or fragment thereof.
  • the invention embraces portions of a pheromone receptor polypeptide that may include, for example, an amino-terminal extracellular domain or a carboxy- terminal intracellular domain coupled to 1, 2, 3, 4, 5, 6, or 7 transmembrane domains.
  • a pheromone receptor polypeptide may include, for example, an amino-terminal extracellular domain or a carboxy- terminal intracellular domain coupled to 1, 2, 3, 4, 5, 6, or 7 transmembrane domains.
  • such polypeptides or fragments thereof are unique fragments and can function as, for example, antigens for making antibodies specific for pheromone receptor family members.
  • the polypeptides of the invention can be used to isolate additional members of the pheromone receptor family or, alternatively, can be used to induce in vivo an immune response to a pheromone receptor, i.e., can be incorporated into a vaccine preparation.
  • Such vaccine compositions are useful for controlling fertility or behavior in an animal by administering to the animal, an effective amount of the vaccine to elicit an immune response to the pheromone receptor.
  • the invention embraces fragments or variants of the foregoing pheromone receptors which exhibit certain detectable activities, e.g., a ligand binding activity, an antigenicity activity.
  • the isolated polypeptide is encoded by a cDNA selected from the group consisting of SEQ ID NO.
  • isolated binding polypeptides which selectively bind a unique amino acid sequence of a pheromone receptor polypeptide or fragment thereof.
  • the isolated binding polypeptide in certain embodiments binds to a polypeptide comprising the extracellular domain and/or 1, 2, 3, 4, 5, 6, or 7 transmembrane domains of a pheromone receptor polypeptide selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52.
  • isolated polypeptide preferably binds to a polypeptide consisting of the amino- terminal extracellular domain and/or one or more portions of the transmembrane region of a pheromone receptor polypeptide sequence selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52.
  • isolated binding polypeptides include antibodies and fragments of antibodies (e.g., Fab, F(ab) 2 , Fd and antibody fragments which include a CDR3 region which binds selectively to the unique sequences of the polypeptides of the invention).
  • the isolated binding peptides do not bind to pheromone receptors that are expressed in vomeronasal organ neurons other than G ⁇ o-protein-expressing neurons.
  • isolated nucleic acids or polypeptides of the invention that are: (a) immobilized to an insoluble support (an affinity matrix containing immobilized pheromone receptor polypeptide or a unique fragment thereof); (b) associated with, covalently coupled to, or encapsulated a drug delivery device (e.g., a microsphere) to effect controlled release of the isolated nucleic acid or polypeptide in vivo or in vitro; (c) covalently coupled to another isolated nucleic acid or protein to form a chimeric molecule; and/or (d) labeled with a detectable agent (e.g., a radiolabel, a fluorescent label).
  • a detectable agent e.g., a radiolabel, a fluorescent label
  • the invention provides chimeric molecules containing at least one first structural domain of one pheromone receptor polypeptide (e.g., an extracellular domain) coupled to a second structural domain (e.g., a transmembrane domain, such as TM1, TM2, etc.) of a different pheromone receptor polypeptide.
  • the invention also provides a method for isolating a pheromone receptor by (1) contacting a composition containing a putative pheromone receptor of the above-described family with an affinity matrix containing immobilized binding polypeptide under conditions to permit the pheromone receptor to selectively bind to the immobilized binding polypeptide, and (2) isolating the polypeptides that bind to the affinity matrix.
  • pharmaceutical compositions containing any of the foregoing compounds of the invention in a pharmaceutically acceptable carrier and methods of producing same by placing the compositions in the carrier also are provided.
  • a pheromone receptor activity e.g., a ligand binding activity, a signal transduction activity
  • the cell can be located in vivo or in vitro and the methods can be used to down regulate (inhibit) or up regulate (stimulate) the pheromone receptor activity.
  • an inhibitor that can be an isolated binding polypeptide that binds to an extracellular portion of the receptor and, thereby, inhibits receptor binding to its cognate ligand.
  • Such binding also can induce conformational changes in the receptor that alter the signal transduction activity of the receptor.
  • the inhibitor can be an isolated antibody (or function equivalent thereof) which binds to an epitope located on an extracellular portion (such as EC2, EC3, EC4) of the pheromone receptor polypeptide, e.g., an amino-terminal extracellular domain or an "extracellular transmembrane region domain", i.e., an extracellular portion of the transmembrane region located between one or more transmembrane domains.
  • the inhibitor can be an agent (e.g., an isolated competitive binding polypeptide) that inhibits receptor-ligand binding.
  • the inhibitor can be an isolated fragment of a pheromone receptor (preferably, a soluble fragment), which fragment contains a ligand (pheromone) binding site.
  • inhibitors can be identified in screening assays which test the ability of a putative inhibitor to inhibit pheromone receptor- mediated signal transduction or which test the ability of the putative inhibitor to inhibit binding of a pheromone receptor to its known cognate ligand.
  • screening assays can be used to identify molecules which stimulate pheromone receptor-mediated signal transduction.
  • Exemplary molecules which stimulate transduction include the naturally-occurring ligands (e.g., isolated from a biological source (e.g., urine, vaginal fluid), as well as synthetic ligands obtained from a non-biological source (e.g., a combinatorial library).
  • methods for inhibiting the binding of a pheromone having a binding domain to a pheromone receptor polypeptide having a ligand binding site that selectively binds to the binding domain involve contacting (in vivo or in vitro) the pheromone receptor polypeptide with an agent which binds to the ligand binding site under conditions to permit binding of the agent to the receptor.
  • the agent can be an isolated binding polypeptide that binds to the ligand binding site of the pheromone receptor.
  • the agent can be an isolated antibody (or functionally equivalent fragment thereof) which selectively binds to the ligand binding site of the receptor.
  • the agent can be a pheromone receptor antagonist, e.g., a molecule that mimics the structure of the naturally-occurring ligand but that does not mimic the function (stimulating the receptor) of the naturally-occurring ligand.
  • Agents which inhibit ligand binding can be identified in screening assays which test the ability of a putative binding inhibitor to inhibit binding of a pheromone receptor to its cognate ligand (e.g., pheromone).
  • Such molecules can be isolated from a biological source or from a non-biological source.
  • methods for modulating pheromone receptor-mediated signal transduction in a subject involve administering to a subject in need of such treatment an agent that selectively binds to any of the above-described isolated nucleic acid molecules which encode a pheromone receptor or unique fragment thereof, or an expression product thereof, in an amount effective to modulate (down regulate or up regulate) pheromone receptor-mediated signal transduction in the subject.
  • agents include antisense nucleic acid molecules and binding polypeptides.
  • methods are provided for identifying lead compounds for an pharmacological agent useful in the diagnosis or treatment of a condition associated with pheromone receptor signal transduction activity or otherwise generally associated with binding of the receptor to its cognate ligand.
  • cells expressing intact pheromone receptor polypeptides or portions thereof are used in the screening assays for identifying lead compounds which modulate pheromone receptor-mediated ligand binding or signal transduction activity.
  • Cells expressing these polypeptides, isolated pheromone receptor polypeptides and fragments of these polypeptides which contain the ligand binding site can be used in the screening assays for identifying lead compounds which modulate binding of the receptor to a known ligand.
  • the screening methods involve forming a mixture of a pheromone receptor polypeptide
  • the mixture is incubated under conditions which, in the absence of the candidate pharmacological agent, permit a first amount of pheromone receptor-ligand binding or receptor-mediated signal transduction by the known ligand.
  • a test amount of the selective binding of the ligand by receptor or of the specific activation of signal transduction is determined.
  • Detection of an increase in the foregoing activities in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which increases specific activation of pheromone receptor-mediated signal transduction or selective binding of the ligand by the ligand binding site of the receptor.
  • Detection of a decrease in the foregoing activities in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which decreases specific activation of pheromone receptor-mediated signal transduction or selective binding of the ligand by the ligand binding site of the receptor.
  • Pheromone receptor polypeptides that are useful in the screening assays are those selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52. Extracellular domains or portions thereof and portions of the transmembrane region, alone or coupled to one another, of these pheromone receptor polypeptides (indicated in the Examples) can be tested for their ability to inhibit receptor-ligand binding.
  • Figure 1 depicts a comparison of the deduced protein sequences encoded by VR cDNA clones.
  • Figure 2 is a schematic comparison of ORs, VNRs, and Vrs.
  • Figure 3 depicts a comparison of the deduced protein sequences encoded by the Go-VN cDNA clones.
  • SEQ ID NO. 1 is the nucleotide sequence of the mouse pheromone receptor VR1 cDNA (GenBank Accession No. AF011411).
  • SEQ ID NO. 2 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VR1 cDNA (GenBank Accession No. AF011411).
  • SEQ ID NO. 3 is the nucleotide sequence of the mouse pheromone receptor VR2 cDNA (GenBank Accession No. AF011412).
  • SEQ ID NO. 4 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VR2 cDNA (GenBank Accession No. AF011412).
  • SEQ ID NO. 5 is the nucleotide sequence of the mouse pheromone receptor VR3 cDNA (GenBank Accession No. AF011413).
  • SEQ ID NO. 6 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VR3 cDNA (GenBank Accession No. AFOl 1413).
  • SEQ ID NO. 7 is the nucleotide sequence of the mouse pheromone receptor VR4 cDNA (GenBank Accession No. AFOl 1414).
  • SEQ ID NO. 8 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VR4 cDNA (GenBank Accession No. AFOl 1414).
  • SEQ ID NO. 9 is the nucleotide sequence of the mouse pheromone receptor VR5 cDNA (GenBank Accession No. AFOl 1415).
  • SEQ ID NO. 10 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VR5 cDNA (GenBank Accession No. AFOl 1415).
  • SEQ ID NO. 11 is the nucleotide sequence of the mouse pheromone receptor VR6 cDNA (GenBank Accession No. AFOl 1416).
  • SEQ ID NO. 12 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VR6 cDNA (GenBank Accession No. AFOl 1416).
  • SEQ ID NO. 13 is the nucleotide sequence of the mouse pheromone receptor VR7 cDNA (GenBank Accession No. AFOl 1417).
  • SEQ ID NO. 14 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VR7 cDNA (GenBank Accession No. AFOl 1417).
  • SEQ ID NO. 15 is the nucleotide sequence of the mouse pheromone receptor VR8 cDNA (GenBank Accession No. AFOl 1418).
  • SEQ ID NO. 16 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VR8 cDNA (GenBank Accession No. AFOl 1418).
  • SEQ ID NO. 17 is the nucleotide sequence of the mouse pheromone receptor VR9 cDNA (GenBank Accession No. AFOl 1419).
  • SEQ ID NO. 18 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VR9 cDNA (GenBank Accession No. AFOl 1419).
  • SEQ ID NO. 19 is the nucleotide sequence of the mouse pheromone receptor VR10 cDNA (GenBank Accession No. AFOl 1420).
  • SEQ ID NO. 20 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VRl 0 cDNA (GenBank Accession No. AFO 11420).
  • SEQ ID NO. 21 is the nucleotide sequence of the mouse pheromone receptor VRl 1 cDNA (GenBank Accession No. AFOl 1421).
  • SEQ ID NO. 22 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VRl 1 cDNA (GenBank Accession No. AFOl 1421).
  • SEQ ID NO. 23 is the nucleotide sequence of the mouse pheromone receptor VRl 2 cDNA (GenBank Accession No. AFOl 1422).
  • SEQ ID NO. 24 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VRl 2 cDNA (GenBank Accession No. AFOl 1422).
  • SEQ ID NO. 25 is the nucleotide sequence of the mouse pheromone receptor VRl 3 cDNA (GenBank Accession No. AFOl 1423).
  • SEQ ID NO. 26 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VRl 3 cDNA (GenBank Accession No. AFO 11423).
  • SEQ ID NO. 27 is the nucleotide sequence of the mouse pheromone receptor VRl 4 cDNA (GenBank Accession No. AFOl 1424).
  • SEQ ID NO. 28 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VRl 4 cDNA (GenBank Accession No. AFOl 1424).
  • SEQ ID NO. 29 is the nucleotide sequence of the mouse pheromone receptor VRl 5 cDNA (GenBank Accession No. AFOl 1425).
  • SEQ ID NO. 30 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VRl 5 cDNA (GenBank Accession No. AFOl 1425).
  • SEQ ID NO. 31 is the nucleotide sequence of the mouse pheromone receptor VRl 6 cDNA (GenBank Accession No. AFOl 1426).
  • SEQ ID NO. 32 is the predicted amino acid sequence of the polypeptide encoded by the mouse pheromone receptor VRl 6 cDNA (GenBank Accession No. AFOl 1426).
  • SEQ ID NO. 33 is the nucleotide sequence of the rat pheromone receptor Go-VNl cDNA (GenBank Accession No. AF016178).
  • SEQ ID NO. 34 is the predicted amino acid sequence of the polypeptide encoded by the rat pheromone receptor Go-VNl cDNA (GenBank Accession No. AF016178).
  • SEQ ID NO. 35 is the nucleotide sequence of the rat pheromone receptor Go-VN2 cDNA (GenBank Accession No. AF016179).
  • SEQ ID NO. 36 is the predicted amino acid sequence of the polypeptide encoded by the rat pheromone receptor Go-VN2 cDNA (GenBank Accession No. AFO 16179).
  • SEQ ID NO. 37 is the nucleotide sequence of the rat pheromone receptor Go-VN3 cDNA (GenBank Accession No. AFOl 6180).
  • SEQ ID NO. 38 is the predicted amino acid sequence of the polypeptide encoded by the rat pheromone receptor Go-VN3 cDNA (GenBank Accession No. AFOl 6180).
  • SEQ ID NO. 39 is the nucleotide sequence of the rat pheromone receptor Go-VN4 cDNA (GenBank Accession No. AF016181).
  • SEQ ID NO. 40 is the predicted amino acid sequence of the polypeptide encoded by the rat pheromone receptor Go-VN4 cDNA (GenBank Accession No. AFOl 6181).
  • SEQ ID NO. 41 is the nucleotide sequence of the rat pheromone receptor Go-VN5 cDNA (GenBank Accession No. AF016182).
  • SEQ ID NO. 42 is the predicted amino acid sequence of the polypeptide encoded by the rat pheromone receptor Go-VN5 cDNA (GenBank Accession No. AF016182).
  • SEQ ID NO. 43 is the nucleotide sequence of the rat pheromone receptor G0-VN6 cDNA (GenBank Accession No. AF016183).
  • SEQ ID NO. 44 is the predicted amino acid sequence of the polypeptide encoded by the rat pheromone receptor G0-VN6 cDNA (GenBank Accession No. AFOl 6183).
  • SEQ ID NO. 45 is the nucleotide sequence of the rat pheromone receptor Go-VN7 cDNA (GenBank Accession No. AFOl 6184).
  • SEQ ID NO. 46 is the predicted amino acid sequence of the polypeptide encoded by the rat pheromone receptor Go-VN7 cDNA (GenBank Accession No. AFOl 6184).
  • SEQ ID NO. 47 is the nucleotide sequence of the rat pheromone receptor Go-VN13C cDNA (GenBank Accession No. AFO 16185).
  • SEQ ID NO. 48 is the predicted amino acid sequence of the polypeptide encoded by the rat pheromone receptor Go-VN13C cDNA (GenBank Accession No. AF016185).
  • SEQ ID NO. 49 is the nucleotide sequence of the rat pheromone receptor Go-VNl 3B cDNA (GenBank Accession No. AF016186).
  • SEQ ID NO. 50 is the predicted amino acid sequence of the polypeptide encoded by the rat pheromone receptor Go-VN13B cDNA (GenBank Accession No. AF016186).
  • SEQ ID NO. 51 is a partial nucleotide sequence of the human pheromone receptor hVRl.
  • SEQ ID NO. 52 is the predicted amino acid sequence of the polypeptide encoded by the partial sequence of the human pheromone receptor hVRl .
  • SEQ ID NO. 53 is a partial nucleotide sequence of the human pheromone receptor hVNOl.
  • SEQ ID NO. 54 is a partial nucleotide sequence of the human pheromone receptor hVNO2.
  • SEQ ID NO. 55 is a partial nucleotide sequence of the human pheromone receptor hVNO3.
  • SEQ ID NO. 56 is the nucleotide sequence of primer AL 1.
  • SEQ ID NO. 57 is the nucleotide sequence of primer AL3.
  • SEQ ID NO. 58 is a fifty amino acid sequence of Go-VN13B (SEQ ID NO. 50) that is absent from Go-VN13C (SEQ ID NO. 48).
  • SEQ ID NO. 59 is the amino acid sequence of a rat kidney extracellular calcium/ polyvalent cation-sensing receptor.
  • SEQ ID NO. 60 is a degenerate oligonucleotide primer from a conserved VR domain.
  • SEQ ID NO. 61 is a degenerate oligonucleotide primer from a conserved VR domain.
  • SEQ ID NO. 62 is a degenerate oligonucleotide primer from a conserved VR domain.
  • SEQ ID NO. 63 is a degenerate oligonucleotide primer from a conserved VR domain.
  • SEQ ID NO. 64 is a degenerate oligonucleotide primer from a conserved VR domain.
  • SEQ ID NO. 65 is a degenerate oligonucleotide primer from a conserved VR domain.
  • SEQ ID NO. 66 is a degenerate oligonucleotide primer from a conserved VR domain.
  • SEQ ID NO. 67 is a degenerate oligonucleotide primer from a conserved VR domain.
  • SEQ ID NO. 68 is the nucleotide sequence of the coding region of the mouse pheromone receptor VRl .
  • SEQ ID NO. 69 is the nucleotide sequence of the coding region of the mouse pheromone receptor VR2.
  • SEQ ID NO. 70 is the nucleotide sequence of the coding region of the mouse pheromone receptor VR3.
  • SEQ ID NO. 71 is the nucleotide sequence of the coding region of the mouse pheromone receptor VR4.
  • SEQ ID NO. 72 is the nucleotide sequence of the coding region of the mouse pheromone receptor VR5.
  • SEQ ID NO. 73 is the nucleotide sequence of the coding region of the mouse pheromone receptor VR6.
  • SEQ ID NO. 74 is the nucleotide sequence of the coding region of the mouse pheromone receptor VR7.
  • SEQ ID NO. 75 is the nucleotide sequence of the coding region of the mouse pheromone receptor VR8.
  • SEQ ID NO. 76 is the nucleotide sequence of the coding region of the mouse pheromone receptor VR9.
  • SEQ ID NO. 77 is the nucleotide sequence of the coding region of the mouse pheromone receptor VR10.
  • SEQ ID NO. 78 is the nucleotide sequence of the coding region of the mouse pheromone receptor VRl 1.
  • SEQ ID NO. 79 is the nucleotide sequence of the coding region of the mouse pheromone receptor VRl 2.
  • SEQ ID NO. 80 is the nucleotide sequence of the coding region of the mouse pheromone receptor VRl 3.
  • SEQ ID NO. 81 is the nucleotide sequence of the coding region of the mouse pheromone receptor VRl 4.
  • SEQ ID NO. 82 is the nucleotide sequence of the coding region of the mouse pheromone receptor VRl 5.
  • SEQ ID NO. 83 is the nucleotide sequence of the coding region of the mouse pheromone receptor VRl 6.
  • SEQ ID NO. 84 is the nucleotide sequence of the coding region of the rat pheromone receptor Go VN1.
  • SEQ ID NO. 85 is the nucleotide sequence of the coding region of the rat pheromone receptor GoVN2.
  • SEQ ID NO. 86 is the nucleotide sequence of the coding region of the rat pheromone receptor GoVN3.
  • SEQ ID NO. 87 is the nucleotide sequence of the coding region of the rat pheromone receptor GoVN4.
  • SEQ ID NO. 88 is the nucleotide sequence of the coding region of the rat pheromone receptor GoVN5.
  • SEQ ID NO. 89 is the nucleotide sequence of the coding region of the rat pheromone receptor GoVN6.
  • SEQ ID NO. 90 is the nucleotide sequence of the coding region of the rat pheromone receptor GoVN7.
  • SEQ ID NO. 91 is the nucleotide sequence of the coding region of the rat pheromone receptor Go VN13C.
  • SEQ ID NO. 92 is the nucleotide sequence of the coding region of the rat pheromone receptor Go VN13B.
  • the present invention in one aspect involves the cloning of cDNAs encoding several members of a multigene family of pheromone receptors. Complete cDNA sequences for selected murine and rat pheromone receptors are provided. Partial sequences of the human gene also are provided.
  • the present invention also relates to the discovery that this family of pheromone receptors is expressed in a G ⁇ 0 protein-expressing vomeronasal organ neurons ("G ⁇ + VNO") or in another olfactory organ neuron in an animal (preferably, a mammal and more preferably, a human) which lacks a vomeronasal organ.
  • G ⁇ + VNO vomeronasal organ neurons
  • the pheromone receptors of the invention alternatively are referred to as "pheromone receptors", "G ⁇ 0 + VNO pheromone receptors" or, simply, "G ⁇ 0 + VNO receptors”.
  • telomere domains include, from amino terminus to carboxyl terminus: (a) an amino-terminal extracellular domain containing from 30 to 600 amino acids; (b) a transmembrane region comprising: (i) seven non-contiguous transmembrane domains designated TMl, TM2, TM3, TM4, TM5, TM6 and TM7, (ii) three non-contiguous extracellular domains designated EC2, EC3 and EC4, and (iii) three non-contiguous intracellular domains designated ICl, IC2, and IC3, wherein the transmembrane domains, the extracellular domains and the intracellular domains are attached to one another from amino terminus to carboxyl terminus in the order TMl -IC 1 -TM2-EC2-TM3- IC2-TM4-EC3-TM5-IC3-TM6-EC4
  • Each polypeptide member of the family is expressed in a G ⁇ 0 protein-expressing vomeronasal organ neuron or are expressed in another olfactory organ neuron in an animal which does not possess a vomeronasal organ.
  • One skilled in the art can readily identify olfactory organs in animals which do not possess a vomeronasal organ.
  • the homology can be calculated using various, publicly available software tools developed by NCBI (Bethesda, Maryland) that can be obtained through the internet (ftp://ncbi.nlm.nih.gov/pub/).
  • Exemplary tools include the BLAST system. Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis can be obtained using the MacVector sequence analysis software (Oxford Molecular Group).
  • GPCR G protein-coupled receptor superfamily
  • G ⁇ 0 + VNO pheromone receptors exhibit seven hydrophobic stretches ("hydrophobic domains") and are similar in structure to other types of GPCRs, the calcium sensing receptor (CSR Ser. ID No. 59) and the metabotropic glutamate receptors (mGluRs).
  • CSR and mGluRs are unusual among the GPCRs in that they have extremely long N-terminal extracellular domain (e.g., 557- 565 amino acids), a feature that is shared by the pheromone receptors of the invention. Despite this similarity, the receptors of the invention do not share substantial primary structure homology with the CSR and mGluRs.
  • the receptors of the invention also are very different structurally from two other G-protein coupled receptors, the odorant receptors and G ⁇ i 2 + vomeronasal receptors, which share none of the characteristic sequence motifs of the receptors of the invention and, moreover, which have very small (-12-28 amino acids) N-terminal extracellular domains.
  • the receptors of the invention differ somewhat in amino acid sequence, with regions of relatively high sequence homology.
  • Examples 1 and 2 for a discussion and illustration of the amino acid sequence homology for the murine and rat G ⁇ 0 + VNO receptors, respectively.
  • Other features of these members of the G ⁇ 0 + VNO receptor family also are discussed and illustrated in the Examples.
  • signal sequences have been identified for several of the G ⁇ 0 + VNO receptors disclosed in the Examples.
  • an aspect of the invention is those nucleic acid sequences (SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 54, and 55) which code for G ⁇ 0 + VNO pheromone receptors and which hybridize to a nucleic acid molecule consisting of the coding region of any one G ⁇ 0 + VNO pheromone receptor selected from the group consisting of SEQ ID NO.
  • high or low stringency conditions refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
  • high stringency conditions refers, for example, to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% Bovine Serum Albumin, 2.5mM NaH 2 PO 4 (pH7), 0.5% SDS, 2mM EDTA).
  • SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid.
  • Low stringency conditions would be the same, but with a lower temperature (e.g., 55 °C).
  • the membrane upon which the DNA is transferred is washed at 2 x SSC at room temperature and then at 0.2 x SSC/0.5% SDS at temperatures of up to 65°C. Additional conditions of varying stringency are provided in the Examples.
  • homologs and alleles typically will share at least 35% nucleotide identity and/or at least 50% amino acid identity to the cDNAs encoding a G ⁇ 0 + VNO pheromone receptor polypeptide selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52, in some instances will share at least 50% nucleotide identity and/or at least 65%) amino acid identity and in still other instances will share at least 60% nucleotide identity and/or at least 75%> amino acid identity.
  • Watson-Crick complements of the foregoing nucleic acids also are embraced by the invention.
  • certain domains within the pheromone receptors may share even greater sequence homology to a pheromone receptor polypeptide selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52.
  • a Southern blot may be performed using the foregoing conditions, together with a radioactive probe. After washing the membrane to which the DNA is finally transferred, the membrane can be placed against X-ray film to detect the radioactive signal.
  • the invention also includes degenerate nucleic acids which include alternative codons to those present in the native materials.
  • serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC.
  • Each of the six codons is equivalent for the purposes of encoding a serine residue.
  • any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into an elongating G ⁇ 0 + VNO pheromone receptor polypeptide.
  • nucleotide sequence triplets which encode other amino acid residues include, but are not limited to,: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons).
  • Other amino acid residues may be encoded similarly by multiple nucleotide sequences.
  • the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.
  • areas of high similarity among pheromone receptors may differ in amino acid sequences such that they share many, but not all, amino acids. Their nucleotide sequences all differ accordingly.
  • the invention also provides isolated unique fragments of the cDNAs encoding a G ⁇ 0 + VNO polypeptide selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52, or complements of these sequences.
  • a unique fragment is one that is a 'signature' for the larger nucleic acid. It, for example, is long enough to assure that its precise sequence is not found in molecules outside of the G ⁇ 0 + VNO pheromone receptor nucleic acids defined above.
  • Unique fragments can be used as probes in Southern blot assays to identify such nucleic acids, or can be used as primers in amplification assays such as those employing PCR. As known to those skilled in the art, large probes such as 200 nucleotides or more are preferred for certain uses such as Southern blots, while smaller fragments will be preferred for uses such as PCR. Unique fragments also can be used to produce fusion proteins for generating antibodies or determining binding of the polypeptide fragments, as demonstrated in the Examples, or for generating immunoassay components.
  • unique fragments can be employed to produce nonfused fragments of the G ⁇ 0 + VNO pheromone receptor polypeptides, useful, for example, in the preparation of antibodies, in immunoassays, and as a competitive binding partner of the pheromones and/or other ligands which bind to the G ⁇ 0 + VNO pheromone receptor polypeptides, for example, in therapeutic applications.
  • Unique fragments further can be used as antisense molecules to inhibit the expression of G ⁇ 0 + VNO pheromone receptor nucleic acids and polypeptides, particularly for the insecticide and other fertility control purposes as described in greater detail below.
  • the size of the unique fragment will depend upon its conservancy in the genetic code.
  • some regions of a cDNA selected from the group consisting of SEQ ID NO. 51, 53, 54, 55, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, and 92, that encodes a G ⁇ 0 + VNO polypeptide, and its complement will require longer segments to be unique while others will require only short segments, typically between 12 and 32 nucleotides (e.g.
  • any segment of the region of the cDNAs encoding the full length G ⁇ 0 + VNO polypeptide or their complements, that is 18 or more nucleotides in length will be unique.
  • Those skilled in the art are well versed in methods for selecting such sequences, typically on the basis of the ability of the unique fragment to selectively distinguish the sequence of interest from non-G ⁇ 0 + VNO pheromone receptor nucleic acids.
  • a comparison of the sequence of the fragment to those on known data bases typically is all that is necessary, although in vitro confirmatory hybridization and sequencing analysis may be performed.
  • the invention embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a G ⁇ 0 + VNO pheromone receptor polypeptide, to decrease a pheromone receptor activity (e.g., a ligand binding activity, a signal transduction activity).
  • a pheromone receptor activity e.g., a ligand binding activity, a signal transduction activity.
  • the compositions of the invention are particularly useful in, for example, controlling fertility in livestock and controlling reproduction in rodents or insects by interrupting the normal behaviors of rodents or insects that result in reproduction.
  • antisense oligonucleotide or “antisense” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA.
  • the antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript.
  • the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon the cDNA sequences of Examples 1 and 2 (SEQ ID NOs.
  • antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides (Wagner et al., Nature Biotechnol. 14:840-844, 1996). Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.
  • oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N- terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3 '-untranslated regions may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol.
  • Examples 1 and 2 disclose cDNA sequences (SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 54, and 55), one of ordinary skill in the art may easily derive the genomic DNA corresponding to the cDNA of these cDNAs.
  • the present invention also provides for antisense oligonucleotides which are complementary to the genomic DNA corresponding to a cDNA sequence selected from the group consisting of SEQ ID NOs.
  • the antisense oligonucleotides of the invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors. In preferred embodiments, however, the antisense oligonucleotides of the invention also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.
  • modified oligonucleotide as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide.
  • a synthetic internucleoside linkage i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide
  • Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
  • modified oligonucleotide also encompasses oligonucleotides with a covalently modified base and/or sugar.
  • modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • modified oligonucleotides may include a 2'-O-alkylated ribose group.
  • modified oligonucleotides may include sugars such as arabinose instead of ribose.
  • the present invention contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding pheromone receptor polypeptides, together with pharmaceutically acceptable carriers.
  • Antisense oligonucleotides may be administered as part of a pharmaceutical composition.
  • Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art.
  • the compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors are typically composed of DNA although RNA vectors are also available.
  • Vectors include, but are not limited to, plasmids, phagemids and virus genomes.
  • a cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., ⁇ -galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).
  • Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • a coding sequence and regulatory sequences are said to be "operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • the precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • RNA heterologous DNA
  • RNA heterologous DNA
  • That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.
  • Preferred systems for mRNA expression in mammalian cells are those such as pRc/CMV (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences.
  • pRc/CMV available from Invitrogen, Carlsbad, CA
  • CMV human cytomegalovirus
  • suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein Barr virus (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element.
  • EBV Epstein Barr virus
  • Another expression vector is the pEF-BOS plasmid containing the promoter of polypeptide Elongation Factor 1 ⁇ , which stimulates efficiently transcription in vitro.
  • the plasmid is described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996).
  • Still another preferred expression vector is an adenovirus, described by Stratford-Perricaudet, which is defective for El and E3 proteins (J Clin. Invest. 90:626-630, 1992).
  • adenovirus as an Adeno.PlA recombinant is disclosed by Warnier et al., in intradermal injection in mice for immunization against PI A (Int. J. Cancer, 67:303-310, 1996).
  • the invention also embraces so-called expression kits, which allow the artisan to prepare a desired expression vector or vectors.
  • expression kits include at least separate portions of each of the previously discussed coding sequences. Other components may be added, as desired, as long as the previously mentioned sequences, which are required, are included.
  • the invention also permits the construction of pheromone receptor gene "knock-outs" in cells and in animals, providing materials for studying certain aspects of pheromone receptor binding, signal transduction activity, or function.
  • the invention also provides isolated polypeptides, which include a pheromone receptor polypeptide selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52 and unique fragments of these pheromone receptor polypeptides.
  • a pheromone receptor polypeptide selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52 and unique fragments of these pheromone receptor polypeptides.
  • Such polypeptides are useful, for example, alone or as fusion proteins to generate antibodies.
  • a unique fragment of a pheromone receptor polypeptide in general, has the features and characteristics of unique fragments as discussed above in connection with nucleic acids. As will be recognized by those skilled in the art, the size of the unique fragment will depend upon factors such as whether the fragment constitutes a portion of a conserved protein domain. Thus, some regions of a pheromone receptor polypeptide selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52 will require longer segments to be unique while others will require only short segments, typically between 5 and 12 amino acids (e.g. 5, 6, 7, 8, 9, 10, 11 and 12 amino acids long).
  • Unique fragments of a polypeptide preferably are those fragments which retain a distinct functional capability of the polypeptide.
  • Functional capabilities which can be retained in a unique fragment of a polypeptide include interaction with antibodies, interaction with other polypeptides (G-proteins) or molecules (e.g., a ligand) or fragments thereof, selective binding of nucleic acids or proteins, and enzymatic activity.
  • G-proteins polypeptides
  • a ligand e.g., a ligand
  • a "variant" of a pheromone receptor polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of a pheromone receptor polypeptide.
  • Modifications which create a pheromone receptor variant can be made to a pheromone receptor polypeptide 1) to reduce or eliminate an activity of a pheromone receptor polypeptide, such as a ligand binding activity or a signal transduction activity; 2) to enhance a property of a pheromone receptor polypeptide, such as protein stability in an expression system or the stability of protein-protein binding; or 3) to provide a novel activity or property to a pheromone receptor polypeptide, such as addition of an antigenic epitope or addition of a detectable moiety.
  • a pheromone receptor polypeptide 1) to reduce or eliminate an activity of a pheromone receptor polypeptide, such as a ligand binding activity or a signal transduction activity
  • 2) to enhance a property of a pheromone receptor polypeptide such as protein stability in an expression system or the stability of protein-protein binding
  • Modifications to a pheromone receptor polypeptide are typically made to the nucleic acid which encodes the pheromone receptor polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids or non-amino acid moieties. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition ofa linker molecule, addition ofa detectable moiety, such as biotin, addition ofa fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the pheromone receptor amino acid sequence.
  • variants include pheromone receptor polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, certain amino acids can be changed to enhance expression of a pheromone receptor polypeptide by eliminating proteolysis by proteases in an expression system. Mutations ofa nucleic acid which encode a pheromone receptor polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the variant polypeptide.
  • Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Variant polypeptides are then expressed and tested for one or more activities to determine which mutation provides a variant polypeptide with the desired properties. Further mutations can be made to variants (or to non- variant pheromone receptor polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli, are well known to those of ordinary skill in the art.
  • variants of pheromone receptor polypeptides can be tested by cloning the gene encoding the variant pheromone receptor polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the variant pheromone receptor polypeptide, and testing for a functional capability of the pheromone receptor polypeptides as disclosed herein.
  • the variant pheromone receptor polypeptide can be tested for a ligand binding activity, wherein a ligand to which the receptor binds is contacted with the variant receptor and the amount of ligand binding to the variant receptor is determined using conventional procedures to measure the binding of one molecule to another. Preparation of other variant polypeptides may favor testing of other activities, as will be known to one of ordinary skill in the art.
  • conservative amino acid substitutions may be made in pheromone receptor polypeptides to provide functionally equivalent variants of the foregoing polypeptides, i.e, the variants retain the functional capabilities of the pheromone receptor polypeptides.
  • a "conservative amino acid substitution” refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J.
  • Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • Conservative amino-acid substitutions in the amino acid sequence of pheromone receptor polypeptides to produce functionally equivalent variants of pheromone receptor polypeptides typically are made by alteration of the nucleic acid encoding pheromone receptor polypeptides.
  • substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method described in Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985, or by chemical synthesis ofa gene encoding a pheromone receptor polypeptide. Where amino acid substitutions are made to a small unique fragment of a pheromone receptor polypeptide, such as a ligand binding site peptide, the substitutions can be made by directly synthesizing the peptide.
  • the activity of functionally equivalent fragments of pheromone receptor polypeptides can be tested by cloning the gene encoding the altered pheromone receptor polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered pheromone receptor polypeptide, and testing for a functional capability of the pheromone receptor polypeptides as disclosed herein.
  • Peptides which are chemically synthesized can be tested directly for function, e.g., for binding to a ligand to which the unaltered pheromone receptor is known to bind.
  • the invention permits isolation of the pheromone receptor polypeptides of the Examples.
  • a variety of methodologies well-known to the skilled practitioner can be utilized to obtain isolated pheromone receptor molecules.
  • the polypeptide may be purified from cells which naturally produce the polypeptide by chromatographic means or immunological recognition.
  • an expression vector may be introduced into cells to cause production of the polypeptide.
  • mRNA transcripts may be microinjected or otherwise introduced into cells to cause production of the encoded polypeptide. Translation of mRNA in cell-free extracts such as the reticulocyte lysate system also may be used to produce polypeptide.
  • the isolation of the pheromone receptor gene also makes it possible for the artisan to diagnose a disorder characterized by expression of pheromone receptor .
  • These methods involve determining expression of the pheromone receptor gene, and/or pheromone receptor polypeptides derived therefrom. In the former situation, such determinations can be carried out via any standard nucleic acid determination assay, including the polymerase chain reaction as exemplified in the examples below, or assaying with labeled hybridization probes.
  • the invention also makes it possible to isolate the naturally occurring ligands (pheromones) and other ligands that have a ligand binding domain, namely, by the binding of such molecules to the pheromone receptor polypeptides (or fragments thereof containing a ligand binding site). Binding of the receptors to a ligand can be accomplished by introducing into a biological system in which the proteins bind (e.g., a cell) a molecule that includes a binding domain (putative ligand) in an amount sufficient to detect the binding.
  • a biological system in which the proteins bind (e.g., a cell) a molecule that includes a binding domain (putative ligand) in an amount sufficient to detect the binding.
  • the invention also provides agents such as binding polypeptides which bind to pheromone receptor polypeptides and/or to complexes of pheromone receptor polypeptides and their ligand binding partners.
  • binding agents can be used, for example, in screening assays to detect the presence or absence of pheromone receptor polypeptides and complexes of pheromone receptor polypeptides and their ligand binding partners and in purification protocols to isolate pheromone receptor polypeptides and complexes of pheromone receptor polypeptides and their ligand binding partners.
  • Such agents also can be used to inhibit the native activity of the pheromone receptor polypeptides or their ligand binding partners, for example, by binding to such polypeptides, or their binding partners or both.
  • the invention therefore, embraces peptide binding agents which, for example, can be antibodies or fragments of antibodies having the ability to selectively bind to pheromone receptor polypeptides.
  • Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology. Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) 77ze Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford).
  • the pFc' and Fc regions are effectors of the complement cascade but are not involved in antigen binding.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.
  • Fab fragments consist ofa covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and F
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDR3 complementarity determining regions
  • non-CDR regions of a mammalian antibody may be replaced with similar regions of nonspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody.
  • This is most clearly manifested in the development and use of "humanized” antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody.
  • PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin.
  • Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies.
  • the present invention also provides for F(ab') 2 , Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab') 2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes so-called single chain antibodies.
  • polypeptides of numerous size and type that bind specifically to pheromone receptor polypeptides, and or complexes of both pheromone receptor polypeptides and their ligand binding partners.
  • polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries.
  • Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties.
  • Phage display can be particularly effective in identifying binding peptides useful according to the invention. Briefly, one prepares a phage library (using e.g. ml3, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures. The inserts may represent, for example, a completely degenerate or biased array. One then can select phage-bearing inserts which bind to the pheromone receptor polypeptide. This process can be repeated through several cycles of reselection of phage that bind to the pheromone receptor polypeptide. Repeated rounds lead to enrichment of phage bearing particular sequences. DNA sequence analysis can be conducted to identify the sequences of the expressed polypeptides.
  • the minimal linear portion of the sequence that binds to the pheromone receptor polypeptide can be determined.
  • Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the pheromone receptor polypeptides.
  • the pheromone receptor polypeptides of the invention, or a fragment thereof can be used to screen peptide libraries, including phage display libraries, to identify and select peptide binding partners of the pheromone receptor polypeptides of the invention.
  • Such molecules can be used, as described, for screening assays, for purification protocols, for interfering directly with the functioning of pheromone receptor and for other purposes that will be apparent to those of ordinary skill in the art.
  • a pheromone receptor polypeptide, or a fragment which contains the ligand binding site also can be used to isolate naturally-occurring ligands and other binding partners of the receptors of the invention.
  • an isolated pheromone receptor can be used to isolate ligands that bind to the receptor binding site by immobilizing a receptor (or fragment containing the ligand binding site) on a chromatographic media, such as polystyrene beads, or a filter, and using the immobilized polypeptide to isolate molecules that bind to this affinity matrix in accordance with standard procedures for affinity chromatography.
  • the invention embraces the use of the pheromone receptor cDNA sequences in expression vectors, as well as to transfect host cells and cell lines, be these prokaryotic (e.g., E. coli), or eukaryotic (e.g., CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells).
  • prokaryotic e.g., E. coli
  • eukaryotic e.g., CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells.
  • oocytes mammalian cells such as mouse, hamster, pig, goat, primate, etc. They may be of a wide variety of tissue types, and include primary cells and cell lines.
  • the expression vectors require that the pertinent sequence, i.e., those nucleic acids described supra, be operably linked to a promoter.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.
  • the therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
  • a preferred route of administration is by pulmonary aerosol.
  • Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences. 18th edition, 1990, pp 1694-1712; incorporated by reference).
  • Those of skill in the art can readily determine the various parameters and conditions for producing antibody aerosols without resort to undue experimentation. When using antisense preparations of the invention, slow intravenous administration is preferred.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the preparations of the invention are administered in effective amounts.
  • An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, produces the desired response in the condition being treated, e.g., modifying fertility or pheromone-mediated behaviors that are related to reproduction or aggression. For example, this can involve the use of the compounds of the invention as pesticides to slow or halt insect or rodent behaviors that result in reproduction.
  • this can involve the use of the compounds of the invention as agents for controlling fertility in animals (e.g., livestock, domestic animals), by providing compounds which inhibit or stimulate the behaviors in such animals that result in reproduction or agression. This can be monitored by routine methods, e.g., observing the behavior in the animal (vertebrate or invertebrate) recipient.
  • the invention also contemplates gene therapy, e.g., to prepare an animal model for studying the conditions and behaviors (e.g., fertility, aggression) that are pheromone receptor- mediated.
  • the procedure for performing ex vivo gene therapy is outlined in U.S. Patent 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents.
  • the invention further provides efficient methods of identifying pharmacological agents or lead compounds for agents active at the level of a pheromone receptor or pheromone receptor fragment modulatable cellular function.
  • functions include ligand binding activity.
  • the screening methods involve assaying for activation of pheromone receptors or assaying for compounds which interfere with a pheromone receptor activity such as pheromone receptor binding to its cognate ligand. Such methods are adaptable to automated, high throughput screening of compounds.
  • target therapeutic indications for pharmacological agents detected by the screening methods that block pheromone receptor activity are limited only in that the target cellular function be subject to modulation by alteration of the formation of a complex comprising a pheromone receptor polypeptide or fragment thereof and one or more natural pheromone receptor ligands.
  • Target indications include cellular processes modulated by pheromone receptor signal transduction following receptor-ligand binding.
  • assays for pharmacological agents including, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays, cell- based assays such as two- or three-hybrid screens, expression assays, activation of G-proteins, etc.
  • three-hybrid screens are used to rapidly examine the effect of transfected nucleic acids on the intracellular binding of pheromone receptor or pheromone receptor fragments to specific extracellular targets (e.g., ligands in biological samples, such as urine, vaginal fluid, or in combinatorial libraries) .
  • Pheromone receptor fragments used in the methods when not produced by a transfected nucleic acid are added to an assay mixture as an isolated polypeptide.
  • the assay can be used to screen putative ligands for their ability to bind to the receptor.
  • Pheromone receptor polypeptides preferably are produced recombinantly, although such polypeptides may be isolated from biological extracts.
  • Recombinantly produced pheromone receptor polypeptides include chimeric proteins comprising a fusion of a pheromone receptor protein with another polypeptide.
  • a polypeptide fused to a pheromone receptor polypeptide or fragment may also provide means of readily detecting the fusion protein, e.g., by immunological recognition or by fluorescent labeling.
  • a screening assay mixture includes a binding partner for the receptor, e.g., a naturally occurring ligand that is capable of binding to the pheromone receptor or, alternatively, is comprised of an analog which mimics the pheromone receptor binding properties of the naturally occurring ligand for purposes of the assay.
  • the screening assay mixture also comprises a candidate pharmacological agent (e.g., a putative receptor agonist or antagonist).
  • a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection.
  • Candidate agents encompass numerous chemical classes, although typically they are organic compounds.
  • the candidate pharmacological agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500.
  • Candidate agents comprise functional chemical groups necessary for structural interactions with polypeptides and or nucleic acids, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups.
  • the candidate agents can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups.
  • Candidate agents also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like.
  • the agent is a nucleic acid
  • the agent typically is a DNA or RNA molecule, although modified nucleic acids as defined herein are also contemplated.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the agents.
  • reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components.
  • reagents that improve the efficiency of the assay such as protease, inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
  • the mixture of the foregoing assay materials is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the pheromone receptor polypeptide specifically binds the cellular binding target, a portion thereof or analog thereof.
  • the order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4°C and 40 °C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours.
  • a separation step is often used to separate bound from unbound components.
  • the separation step may be accomplished in a variety of ways. Conveniently, at least one of the components is immobilized on a solid substrate, from which the unbound components may be easily separated.
  • the solid substrate can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc.
  • the substrate preferably is chosen to maximum signal to noise ratios, primarily to minimize background binding, as well as for ease of separation and cost.
  • Separation may be effected for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent.
  • the separation step preferably includes multiple rinses or washes.
  • the solid substrate is a microtiter plate
  • the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein, etc.
  • the solid substrate is a magnetic bead
  • the beads may be washed one or more times with a washing solution and isolated using a magnet.
  • Detection may be effected in any convenient way for cell-based assays such as two- or three-hybrid screens.
  • the transcript resulting from a reporter gene transcription assay of Pheromone receptor polypeptide binding to a target molecule typically encodes a directly or indirectly detectable product, e.g., ⁇ -galactosidase activity, luciferase activity, and the like.
  • a wide variety of cell based assays for G-protein coupled receptors could also be employed for detection of molecules that stimulate (agonsists) pheromone receptors or block (agonists) that stimulation by natural ligands or agonists.
  • Pheromone receptor polypeptides or chimeric receptors composed only in-part of a pheromone receptor could be employed in these assays.
  • the chimeric receptors might, for example, contain part of another G-protein coupled receptor such that binding ofa ligand to the pheromone receptor binding domain results in coupling to a particular G-protein where activation could be easily assayed.
  • one of the components usually comprises, or is coupled to, a detectable label.
  • labels can be used, such as those that provide direct detection (e.g., radioactivity, luminescence, optical or electron density, etc), or indirect detection (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, etc.).
  • the label may be bound to a pheromone receptor binding partner (ligand), or incorporated into the structure of the binding partner.
  • the label may be detected while bound to the solid substrate or subsequent to separation from the solid substrate.
  • Labels may be directly detected through optical or electron density, radioactive emissions, nonradioactive energy transfers, etc. or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, etc.
  • Methods for detecting the labels are well known in the art.
  • the invention provides pheromone receptor -specific binding agents, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development, including the development of pesticides and other agents for controlling fertility and reproduction (or related behaviors) in animals.
  • pheromone receptor-specific pharmacological agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with improper utilization of a pathway involving pheromone receptor.
  • Novel pheromone receptor-specific binding agents include pheromone receptor-specific antibodies and other natural intracellular binding agents identified with assays such as two hybrid screens, and non-natural intracellular binding agents identified in screens of chemical libraries and the like.
  • the specificity of pheromone receptor binding to a binding agent is shown by binding equilibrium constants.
  • Targets which are capable of selectively binding a pheromone receptor polypeptide preferably have binding equilibrium constants of at least about IO 7 M "1 , more preferably at least about 10 8 M "1 , and most preferably at least about IO 9 M "1 .
  • the wide variety of cell based and cell free assays may be used to demonstrate pheromone receptor - specific binding.
  • Cell based assays include one, two and three hybrid screens, assays in which pheromone receptor -mediated transcription is inhibited or increased activation of G-proteins, etc.
  • Cell free assays include pheromone receptor -protein binding assays, immunoassays, etc.
  • Other assays useful for screening agents which bind pheromone receptor polypeptides include fluorescence resonance energy transfer (FRET), and electrophoretic mobility shift analysis (EMSA).
  • FRET fluorescence resonance energy transfer
  • ESA electrophoretic mobility shift analysis
  • nucleic acids of the invention may be introduced in vitro or in vivo in a host.
  • Such techniques include transfection of nucleic acid-CaPO 4 precipitates, transfection of nucleic acids associated with DEAE, transfection with a retrovirus including the nucleic acid of interest, liposome mediated transfection, and the like.
  • a vehicle used for delivering a nucleic acid of the invention into a cell e.g., a retrovirus, or other virus; a liposome
  • a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid delivery vehicle.
  • proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake.
  • proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like.
  • Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acids. Examples
  • VNOs Male mouse (C57BL/6J) VNOs were minced, incubated in Trypsin-EDTA (Gibco- BRL/LTI, Rockville, Maryland), and triturated to obtain dissociated cells. The cells were centrifuged (1000 RPM, 5 min) and resuspended in phosphate buffered saline + 0.1% bovine serum albumin. Individual cells that appeared to be neurons were transferred to separate tubes with a microcapillary pipet. cDNAs were prepared from each cell and amplified according to Brady and Iscove (Methods in Enzymology, 1993, 225:611-621) with minor modifications.
  • cDNAs were prepared from the 3' ends of mRNAs by reverse transcription with an oligo (dT) primer, and a poly dA stretch was added to each cDNA with terminal transferase.
  • the cDNAs were then amplified by PCR with one of two primers, ALl (ATTGGATCCAGGCCGCTCTGGACAA AATATGAA TTC(T) ( SEQ. ID. No. 56) (Dulac and Axel, Cell, 1995, 83:195-206 or AL3 (GGCACATGG ACGAAATCTTGGTACTCTTCAGAATTC(T), (SEQ. ID. No.
  • Hyb Buffer 0.5M sodium phosphate buffer (pH7.3), 4% SDS, 1% bovine serum albumin (BSA)
  • BSA bovine serum albumin
  • the region (D10-TM7) of one clone (D10) that showed homology to TM7 of the CSR (SEQ ID NO. 59) was then used to screen IVNO (55°C, Hyb Buffer), yielding a variety of VR cDNA clones.
  • Additional clones were obtained from IVNO using probes prepared from clones previously isolated, or from PCR products obtained by amplification of mouse genomic DNA or VNO cDNA with degenerate primers (Buck, L., et al., Cell, 1991, 65:175-187) matching conserved motifs in the VRs. Some PCR products were also cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced.
  • Random-primed cDNA prepared from male or female C57BL/6J mouse VNO RNAs were used in PCR reactions with degenerate primers (Buck and Axel, Cell 1991, 65:175-187) matching conserved VR motifs to amplify VR sequences corresponding to amino acids 33-772 in VRl (SEQ ID NO.2).
  • Nested PCR was performed with a 1/1000 dilution of the first PCR reaction and primer pairs matching regions of putative exons 1 and 6 in specific VR cDNA clones.
  • Blots prepared from size-fractionated, nested PCR products were hybridized (70°C, Hyb buffer containing lOO ⁇ g/ml herring sperm DNA (Sigma, St Louis, MO)) to probes prepared from the PCR products of the cDNA clones.
  • Northern and Southern blots and genomic library screens Northern Blots: One ⁇ g of PolyA + RNA prepared from mouse VNO and OE, or purchased from Clontech (other tissue RNAs), was size fractionated on formaldehyde gels, and blotted (see above) (Berghard and Buck, J Neurosci, 1996, 16:909-918). The blot was hybridized (70°C, Hyb Buffer) with a 32 P-labeled probe prepared from the regions of cDNAs VRl , VR2, VR4, and VRl 5 corresponding to that encoding amino acids 33-772 in VRl (SEQ ID NO. 1).
  • Genomic library screens to determine VR gene number A mouse genomic library was screened separately at 70°C or 55°C (see above) with different 32 P-labeled probes.
  • Probe 1 a mix of segments of cDNAs VRl (SEQ ID NO. 1), VR2 (SEQ ID NO. 3), VR4 (SEQ ID NO. 7), and VRl 5 (SEQ ID NO. 29) encoding the region corresponding to amino acids 619-772 of VRl (SEQ ID NO. 2).
  • Probes 2-6 Segments of VR genes obtained from mouse genomic DNA by PCR with degenerate primers matching conserved VR sequence motifs. The PCR segments corresponded to the following amino stretches in VRl (SEQ ID NO.
  • amino acids 191-397, 565-825, 637-825, 637-804, and 619-784 amino acids 191-397, 565-825, 637-825, 637-804, and 619-784.
  • degenerate oligonucleotide primer pairs used included: for amino acids 191-397: 5' prime ⁇ (GCT)TI(CT)A(CT) CA(AG)(AG)TIGCI(AC)CIAA(AG)GA(CT)AC (SEQ ID NO. 60),
  • 5' primer ATI(AT)(GC)I(CT)TI(AG)TITT(CT)TG(CT)TT(CT)(CT)TITG (SEQ ID NO. 64),
  • 3' primer AAIGTIA(CT)CCAIACI(GC)(AT)(AG)CA(AG)AAIAC (SEQ ID NO. 67), wherein all primers are in a 5'-3' direction, Inosine.
  • Southern blots of genomic DNA from C57BL/6J and Mus spretus (Jackson Labs) digested with different restriction enzymes were prepared and probed with specific VR cDNA probes as described above.
  • G ao and G ai2 probes hybridized to different OMP+ samples, allowing us to identify samples that were derived from G ao + VNs.
  • VN14 G m + single-cell cDNA samples
  • the 5' end of the D10 cDNA contained a short open reading frame, which encoded a protein fragment with homology to transmembrane domain 7 (TM7) of the calcium sensing receptor (CSR), a G protein-coupled receptor (GPCR) (Brown et al, Nature, 1993, 366:575-580).
  • TM7-related region of D10 D10-TM7 was hybridized at reduced stringency (55°C) to the original panel of single-cell cDNAs, it labeled many of the G m + samples, but none of G ⁇ ones (except the one that was also G ao +, and was probably derived from two cells). Since D10 labeled only a small percentage of VNs in tissue sections under high stringency conditions, this suggested that many G ao + neurons express a gene related to D10, but not identical to it.
  • GPCR G protein- coupled receptor
  • VRs VNO receptors
  • CSR calcium sensing receptor
  • mGluRs metabotropic glutamate receptors
  • the most highly related molecule is the CSR; for example, VRl is 31% identical to rat CSR (Riccardi et al., Proc. Natl.
  • the VRs comprise a distinct family of receptors, which share novel sequence motifs, and are more related to one another than they are to other receptors.
  • two divergent VRs VRl (SEQ ID NO. 1, 2) and VR4 (SEQ ID NO. 7, 8), are 70% identical in TM1- TM7, and 48% identical overall.
  • the VRs are unusual among GPCRs in having an extremely long N-terminal extracellular domain ( Figures 1 and 2).
  • the size of the N-terminal extracellular domain of VRs far exceeds that of ORs and VNRs (-12-28 amino acids) ( Figure 2).
  • the VRs are most variable in the N-terminal domain (25% identical residues compared to 51% in TM1-TM7).
  • the ligand binding site is thought to reside in the large N- terminal domain (O'Hara et al., Neuron, 1993, 11 :41-52; Takahashi et al, J Biol. Chem., 1993, 268:19341-19345). If this is also true of VRs, the accentuated diversity of the N-terminal domain may reflect an ability to recognize diverse pheromonal ligands.
  • VRl SEQ ID NOs. 1 , 2)
  • VR2 SEQ ID NOs. 3, 4
  • VR3 SEQ ID NOs. 5, 6
  • VR4 SEQ ID NOs. 7, 8
  • VR5 SEQ ID NOs. 9, 10
  • VR6 SEQ ID NOs. 11, 12
  • VR7 SEQ ID NOs. 13, 14
  • RNA used for library construction and PCR came from an inbred mouse strain (C57BL/6J), so they cannot be allelic variants.
  • the error rates of reverse transcriptase (or Taq polymerase) cannot account for the extent to which the cDNAs differ.
  • VR4 SEQ ID NOs. 7, 8
  • VR5 SEQ ID NOs. 9, 10
  • cDNAs are 99% identical in nucleotide sequence, but the reverse transcriptase used to prepare them has an error rate of only 3.6 x 10 5 /bp (Ji, J., et al., Biochemistry, ,1992, 31 :954-958).
  • Variant VR mRNAs could derive either from different genes, or from the same gene by alternative RNA splicing. Consistent with the latter possibility, two pairs of cDNAs that we sequenced VR8 (SEQ ID NOs. 15, 16) and VR9 (SEQ ID NOs. 17, 18), and VR10 (SEQ ID NOs. 19, 20) and VRl 1 (SEQ ID NOs. 21, 22) were identical in nucleotide sequence, but were missing different segments. However, when we used RT-PCR to amplify VNO mRNA sequences encoding 5 different VRs, we obtained one major PCR product in each case, regardless of whether the RNA used was from male or female mice.
  • the size of the major product corresponded to a complete VR, even though one of the cDNAs (but not the PCR product) contained an intron (#5).
  • the major PCR product was even smaller, and was found to lack two exons. Although PCR products of a smaller size were also seen in these experiments, they were much less abundant.
  • VR genes may be expressed pseudogenes, which either lack one or more exons, or have mutations that prevent proper RNA splicing.
  • pseudogenes which either lack one or more exons, or have mutations that prevent proper RNA splicing.
  • some truncated VRs that lack transmembrane domains could conceivably be secreted pheromone-binding proteins.
  • the G ⁇ , and G ⁇ probes gave patterns of hybridization similar to those we had previously seen (Berghard, A., et al, J Neurosci, 1996, 16:909-918).
  • the G ao probe hybridized to a wavy stripe of VNO neurons in the basal (lower) region of the VNO neuroepithleium, whereas the G ai2 probe hybridized to an adjacent stripe of neurons in the apical (upper) part of the neuroepithelium.
  • the waviness of the two zones appears to be caused by the periodic presence of blood vessels near the base of the epithelium (Berghard, A., et al, J Neurosci, 1996, 16:909- 918).
  • Approximately 57% of VNs were labeled by the G ai2 probe and 43% were labeled by the G ⁇ probe.
  • the single layer of supporting cells located just beneath the epithelial surface was not labeled by either probe.
  • Each of the VR probes hybridized to a small percentage (2.4-5.7%)) of VNs that appeared to be restricted to the basal, G ao + zone of the VNO neuroepithelium. Labeled neurons were scattered throughout the anterior-posterior and dorsal-ventral extent of the G ao + zone. Small clusters of labeled cells were somtimes seen, particularly with the VR2 probe The mixed probe labeled a larger percentage of VNs (10.6%) that was almost equal to the sum of the percentages labeled by its individual components (10.8%). Thus different G ao + neurons must express different VRs.
  • the size of the VR multigene family To investigate the size of the VR gene family, we hybridized several different mixed VR gene probes to a mouse genomic library, using high (70°C) or low (55°C) stringency conditions. A probe prepared from the membrane spanning regions (putative exon 6) of several different cDNA clones hybridized to 59 and 98 clones per haploid genome equivalent, at high and low stringency, respectively. To obtain probes that were potentially more diverse, we amplified internal segments of putative exon3 or 6 from genomic DNA by PCR with degenerate primers. At high stringency, these probes hybridized to 60-140 clones per haploid equivalent. These results indicate that there are as many as 140 VR genes in the mouse genome.
  • the VR probes that we used for in situ hybridization each labeled a small percentage of neurons.
  • each VR gene may be expressed in only -1.1-1.9% of G ao + VNs. Since there appear to be 60-140 VR genes in the mouse genome, this suggests that each G ao + VNO neuron may express only one, or at most a few, VR genes.
  • DNA Mapping Panel which allows the mapping of mouse genes using interspecies mouse crosses.
  • Probes prepared from the 3' untranslated regions of VR2 (SEQ ID NO. 3) or VR4 cDNAs were first hybridized to Southern blots of genomic DNAs from two mouse species, C57BL/6J and Mus spretus, which had been digested with different restriction enzymes. Eco RI digests showed a number of restriction length polymorphisms with both VR probes. The VR probes were then hybridized to Eco Rl-digested DNAs from a large panel of different backcross mice ((C57BL/6J x M. spretus) x M. spretus).
  • the patterns of inheritance of the polymorphic fragments recognized by the two VR probes allowed us to assign chromosomal locations to approximately 9 VR genes.
  • Using the VR4 (SEQ ID NO. 7) probe we could follow the inheritance of 4 polymorphic restriction fragments. All of these cosegregated in the backcrosses, and mapped to the proximal end of chromosome 7 (near D7Bir5). Five restriction fragments were followed for the VR2 (SEQ ID NO. 3) probe. Again, all of the restriction fragments cosegregated, allowing us to map the VR2 (SEQ ID NO. 3) fragments to the distal end of chromosome 4 (near D4Birl).
  • the cosegregating fragments can be no more than 3.8 cM from one another.
  • VR genes are located near the ends of at least two different mouse chromosomes. They also indicate that highly related VR genes are clustered at the same chromosomal locus, as previously seen in our studies and others (Ben-Arie et al, Human Molecular Genetics, 1994, 3:229-235.).
  • the VR4 gene subfamily appears to be closely linked to one OR gene locus, (olfR5 )
  • VNOs were dissected from adult (7- to 8-week-old) male Lewis rats (Sprague-Dawley).
  • Single-cell cDNA synthesis and amplification were performed and checked according to Dulac and Axel (Ce/7,1995, 83:195-206). Southern blot analysis of single-cell cDNA was used to detect expression of tubulin, OMP, Go, and Gi 2 ⁇ (Dulac and Axel, Cell, 1995, 83:195-206). Eighteen cDNAs showed strong hybridization with tubulin and OMP probes, indicating that they originated from mature neurons, and were selected for further study. Cells VN3 and VN13 exhibited high levels of Go expression, whereas VN10 showed presence of Gi 2 ⁇ , indicating the origin of these cells from two distinct regions of the VNO neuroepithelium. VN13 single-cell cDNA library was prepared according to Dulac and Axel (Cell, 1995, 83:195-206).
  • Plaque-forming units (12 x 10 3 ) from the VN13 library were plated at low density, and duplicate filters (Hybond N + , Amersham) were hybridized with probes generated from VN 10 and VN13 single-cell cDNAs, following the procedure described in Dulac and Axel, Cell, 1995, 83 : 195-206.
  • Ten phage plaques were detected that showed a positive signal unique to the VN13 probe. These plaques were purified, and the corresponding phage inserts were amplified by PCR, run on 1.5% agarose gel, blotted onto nylon filter, and hybridized with the VN10, VN3, and VN13 single-cell cDNA probes.
  • Go-VNl 3 A present at the frequency of 0.1% in the VN13 single-cell cDNA library, was selected and in vivo excised to generate the pBlueScriptSK(-) phagemid.
  • Phages (7.2 x 10 5 ) of the female rat VNO library were further screened with the Go-VN13B (SEQ ID NO.
  • cDNA probe under low stringency conditions hybridization was carried out at 55 °C for 24 hr, and the filters were washed three times at 55 °C for 30 min in 0.5x SSC and 0.5% SDS. A total of 75 positive phages were identified and the corresponding inserts were amplified by PCR and analyzed by Southern blot using the Go-VNl 3B (SEQ ID NO. 49) probe at both high (65 °C) and low (55 °C) stringency. This led to the identification of 22 cDNA clones with insert sizes longer than 3 kb. Among those, six distinct subfamilies were defined by absence of cross-hybridization under stringent conditions of hybridization and washing.
  • Go-VNl to Go-VN ⁇ SEQ ID NOs. 33, 35, 37, 39, 41, 43
  • Go-VN13C SEQ ID NO. 47
  • Go-VN13B SEQ ID NO. 49
  • NMDQCANCPEYQYANTEKNKCIQKGVIVLSYEDPLGMALALIAFCFSAFTV (SEQ ID NO. 58) in Go-VN13B (SEQ ID NO. 49) and is replaced by an M at position 552 in Go-VN13C (SEQ ID NO. 48).
  • DNA sequencing was performed using ABI Prism dye terminator cycle ready reaction (Perkin Elmer, Norwalk, CT ) according to manufacturer's protocol. Samples were run on an ABI Prism 310 Genetic Analyzer (Perkin Elmer, Norwalk, CT). Sequence homologies were determined using the BLAST system (NIH network service). Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis were obtained with the Mac Vector sequence analysis software (Oxford Molecular Group).
  • VNOs were dissected from adult male (8- to 9-week-old), adult female (9- to 11 -week-old), and young (1 -week-old) rats. Tissues were embedded in Tissue-Tek OCT. Antisense and sense digoxigenin-labeled probes were generated from the full-length cDNAs encoding for Go, Gi 2o , Go-VN13B (SEQ ID NO. 49), and Go-VNl to G0-VN6 (SEQ ID NOs. 33, 35, 37, 39, 41, 43), as well as from the 3' untranslated regions of the Go-VNl to G0-VN6 clones.
  • Average data for Go-VNl and Go-VN3 to G0-VN6 were obtained from six to eight VNO sections, corresponding to four individuals analyzed in two independent experiments.
  • Genomic DNA prepared from Lewis rat (Sprague-Dawley) liver, was digested with the restriction enzymes EcoRI and BamHI, size fractionated on 0.8% agarose gels, and blotted onto nylon membrane (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989). Membranes were cross-linked under UV light, hybridized overnight at both high (68 °C) and low (55 °C) stringency in hybridization buffer, and washed as described above.
  • 32 P-labeled probes were generated by random priming, using the following DNA templates: EcoRI-EcoRV, Notl-Nsil, EcoRI-Sall, Pstl-Ndel, Xbal-HincII, and EcoRI-Nsil fragments of Go-VNl to Go-VN ⁇ (SEQ ID NOs. 33, 35, 37, 39, 41, 43), respectively; a full-length (425 bp) insert of Go-VN13A; and a cDNA fragment including the seven transmembrane domains of Go-VN 13B (SEQ ID NO. 49).
  • Plaque-forming units (3 x 10 5 ) from rat and human genomic libraries (Stratagene, La Jolla, CA) were screened at low stringency (55 °C) using a mix of 32 P-labeled probes prepared from fragments of Go-VNl to Go-VN ⁇ (SEQ ID NOs. 33, 35, 37, 39, 41, 43) encompassing the transmembrane domains 2 to 7.
  • VNO Neuroepithelium Expresses Two Independent Families of Pheromone Receptors
  • VN10 VN10
  • VN10 VNO neuron cDNA
  • a 425 bp cDNA (Go-VN 13 A) present at a frequency of 0.1% in the VN13-cDNA library showed selective hybridization with VN13 cell probe.
  • Two cDNAs of longer size, Go-VNl 3B (SEQ ID NO. 49) and Go-VNl 3C (SEQ ID NO. 47) were subsequently isolated from a cDNA library prepared from dissected adult VNOs and showed 90% sequence similarity with Go-VN 13 A.
  • Recombinant phages from a VNO cDNA library were screened at low stringency with the Go-VNl 3B (SEQ ID NO. 49) DNA probe. Six distinct gene subfamilies were isolated that showed no cross-hybridization under stringent conditions of hybridization and washing. cDNAs Go-VNl to G0-VN6, each representative ofa subfamily, were fully sequenced (SEQ ID Nos 33, 35, 37, 39, 41 and 43).
  • sequence (amino acid 532; indicated by slash bar in Fig. 3) indicated that this transcript is unable to generate a functional protein.
  • Go-VN4 (SEQ ID NO. 40), Go-VN7 (SEQ ID NO. 46), and Go-VNl 3C (SEQ ID NO. 50) by an initial hydrophobic 21 amino acid segment characteristic of eukaryotic signal sequences.
  • a cluster of seven hydrophobic regions representing potential membrane-spanning helices and typical of the G protein-coupled receptor superfamily is followed by a short hydrophilic sequence that indicates a potential intracytoplasmic C-terminal domain.
  • a database search indicated the presence of sequence motifs common to Ca2 + -sensing and metabotropic glutamate (mGluR) receptors (Houamed, K., et al., Science, 1991, 252:1318-1321; Masu, M., et al., Nature, 1991, 349:760-765; Brown, E., et al., Nature, 1993, 366:575-580 ; Pollak, M., et al., Cell, 1993 75 : 1297- 1303).
  • mGluR metabotropic glutamate
  • Pairwise sequence alignments reveal 18% to 23%) sequence identity between the rat Ca2 + -sensing receptor and the most distant (Go-VN3, SEQ ID Nos.37, 38) and the closest (Go-VNl, SEQ ID NOs. 33, 34) Go-VN sequences, respectively. Sequences of rat mGluRl and Go-VN cDNAs appear more distantly related. Several localized regions showed a more pronounced degree of similarity, including a cysteine-rich sequence just preceding the first transmembrane domain (amino acid 206 to 260 in Go-VNl, SEQ ID NO.
  • the N-terminal and first transmembrane domains show little degree of homology.
  • the second intracellular loop is involved in providing specificity for G-protein coupling (Gomeza, J., et al., J. Biol. Chem., 1996, 271:2199-2205), enabling different classes of mGluR receptors to activate phospholipase C or to inhibit adenylyl cyclase.
  • Go-VN this domain is rich in basic residues, as expected for potential G-protein coupling, and shows closer resemblance to the class II and III mGluRs that were shown to couple to Go and Gi subunits.
  • the six Go-VN sequences share between 42%o and 75% sequence identity.
  • Regions of Go-VN proteins downstream of transmembrane domain 2 are nearly identical in all VNO receptor sequences. In contrast, N-terminal extracellular regions and first transmembrane domains are quite divergent.
  • Go-VN cDNA Sequences Two unusual features were observed in the sequence of some Go-VN cDNAs.
  • Go-VNl SEQ ID NO. 33
  • Go-VN3 SEQ ID NO. 37
  • stretches of open reading frame can be found in the 5' extremity of the cDNAs that generate polypeptide sequences of 310 and and 152 amino acids, respectively, which are interrupted by a frameshift in Go-VNl and by an insertion of 500 nucleic acids in Go-VN3.
  • the prospective receptor protein sequences indicated for Go-VNl (SEQ ID NO. 33) and Go-VN3 (SEQ ID NO. 37) Fig.
  • Go-VN7 (SEQ ID NO. 45) and Go-VNl 3C (SEQ ID NO. 47) cDNAs show a similar deletion of 150 bp located at the exact same position in the sequence. Strikingly, the 150 bp deletion does not alter the open reading frame but generates a gap that encompasses 34 amino acids upstream of the first transmembrane domain and most of the first transmembrane domain itself. Hydropathy analysis of Go-VN7 (SEQ ID NO. 46) and Go-VNl 3C (SEQ ID NO.
  • a direct estimate of the size of the Go-VN receptor gene family was obtained by low stringency screening of a rat genomic library. PCR amplification on genomic DNA had indicated that receptor genes are devoid of introns in the region encompassing transmembrane domains 2 to 7, enabling us to deduce directly the number of genes present in the rat genome.
  • a mix of 32 P-labeled DNA probes prepared from the six Go-VN cDNA fragments identified 110 positive clones per haploid genome, indicating that the family of Go-VN receptors may consist of 100 genes.
  • the pattern of expression of the Go-VN receptor genes was examined by in situ hybridization with digoxigenin-labeled RNA antisense probes. No signal was observed after hybridizing the mix of Go-VNl to G0-VN6 (SEQ ID NOs. 33, 35, 37, 39, 41 and 43) receptor probes to sections of muscle, testis, brain, or whole head.
  • the adult olfactory epithelium was also consistently negative, although rare positive cells (one to three cells per section) were observed in the olfactory neuroepithelium of El 9 rat embryo.
  • strong signals were observed when antisense receptor RNA probes were hybridized to VNO neuroepithelium.
  • each one of the Go-VN probes detects small subsets of VNO sensory neurons. When hybridization and washing were performed at lower temperature, the number of faintly labeled neurons increased, revealing cross- hybridization to more distant receptor genes.
  • cDNA clones Go-VNl to G0-VN6 label 1.9%, 3.6%, 6.1%), 0.4%), 3.5%), and 1.3% of the VNO sensory neurons, respectively.
  • the mix of all six Go-VN RNA probes labels 19% of the cells. This number is similar to the sum of labeled neurons detected with the six individual Go-VN probes (17%), indicating that probes representing the six receptor subfamilies recognize distinct populations of VNO sensory neurons. Spatial Distribution of Go-VN Receptor Transcripts Positive neurons identified with each of the Go-VN probes were randomly distributed along the anteroposterior and dorso-ventral axis of the VNO neuroepithelium.
  • RNA probes recognize cells that are preferentially localized in the most basal two-thirds of the neuroepithelium corresponding to the zone of Go expression.
  • careful examination of adjacent cross-sections of vomeronasal neuroepithelium labeled with each of the Go-VN probes reveals a well-organized spatial distribution of receptor expression. Different receptors appear preferentially localized in radial zones that define a series of hemiconcentric rings of distinct diameters. This pattern is observed along the entire length of the VNO and is conserved in all animals analyzed.
  • the Go-VN3 (SEQ ID NO. 37) probe recognizes a subset of neurons that are confined to the most basal third of the VNO neuroepithelium.
  • Go-VNl SEQ ID NO. 33
  • Go-VN4 SEQ ID NO. 39
  • Go-VN5 SEQ ID NO. 41
  • RNA probes identify cells restricted to a hemiconcentric zone immediately apical to the area of Go-VN3 expression
  • Go-VN2 identifies cells apposed to the apical layer of supporting cells.
  • G0-VN6 in turn is found only in sparse cells immediately apposed to the basal membrane. This is best seen in a statistical representation of Go-VN receptor localization collected from VNO sections and multiple animals that shows a striking conservation of these patterns.
  • Go-VN cDNAs appears restricted to one of three circumscribed areas of the VNO neuroepithelium in a manner quite reminiscent of the odorant receptor gene expression in four zones of the MOE (Ressler, K., et al., Cell, 1993, 73:597-609 ; Vassar, R., et al., Cell, 1993, 74:309-318).
  • Go-VN3 (SEQ ID NO. 37) and G0-VN6 (SEQ ID NO. 43) transcripts show a clear segregation in the most basal region of the VNO neuroepithelium, the sequence anomalies found in both transcripts leave the functionality of this area of the neuroepithelium as an open question.
  • Go-VN2 appears expressed in a large and centrally located region comprising one-third of the neuroepithelium.
  • the same probe recognizes in males a cohort of cells in the most apical side of the neuroepithelium, closely apposed to the VNO lumen, and most likely intermingled with Gi 2 ⁇ VNO sensory neurons.
  • Go-VN2 Such a difference in the Go-VN2 expression pattern in males and females might result from the expression of the same receptor gene in a different zone of the VNO epithelium or from a differential expression of two distinct but closely related genes of the Go-VN2 subfamily.
  • Go-VN2 In females, Go-VN2 generates a very intense hybridization signal to most positive neurons and a fainter staining on a second set of labeled cells. The population of faintly labeled cells was never detected in males, indicating the existence of a female-specific neuronal subpopulation expressing either a lower level of the Go-VN2 transcript or a female-specific receptor significantly different but still cross-hybridizing to the Go-VN2 probe.
  • Go-VN Receptors Is Restricted to Go+ VNO Neurons
  • the expression of some of the Go-VN receptors in neurons lining the VNO lumen in an area mainly occupied by Gi 2a + cells raises the obvious question as to whether the expression of this family of genes is strictly restricted to Go+ VNO neurons.
  • Single-cell cDNA prepared from 23 individual VNO neurons was analyzed by Southern blots with probes representing the six divergent subfamilies of Go-VN receptors and was PCR amplified with degenerated primers based on conserved motifs between Go-VN receptor sequences. Both approaches confirmed that none of the 19 cell cDNAs prepared from Gi 2a + neurons contained any sequence of the Go-VN receptor family.
  • ADDRESSEE Wolf, Greenfield & Sacks, P.C.
  • AAA CAC ATT ATG ACA TCT TCA GCA AAG GTT GTT ATC ATT TAT GGT GAA 875 Lys His He Met Thr Ser Ser Ala Lys Val Val He He Tyr Gly Glu 260 265 270
  • AAC ACT GCC AAA TAC CCA GTA GAT ATT TCT CAT ACT ATA TTG GAG TGG 1115 Asn Thr Ala Lys Tyr Pro Val Asp He Ser His Thr He Leu Glu Trp 340 345 350
  • ATC CAA CTT GTT CTC TGT GGA ATC TGG TTG GTC ACA TCT CCT CCC TTT 2219 He Gin Leu Val Leu Cys Gly He Trp Leu Val Thr Ser Pro Pro Phe 710 715 720
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • GCC AAT TTC ATT GAT CCC AGG TGC TTT TGG AGA ATA AAT TTG GAT GAA 215 Ala Asn Phe He Asp Pro Arg Cys Phe Trp Arg He Asn Leu Asp Glu 20 25 30
  • CACACCTAAC CCCAGAGAGA CTTAAGTCCC CAGGGATTGG GAAGTGCTGG GCATTGGGGA 3501
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal

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Abstract

L'invention concerne une famille multigènes codant une collection de nouveaux récepteurs de phéromones mammifères. L'invention concerne également des acides nucléiques codant les polypeptides récepteurs de phéromones, notamment des fragments et des variants biologiquement fonctionnels de ceux-ci. L'invention concerne également des polypeptides et leurs fragments codés par ces acides nucléiques, et des anticorps afférents. En outre, l'invention concerne des procédés et des produits d'utilisation de ces acides nucléiques et polypeptides.
PCT/US1998/013680 1997-06-30 1998-06-30 Nouvelle famille de recepteurs de pheromones WO1999000422A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP98933045A EP0996635A4 (fr) 1997-06-30 1998-06-30 Nouvelle famille de recepteurs de pheromones
JP50590499A JP2002511871A (ja) 1997-06-30 1998-06-30 フェロモンレセプターの新規なファミリー
CA002294473A CA2294473A1 (fr) 1997-06-30 1998-06-30 Nouvelle famille de recepteurs de pheromones

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US5128497P 1997-06-30 1997-06-30
US60/051,284 1997-06-30

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WO1999000422A9 WO1999000422A9 (fr) 1999-04-15

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WO1999000422A9 (fr) 1999-04-15
EP0996635A4 (fr) 2003-08-27
EP0996635A1 (fr) 2000-05-03
JP2002511871A (ja) 2002-04-16
CA2294473A1 (fr) 1999-01-07

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