WO2001098526A2 - Cartographie de recepteurs, perception sensorielle et biocapteurs d'agents sensoriels chimiques - Google Patents

Cartographie de recepteurs, perception sensorielle et biocapteurs d'agents sensoriels chimiques Download PDF

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WO2001098526A2
WO2001098526A2 PCT/US2001/020122 US0120122W WO0198526A2 WO 2001098526 A2 WO2001098526 A2 WO 2001098526A2 US 0120122 W US0120122 W US 0120122W WO 0198526 A2 WO0198526 A2 WO 0198526A2
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seq
odorant
receptor
sensory
receptors
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PCT/US2001/020122
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WO2001098526A9 (fr
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Sergey Zozulya
Lubert Stryer
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Senomyx, Inc.
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Application filed by Senomyx, Inc. filed Critical Senomyx, Inc.
Priority to AU2001270121A priority Critical patent/AU2001270121A1/en
Publication of WO2001098526A2 publication Critical patent/WO2001098526A2/fr
Publication of WO2001098526A9 publication Critical patent/WO2001098526A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the invention relates to the use of sensory G protein-coupled receptor complexes that recognize chemical sensants, particularly those involving olfactory and taste receptors; polypep-tide fragments and mutants thereof; classes of such receptors; polynucleotides encoding such receptors, fragments and mutants thereof, and representatives of receptor classes; genetic vectors including such polynucleotides; and cells and non-human organisms engineered to express such receptor complexes, fragments and mutants of an olfactory or taste receptor, and representatives of receptor classes to simulate sensory perception of odorants and tastants.
  • the invention also relates to the use of such products as a biosensor or component thereof to detect, to identify, to measure, or otherwise process the event of binding between the receptor and its cognate ligand (i.e., chemical sensant).
  • the invention has application, for example, in the design and formu-lation of odorant and tastant compositions.
  • the olfactory and taste systems provide sensory information about the chemical environ-ment.
  • Olfactory receptors and taste receptors recognize, respectively, "odorants” and “tastants,” collectively referred to as “sensants” or “sensory receptor ligands” herein.
  • a "primary” sensant is an odorant or tastant ligand that substantially binds to sensory receptors with a ligand-binding site of a single amino acid sequence.
  • Olfactory and taste receptors belong to the superfamily of seven-transmembrane guanyl nucleotide-binding proteins: such receptors are, however, also recognized as distinct families, or sub-genuses, of olfactory or taste receptors (see Raming Nature 361 :353, 1993). These receptors control diverse physiological functions such as media-ting signaling from an external chemical stimulus across the membrane containing the receptor into a cell, endocrine function, exocrine function, heart rate, lipolysis, and carbohydrate metabolism. Thus, the dissection of these diverse functions into component signals is needed.
  • WO 00/15269 discloses methods and apparatus for odor reproduction.
  • the total affinities of a specific odorant with a group of receptors was called the affinity fingerprint of the odorant.
  • This odorant fingerprint was represented by a vector of affinity values. It was proposed to repro-duce an arbitrary odor by inputting its sensed odorant fingerprint into a device, which has a palate of predetermined odorants and produces a composite odor using predetermined odorant finger-prints by minimizing the difference between vectors representing the sensed odorant fingerprint and the predetermined odorant fingerprints.
  • This reference does not teach or suggest the human olfactory receptors disclosed herein.
  • WO 00/70343 discloses biosensors and sense replication systems using G- protein coupled receptors (GPCR). It was proposed to mimic the response of the G- protein signal transduction system by detecting the affinity of a stimulus to a plurality of GPCR, codifying such information into electronic signals, and reproducing the stimulus by converting the codified information into a combination of stimulant entities. But this reference also does not teach or suggest the human olfactory receptors disclosed herein.
  • WO 01/27158 discloses olfactory receptors and their use to determine the correspondence between individual odorant receptors and particular odors. It was proposed that the interactions between an odor and olfactory receptors can be used to represent the odor and to re-create it. This reference, however, does not teach or suggest the human olfactory receptors disclosed herein.
  • the present invention addresses the need for better understanding of these ligand-receptor interactions by using a large set of identified sensory receptors. Even if the receptor set used is incomplete ( . e. , a partial set of all sensory receptors encoded in the genome), a large number of sensants will be detected. Moreover, redundancy in the chemical structures recognized by the sensory receptors or combinatorial processing of signals from different sensory receptors would allow broad coverage of chemically diverse sensants (e.g., by selection of a representative class of sensory receptors). Also provided are, ter alia, methods for utilizing such sensory receptors and biosensors to simulate sensory perception.
  • OLFR mammalian olfactory G-protein-coupled receptors
  • TASR mammalian taste G-protein-coupled receptors
  • chimeric proteins or other polypeptides with altered ligand-binding and/or signaling properties can be made from two or more different sensory receptors by mixing domains.
  • the invention provides methods for representing the sensory perception of one or more chemicals (e.g., a primary sensant or mixture thereof) and/or for predicting the sensory percep-tion of one or more chemicals in a mammal (e.g., human) using the aforementioned products. Given a known member of a ligand-receptor binding pair, one or both members of the pair (i.e., ligand, receptor, or both) may be detected, identified, and/or measured under binding conditions.
  • Novel molecules or combinations of molecules which elicit a desired and predetermined sensory perception in a mammal can be generated by determining a value of sensory perception in a mammal for a known molecule or combination thereof; determining a value of sensory perception in a mammal for one or more unknown molecules or combinations thereof; comparing the value of sensory perception in a mammal for one or more unknown corn-positions to the value of sensory perception in a mammal for one or more known compositions; selecting a molecule or combination of molecules that elicits a predetermined sensory perception in a mammal; and mixing two or more unknown molecules or combinations thereof to form a molecule or combination thereof that elicits a predetermined sensory perception in a mammal.
  • the combining step yields a single molecule or a combination thereof that elicits a predetermined sensory perception in a mammal.
  • primary odorants that uniquely bind to olfactory receptors with a single ligand-binding domain may be such novel molecules.
  • Variants thereof may be used (1) to enhance stimulation of a limited response by olfactory receptors with a single ligand-binding domain or (2) to block stimulation of olfactory receptors with a single ligand-binding domain to reduce or inhibit olfactory perception.
  • An alternative method for identifying primary odorants is to identify the mutated receptors in genetic anosmias because that odorant would be expected to be recognized by only one or a few olfactory receptors affected by the mutation.
  • U.S. Patent No. 5,691,188 describes how upon binding of ligand to receptor, the receptor presumably undergoes a conformational change leading to activation of the G protein.
  • the G proteins are comprised of three subunits: a guanyl nucleotide binding ⁇ subunit, a ⁇ subunit, and a ⁇ subunit. G proteins cycle between two forms, depending on whether GDP or GTP is bound to the ⁇ subunit. When GDP is bound, the G protein exists as a heterotrimer: the G ⁇ complex. When GTP is bound, the ⁇ subunit dissociates from the heterotrimer, leaving a G ⁇ complex.
  • G ⁇ complex When a G ⁇ complex operatively associates with an activated G protein coupled receptor in a cell membrane, the rate of exchange of GTP for bound GDP is increased and the rate of disso-ciation of the bound G ⁇ subunit from the G ⁇ complex increases.
  • the free G ⁇ subunit and G ⁇ complex are thus capable of transmitting a signal to downstream elements of a variety of signal transduction pathways.
  • a high-resolution X-ray crystal structure is available for rhodopsin, a guanyl nucleotide binding protein, has been solved (Palczewski et al, Science 289:739, 2000). Using this structure, the portions of the amino acid sequence of sensant receptors that are responsible for ligand binding can be identified.
  • Sensory Perception - Olfaction Genes encoding the olfactory receptors are active primarily in olfactory neurons (Axel Sci. Amer. 273:154, 1995). Individual olfactory receptor types are expressed in subsets of cells distributed in distinct zones of the olfactory epithelium (Breer Semin. Cell Biol. 5:25, 1994). The human genome contains thousands of genes that encode a diverse repertoire of olfactory receptors (Rouquier Nat. Genet. 18:243, 1998; Trask Hwm. Mol. Genet. 7:2007, 1998).
  • Mammals are believed to have five basic taste modalities: sweet, bitter, sour, salty, and umami (the taste of monosodium glutamate). See, e.g., Kawamura et al, Introduction to Umami: A Basic Taste (1987); Kinnamon et al, Ann. Rev. Physiol, 54:715, 1992; Lindemann, Physiol. Rev., 76:718, 1996; Stewart et al, Am. J. Physiol, 272:1, 1997. Numerous physiological studies in animals have shown that taste receptor cells may selectively respond to different chemical stimuli. See, e.g., Akabas et al, Science, 242:1047, 1988; Gilbertson et al, J. Gen. Physiol, 100:803, 1992; Bernhardt et al, J. Physiol, 490:325, 1996; Cummings et al, J. Neurophysiol, 75:1256, 1996.
  • taste receptor cells are assembled into taste buds that are distributed into different papillae in the tongue epithelium.
  • Cirristopapillae found at the very back of the tongue, contain hundreds, e.g., mice, to thousands, e.g., human, of taste buds.
  • foli-ate papillae localized to the posterior lateral edge of the tongue, only contain dozens to hundreds of taste buds.
  • fungiform papillae contain only a single or a few taste buds, and are at the front of the tongue. r
  • nucleic acids encoding the sensory receptors and other related polypep-tides can be isolated from a variety of sources, genetically engineered, amplified, synthesized, and or expressed recombinantly according to the methods disclosed in WO 00/35374, which is herein incorporated by reference in its entirety. These nucleic acids provide probes for the identification of cells expressing sensory receptors, as the nucleic acids are specifically expressed in such cells. They can also serve as tools for the generation of sensory topographical maps that elucidate the relationship between cells expressing sensory receptors and sensory neurons leading to particular regions of the brain. Furthermore, the nucleic acids and the polypeptides they encode can be used as probes to elucidate olfactant- or tastant- induced behaviors.
  • Nucleic acid molecules encoding a sensory receptor comprising a nucleic acid sequence that is at least 75%, 85%, 90%, 95%, or 99% identical to a nucleic acid sequence selected from those known in the prior art or disclosed herein are considered variants.
  • Other nucleic acid molecules comprising a nucleic acid sequence that encodes a polypeptide having an amino acid sequence at least 75%, 85%, 90%, 95%, or 99% identical to an amino acid sequence selected from those known in the prior art or disclosed herein are also considered variants. Further variants contain amino acid sequence differences in at most ten, five, four, three, two, or one amino acid residue(s).
  • nucleic acid sequences may be selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ D NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ D NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO:
  • Nucleic acid molecule comprising a nucleic acid sequence that encodes a fragment of a polypeptide having an amino acid sequence selected from those known in the prior art or dis-closed herein; wherein the fragment is at least ten, 20, 30, 50, 70, 100, or 150 amino acid resi-dues in length, are useful as probes, primers, and to construct hybrids or chimerae.
  • Polypeptides comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from those known in the prior art or disclosed herein are considered variants. Further variants contain amino acid sequence differ-ences in at most ten, five, four, three, two, or one amino acid residue(s).
  • Other polypeptides comprising a fragment of a polypeptide having an amino acid sequence selected from those known in the prior art or disclosed herein; wherein the fragment is at least 40, 60, 80, 100, 150, 200, or 250 amino acid residues in length, are useful as specific binders of sensants, competitive binders, antigens, and to construct hybrids or chimerae.
  • Exemplary amino acid sequences may be selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ DD NO: 7, SEQ DD NO: 9, SEQ ED NO: 11, SEQ DD NO: 13, SEQ ID NO: 15, SEQ TD NO: 17, SEQ ID NO: 19, SEQ TD NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ TD NO: 27, SEQ ED NO: 29, SEQ TD NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ED NO: 37, SEQ DD NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ TD NO: 49, SEQ ED NO: 51, SEQ DD NO: 53, SEQ ED NO: 55, SEQ DD NO: 57, SEQ ED NO: 59, SEQ DD NO: 61, SEQ ID NO: 63, SEQ TD NO: 65, S
  • modulators e.g., activators, inhibitors, stimu-lators, enhancers, agonists, and antagonists
  • Such modulators of signal transduction are useful for pharmacological or genetic modu-lation of signaling pathways.
  • These methods of screening can be used to identify high affinity agonists and antagonists of sensory cell activity.
  • modulator compounds can then be used in the pharmaceutical, food, and cosmetic industries to customize odorants or tastants.
  • the invention provides assays for sensory modulation, where the sensory receptors, or fragments or variants thereof, act as direct or indirect reporter molecules for the effect of modulators on signal transduction.
  • Sensory receptors, or fragments or variants thereof can be used in assays, e.g., to measure changes in ion concentration, membrane potential, current flow, ion flux, transcription, signal transduction, receptor-ligand interaction, second messenger concen-trations, in vitro, in vivo, and ex vivo.
  • sensory receptors, or fragments or variants thereof can be used as an indirect reporters via attachment to second reporter molecules, such as green fluorescent protein (see, e.g., Mistili et al, Nature Biotech., 15:961, 1997).
  • the sensory receptors, or fragments or variants thereof can be expressed in host cells, and modulation of signal transduction via sensory receptor activity can be assayed by measuring changes in Ca 2+ levels.
  • Methods of assaying for modulators of signal transduction include in vitro ligand binding assays using the sensory receptors, or fragments or variants thereof. More particularly, such assays can use the sensory receptors; portions thereof such as the extracellular or transmembrane domains; chimeric proteins comprising one or more of such domains; oocyte receptor expression; tissue culture cell receptor expression; transcriptional activation of the receptor; G-protein binding to the receptor; ligand binding assays; voltage, membrane potential and conductance changes; ion flux assays; changes in intracellular second messengers such as cAMP and inositol triphosphate; changes in intracellular Ca levels; and neurotransmitter release.
  • the invention also provides for methods of detecting sensory receptor nucleic acid and protein expression, allowing for the investigation of taste transduction regulation and specific identification of sensory receptor cells.
  • the sensory receptors, fragments, and variants of the invention can also be used to generate monoclonal and polyclonal antibodies useful for identi-fying a sensory receptor cells.
  • Sensory receptor cells can be identified using techniques such as reverse transcription and amplification of mRNA, isolation of total RNA or poly A + RNA, Northern blotting, dot blotting, in situ hybridization, RNase protection, SI digestion, probing DNA microchip arrays, Western blots, and the like.
  • amino acid sequences of the sensory receptors and polypeptides of the invention can be identified by putative translation of the coding nucleic acid sequences. These various amino acid sequences and the coding nucleic acid sequences may be compared to one another or to other sequences according to a number of methods. For example, in sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, as described below for the BLASTN and BLASTP programs, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of. contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g. , by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al, Altschul et al, Nucl. Acids Res. 25:3389, 1977 and Altschul et al, J. Mol. Biol. 215:403, 1990).
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a so- called “tree” or “dendogram” showing the clustering relationships used to create the alignment (see, e.g., Figure 2).
  • PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, JMol. Evol. 35:351, 1987. The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151, 1989.
  • the program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7. 0
  • the nucleic acid molecules of the present invention are typically intronless and encode putative sensory receptor proteins generally having lengths of about 300 to about 400 amino acid residues that contain seven transmembrane domains, as predicted by hydrophobicity plotting analysis, indicating that they belong to the G protein-coupled receptor superfamily, which includes the subset of taste and olfactory receptors.
  • putative sensory receptor proteins generally having lengths of about 300 to about 400 amino acid residues that contain seven transmembrane domains, as predicted by hydrophobicity plotting analysis, indicating that they belong to the G protein-coupled receptor superfamily, which includes the subset of taste and olfactory receptors.
  • each of the 256 sensory receptors identified herein has a characteristic sequence signature of an olfactory receptor.
  • novel human receptors have amino acid sequences distinctly different from the previously known human sensory receptors, which suggests their different specificity in sensant recognition. Therefore, these novel sensory receptors and their genes can be used, alone or in combination with known sensory receptors, in developing detec-tion systems and assays for chemically distinct types of sensants not recognized by the known sensory receptors, as well as for diagnostic and research purposes.
  • purified refers to the state of being free of other, dissimilar compounds with which the compound of the invention is normally associated in its natural state, so that the “purified,” “substantially purified,” and “isolated” subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample. In one preferred embodiment, these terms refer to the compound of the invention comprising at least 95% of the mass, by weight, of a given sample.
  • nucleic acid or protein when referring to a nucleic acid or protein, of nucleic acids or proteins, also refers to a state of purification or concentration different than that which occurs naturally in the mammalian, especially human, body.
  • nucleic acid or protein or classes of nucleic acids or proteins, described herein may be isolated, or otherwise associated with structures or compounds to which they are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.
  • amplifying and amplification refer to the use of any suitable amplification methodology for generating or detecting recombinant or naturally expressed nucleic acid, as described in detail, below.
  • the invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs) for amplifying (e.g. , by polymerase chain reaction, PCR) naturally expressed (e.g. , genomic or mRNA) or recombinant (e.g., cDNA) nucleic acids of the invention (e.g., sensant-binding sequences of the invention) in vivo or in vitro.
  • amplifying e.g. , by polymerase chain reaction, PCR
  • naturally expressed e.g. , genomic or mRNA
  • recombinant e.g., cDNA
  • 7- transmembrane receptor means a polypeptide belonging to a superfamily of transmembrane proteins that have seven domains that span the plasma membrane seven times (thus, the seven domains are called “transmembrane” or "TM" domains TM I to TM Nil).
  • the families of olfactory and taste receptors each belong to this super-family.
  • Seven-transmembrane receptor polypeptides have similar and characteristic primary, secondary and tertiary structures, as discussed in further detail below.
  • expression vector refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell.
  • the term includes linear or circular expression systems.
  • the term includes expression systems that remain episomal or integrate into the host cell genome.
  • the expression systems can have the ability to self-replicate or not, i.e., drive only transient expression in a cell.
  • the term includes recombinant expression "cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid.
  • the term “library” means a preparation that is a mixture of different nucleic acid or polypeptide molecules, such as the library of recombinantly generated sensory, particularly olfactory or taste, receptor ligand-binding domains generated by amplification of nucleic acid with degenerate primer pairs, or an isolated collection of vectors that incorporate the amplified sensant-binding domains, or a mixture of cells each randomly transfected with at least one vector encoding a sensory receptor.
  • nucleic acid or “nucleic acid sequence” refers to a deoxy- ribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form.
  • the term encompasses nucleic acids, i.e., oligonucleotides, containing known analogs of natural nucleotides.
  • the term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Oligonucleo-tides and Analogues, a Practical Approach, ed. F. Eckstein, Oxford Univ. Press (1991); Anti-sense Strategies, Annals of the N.Y. Academy of Sciences, Vol. 600, Eds. Baserga et al. (NYAS 1992); Milligan (1993) J. Med. Chem.
  • sensory receptor refers to sequences derived from a sensory receptor that substantially incorporates transmembrane domains II to N ⁇ (TM ⁇ to NH).
  • the domain may be capable of binding a sensant.
  • conservative variant refers to a polypeptide which has a modified amino acid sequence, such that the change(s) do not substantially alter the poly-peptide's (the conservative variant's) structure and/or activity, as defined herein.
  • conservatively modified variations of an amino acid sequence i.e., amino acid substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity.
  • Conservative substitution tables providing function-ally similar amino acids are well known in the art.
  • one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gin/ Asn; Gly/ Asp; Gly/ Ala or Pro; His/ Asn or Gin; Ile/Leu or Nal; Leu/Ile or val; Lys/ Arg or Gin or Glu; Met/Leu or Tyr or He; Phe/Met or Leu or Tyr; Ser/Thr; Thr/Ser; Trp/Tyr; Tyr/Trp or Phe; Nal/Ile or Leu.
  • An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine ( ⁇ ), Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (N); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H. Freeman, 1984; Schultz & Schimer, Principles of Protein Structure, Springer-Nerlag, 1979).
  • substitutions are not the only possible conservative substitutions. For example, for some purposes, one may regard all charged amino acids as conservative substitutions for each other whether they are positive or negative. In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered “conservatively modified variations.”
  • the terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound that has substantially the same structural and/or functional characteristics of the polypeptides, e.g., translocation domains or sensant-binding domains or chimeric receptors of the invention.
  • the mimetic can be either entirely composed of synthetic, non-natural analogs of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity.
  • routine experi-mentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered.
  • Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
  • a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
  • peptide bonds can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbod ⁇ mide (DCC) or N,N'- diisopropylcarbodiimide (DIC).
  • a polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature.
  • recombinant refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide.
  • transmembrane domain means a polypeptide domain that can completely span the plasma membrane.
  • the general secondary and tertiary structure of transmembrane domains, in particular the seven transmembrane domains of 7- transmembrane receptors such as olfactory receptors, are well known in the art. Thus, primary structure sequence can be designed or predicted based on known transmembrane domain sequences, as described in detail below.
  • Isolation and expression of the sensory receptors, or fragments or variants thereof, of the invention can be performed as described below.
  • PCR primers can be used for the amplification of nucleic acids encoding olfactory receptor ligand binding regions and libraries of these nucleic acids can thereby be generated.
  • Libraries of expression vectors can then be used to infect or transfect host cells for the functional expression of these libraries. These genes and vectors can be made and expressed in vitro or in vivo.
  • nucleic acid sequences of the invention and other nucleic acids used to practice this invention may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly. Any recombinant expression system can be used, including, in addition to mammalian cells, e.g., bacterial, yeast, insect or plant systems.
  • these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Carruthers, Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982); Adams, Am. Chem. Soc. 105:661 (1983); Belousov, Nucleic Acids Res. 25:3440-3444 (1997); Frenkel, Eree Radio. Biol Med. 19:373-380 (1995); Blommers, Biochemistry 33:7886-7896 (1994); Narang, Meth. Enzymol 68:90 (1979); Brown, Meth. Enzymol 68:109 (1979); Beaucage, Tetra. Lett. 22:1859 (1981); U.S. Patent No. 4,458,066. Double-stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence..
  • nucleic acids such as, for example, for generating mutations in sequences, subcloning, labeling probes, sequencing, hybridization and the like are well described in the scientific and patent literature. See, e.g., Sambrook, ed., Molecular Cloning: a Laboratory manual (2nd ed.), Nols. 1- 3, Cold Spring Harbor Laboratory (1989); Current Protocols in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); Laboratoi ⁇ Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I, Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
  • Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromato-graphy (TLC), and hyperdiffusion chromatography, various immunological methods, e.g., fluid or gel precipitin reactions, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescent assay, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantita-tive PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromat
  • Oligonucleotide primers are used to amplify nucleic acid encoding an olfactory receptor ligand-binding region.
  • the nucleic acids described herein can also be cloned or measured quan-titatively using amplification techniques.
  • primer pair sequences (see below), the skilled artisan can select and design suitable oligonucleotide amplification primers.
  • Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (PCR Protocols, a Guide to Methods and Applications, ed. Innis. Academic Press, NY, 1990 and PCR Strategies, ed.
  • LCR ligase chain reaction
  • transcription amplification see, e.g., Kwoh, Proc. Natl. Acad. Sci. USA 86:1173, 1989
  • self-sustained sequence replication see, e.g., Guatelli, Proc. Natl. Acad. Sci. USA 87:1874, 1990
  • Q Beta replicase amplification see, e.g., Smith, J. Clin. Microbiol.
  • the nucleic acids may be cloned according to methods known in the art, if desired, into any of a variety of vectors using routine molecular biological methods; methods for cloning in vitro amplified nucleic acids are described, e.g., U.S. Patent No. 5,426,039.
  • restriction enzyme sites can be "built into” the PCR primer pair. For example, Pst I and Bsp El sites were designed into the exemplary primer pairs of the invention.
  • restriction sites have a sequence that, when ligated, are "in-frame” with respect to the 7-membrane receptor "donor" coding sequence into which they are spliced (the sensant-binding region-coding sequence is internal to the 7-membrane polypeptide, thus, if it is desired that the construct be translated downstream of a restriction enzyme splice site, out of frame results should be avoided; this may not be necessary if the inserted sensant-binding domain comprises substantially most of the transmembrane N ⁇ region).
  • the primers can be designed to retain the original sequence of the "donor" 7-membrane receptor (the Pst I and Bsp El sequence in he primers of the invention generate an insert that, when ligated into the Pst I/Bsp El cut vector, encode residues found in the "donor" mouse olfactory receptor M4 sequence).
  • the primers can encode amino acid residues that are conservative substitutions (e.g., hydrophobic for hydrophobic residue, see above discussion) or functionally benign substitutions (e.g., do not prevent plasma membrane insertion, cause cleavage by peptidase, cause abnormal folding of receptor, and the like).
  • the primer pairs are designed to selectively amplify sensant-binding regions of olfactory receptor proteins. These domain regions may vary for different sensnants, and more particularly odorants; thus, what may be a minimal binding region for one sensant, and more particularly odorants, may be too limiting for a second potential ligand. Thus, domain regions of different sizes comprising different domain structures may be amplified; for example, transmembrane (TM) domains H through N ⁇ , HI through N ⁇ , HI through NI or H through VI, or variations thereof (e.g., only a subsequence of a particular domain, mixing the order of the domains, and the like), of a 7-transmembrane sensory receptor.
  • TM transmembrane
  • a nucleic acid sequence encoding domain regions ⁇ through N ⁇ can be generated by PCR ampli-fication using a primer pair.
  • a degenerate primer can be designed from a nucleic acid that encodes the amino acid sequence LFLLYL.
  • Such a degenerate primer can be used to generate a binding domain incorpo-rating TM I through TM HI, TM I through TM IN, TM I through TM N, TM I through TM VI or TM I through TM VH.
  • a degenerate primer (of at least about 17 residues) can be designed from a nucleic acid that encodes the amino acid sequence M(A G)(Y/F)DRYVAI (encoded by a nucleic acid sequence such as
  • Such a degenerate primer can be used to generate a binding domain incorporating TM HI through TM IN, TM m through TM N, TM m through TM NI or TM m through TM V ⁇ .
  • a degenerate primer (of at least about 17 residues) can be designed from nucleic acid encoding an amino acid sequence TC(G/A)SHL, encoded by a sequence such as 5'- AG(G/A)TG ⁇ (G/C)(T/A) ⁇ (G/C)C(G/A)CA- ⁇ GT-3'.
  • Such a degenerate primer can be used to generate a binding domain incorporating TM I through TM VI, TM H through TM VI, TM m through TM VI or TM TV * through TM VI).
  • CODEHOP COnsensus-DEgenerate Hybrid Oligonucleotide Primer
  • oligonucleotide primer pairs are well known in the art. "Natural" base pairs or synthetic base pairs can be used. For example, use of artificial nucleobases offers a versatile approach to manipulate primer sequence and generate a more complex mixture of amplification products. Various families of artificial nucleobases are capable of assuming multiple hydrogen bonding orientations through internal bond rotations to provide a means for degenerate molecular recognition. Incorporation of these analogs into a single position of a PCR primer allows for generation of a complex library of amplification products. See, e.g., Hoops, Nucleic Acids Res. 25:4866, 1997. Nonpolar molecules can also be used to mimic the shape of natural DNA bases.
  • a non-hydrogen-bonding shape mimic for adenine can replicate efficiently and selectively against a nonpolar shape mimic for thymine (see, e.g., Morales, Nat. Struct. Biol. 5:950, 1998).
  • two degenerate bases can be the pyrimidine base 6H, 8H-3,4-dihydropyrimido[4,5-c][l,2]oxazin-7-one or the purine base ⁇ 6-methoxy-2,6-diaminopurine (see, e.g., Hill, Proc. Natl, Acad. Sci. USA 95:4258, 1998).
  • Exemplary degenerate primers of the invention incorporate the nucleobase analog 5'-Dimethoxytrityl-N-benzoyl-2'-deoxy-Cytidine, 3'-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (the term "P" in the sequences, see above).
  • This pyrimidine analog hydrogen bonds with purines, including A and G residues.
  • Exemplary primer pairs for amplification of olfactory receptor transmembrane domains ⁇ through NH include:
  • Nucleic acids that encode ligand-binding regions of olfactory receptors are generated by amplification (e.g., PCR) of appropriate nucleic acid sequences using degenerate primer pairs.
  • the amplified nucleic acid can be genomic DNA from any cell or tissue or mRNA or cDNA derived from olfactory receptor-expressing cells, e.g., olfactory neurons or olfactory epithelium.
  • Isolation from olfactory receptor-expressing cells is well known in the art (cells expressing naturally or inducibly expressing olfactory receptors can be used to express the hybrid olfactory receptors of the invention to screen for potential odorants and odorant effect on cell physiology, as described below).
  • cells can be identified by olfactory marker protein (OMP), an abundant cytoplasmic protein expressed almost exclusively in mature olfactory sensory neurons (see, e.g., Buiakova, Proc. Natl. Acad. Sci. USA 93:9858, 1996). Shirley, Eur. J. Biochem.
  • OMP olfactory marker protein
  • Hybrid protein-coding sequences comprising nucleic acids sensory receptors fused to the translocation sequences described herein may be constructed. Also provided are hybrid receptors comprising the translocation motifs and ligand-binding domains of sensory receptors. These nucleic acid sequences can be operably linked to transcriptional or translational control elements, e.g., transcription and translation initiation sequences, promoters and enhancers, transcription and translation terminators, polyadenylation sequences, and other sequences useful for transcribing DNA into RNA.
  • transcriptional or translational control elements e.g., transcription and translation initiation sequences, promoters and enhancers, transcription and translation terminators, polyadenylation sequences, and other sequences useful for transcribing DNA into RNA.
  • vectors, transgenics, and a promoter fragment can be employed to direct expression of the desired nucleic acid in all tissues.
  • Olfactory cell-specific transcriptional elements can also be used to express the fusion polypeptide receptor, including, e.g., a 6.7 kb region upstream of the M4 olfactory receptor coding region. This region was sufficient to direct expression in olfactory epithelium with wild type zonal restriction and distributed neuronal expression for endogenous olfactory receptors (Qasba, J. Neurosci. 18:227, 1998).
  • Receptor genes are normally expressed in a small subset of neurons throughout a zonally restricted region of the sensory epithelium.
  • the transcriptional or translational control elements can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods.
  • Fusion proteins may also comprise the translocation motif described herein. However, these fusion proteins can also comprise additional elements for, e.g., protein detection, purification, or other applications.
  • Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts or histidine-tryptophan modules or other domains that allow purification on immobilized metals; maltose binding protein; protein A domains that allow purification on immobilized immunoglobulin; or the domain utilized in the FLAGS extension/ affinity purification system (Immunex Corp, Seattle WA).
  • cleavable linker sequences such as Factor Xa (see, e.g., Ottavi, Biochi-mie 80:289, 1998), subtilisin protease recognition motif (see, e.g., Polyak, Protein Eng. 10:615, 1997); enterokinase (Invitrogen, San Diego, CA), and the like, between the translocation domain (for efficient plasma membrane expression) and the rest of the newly translated polypeptide may be useful to facilitate purification.
  • one construct can include a nucleic acid sequence encoding a polypeptide linked to six histidine residues followed by a thioredoxin, an enterokinase cleavage site (see, e.g., Williams, Biochemistry 34:1787, 1995), and an amino terminal translocation domain.
  • the histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the desired protein(s) from the remainder of the fusion protein.
  • Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see, e.g., Kroll, DNA Cell. Biol. 12:441, 1993).
  • Expression vectors either as individual expression vectors or as libraries of expression vectors, comprising the olfactory binding domain-encoding sequences may be introduced into a genome or into the cytoplasm or a nucleus of a cell and expressed by a variety of conventional techniques, well described in the scientific and patent literature. See, e.g., Roberts, Nature 328:731, 1987; Berger supra; Schneider, Protein Expr. Purif. 6435:10, 1995; Sambrook; Tijssen; Ausubel. Product information from manufacturers of biological reagents and experimental equipment also provide information regarding known biological methods.
  • the vectors can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods.
  • the nucleic acids can be expressed in expression cassettes, vectors or viruses which are stably or transiently expressed in cells (e.g., episomal expression systems).
  • Selection markers can be incorporated into expression cassettes and vectors to confer a selectable phenotype on transformed cells and sequences. For example, selection markers can code for episomal maintenance and replication such that integration into the host genome is not required.
  • the marker may encode antibiotic resistance (e.g., chloramphenicol, kanamycin, G418, bleomycin, hygromycin) or herbicide resistance (e.g., chlorosulfuron or Basta) to permit selection of those cells transformed with the desired DNA sequences (see, e.g., Blondelet-Rouault, Gene 190:315, 1997; Aubrecht, J. Pharmacol. Exp. Ther. 281:992, 1997). Because selectable marker genes conferring resistance to substrates like neomycin or hygromycin can only be utilized in tissue culture, chemoresistance genes are also used as selectable markers in vitro and in vivo.
  • antibiotic resistance e.g., chloramphenicol, kanamycin, G418, bleomycin, hygromycin
  • herbicide resistance e.g., chlorosulfuron or Basta
  • a chimeric nucleic acid sequence may encode a sensant-binding domain within any 7-transmembrane polypeptide.
  • Seven-transmembrane receptors belong to a superfamily of trans-membrane (TM) proteins having seven domains that traverse a plasma membrane seven times. Each of the seven domains spans the plasma membrane (TM I to TM VII). Because 7-trans-membrane receptor polypeptides have similar primary sequences and secondary and tertiary structures, structural domains (e.g., TM domains) can be readily identified by sequence analysis. For example, homology modeling, Fourier analysis and helical periodicity detection can identify and characterize the seven domains with a 7-transmembrane receptor sequence.
  • FFT Fast Fourier Transform
  • the library sequences include receptor sequences that correspond to TM ligand-binding domains, including, e.g., TM H to VH, TM H to VI, TM in to VH, and TM m to V ⁇ , that have been amplified (e.g., PCR) from mRNA of or cDNA derived from, e.g., olfactory receptor-expressing neurons or genomic DNA.
  • TM ligand-binding domains including, e.g., TM H to VH, TM H to VI, TM in to VH, and TM m to V ⁇ , that have been amplified (e.g., PCR) from mRNA of or cDNA derived from, e.g., olfactory receptor-expressing neurons or genomic DNA.
  • TM domain sequences can include a various TM domains or variations thereof, as described above. These sequences can be derived from any 7-transmembrane receptor. Because these polypeptides have similar primary sequences and secondary and tertiary structures, the seven domains can be identified by various analyses well known in the art, including, e.g., homology modeling, Fourier analysis and helical periodicity (see, e.g., Pilpel supra), as described above. Using this information sequences flanking the seven domains can be identified and used to design degenerate primers for amplification of various combinations of TM regions and subsequences.
  • the present invention also includes not only the DNA and proteins having the specified amino acid sequences, but also DNA fragments, particularly fragments of, for example, 40, 60, 80, 100, 150, 200, or 250 nucleotides, or more, as well as protein fragments of, for example, 10, 20, 30, 50, 70, 100, or 150 amino acids, or more.
  • chimeric proteins comprising at least 10, 20, 30, 50, 70, 100, or 150 amino acids, or more, of one of at least one of the sensory receptors described herein, coupled to additional amino acids representing all or part of another G protein receptor, preferably a member of the 7-transmembrane superfamily.
  • These chimerae can be made from the instant receptors and a G protein receptor described herein, or they can be made by combining two or more of the present proteins.
  • one portion of the chimera corresponds to and is derived from one or more of the domains of the 7-transmembrane protein described herein, and the remaining portion or portions come from another G protein-coupled receptor.
  • Chimeric receptors are well known in the art, and the techniques for creating them and the selection and boundaries of domains or fragments of G protein-coupled receptors for incorpo-ration therein are also well known. Thus, this knowledge of those skilled in the art can readily be used to create such chimeric receptors.
  • the use of such chimeric receptors can provide, for example, an olfactory selectivity characteristic of one of the receptors specifically disclosed herein, coupled with the signal transduction characteristics of another receptor, such as a well known receptor used in prior art assay systems.
  • a domain such as a ligand binding domain, an extracellular domain, a transmembrane domain (e.g., one comprising seven transmembrane regions and corresponding extracellular and cytosolic loops), the transmembrane domain and a cytoplasmic domain, an active site, a subunit association region, etc., can be covalently linked to a heterologous protein.
  • an extracellular domain can be linked to a heterologous GPCR transmembrane domain, or a heterologous CGPCR extracellular domain can be linked to a transmembrane domain.
  • heterologous proteins of choice can include, e.g., green fluorescent protein, ⁇ -gal, glutamtate receptor, and the rhodopsin presequence.
  • Polymorphic variants, alleles, and interspecies homologs that are substantially identical to a sensory receptor disclosed herein can be isolated using the nucleic acid probes described above.
  • expression libraries can be used to isolate sensory receptors and polymorphic variants, alleles, and interspecies homologs thereof, by detecting expressed homologs immunologically with antisera or purified antibodies made against a sensory receptor-derived polypeptide, which also recognize and selectively bind to the sensory receptor homolog.
  • host cells for expressing the sensory receptors, fragments, or variants of the invention.
  • a cloned gene or nucleic acid such as cDNAs encoding the sensory receptors, fragments, or variants thereof
  • the nucleic acid sequence of interest is subcloned into an expression vector that contains a strong promoter to direct transcription, a transcription translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation.
  • Suitable prokaryotic and eukaryotic expression systems are well known in the art and described, e.g., in Sambrook et al.
  • Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasmid vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at lest one gene into the host cell capable of expressing the olfactory receptor, fragment, or variant of interest.
  • the transfected cells are cultured under conditions favoring expression of the receptor, fragment, or variant of interest, which is then recovered from the culture using standard techniques. Examples of such techniques are well known in the art. See, e.g., WO 00/06593, which is incorporated by reference in a manner consistent with this disclosure.
  • Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al, Science, 246:1275, 1989; Ward et al, Nature, 341:544, 1989).
  • a number of sensory receptor-comprising immunogens may be used to produce antibody specifically reactive with a sensory receptor family member.
  • a recombinant sensory receptor protein, or an antigenic fragment thereof can be isolated as described herein. Suitable antigenic regions include, e.g., the conserved motifs that are used to identify members of the sensory receptor family.
  • Recombinant proteins can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above.
  • Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies.
  • a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen.
  • Naturally occurring protein may also be used either in pure or impure form.
  • the product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.
  • mice is immunized with the protein using an adjuvant (e.g., Freund's adjuvant) and a standard immunization protocol with periodic boosts.
  • an adjuvant e.g., Freund's adjuvant
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the sensory receptor.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see Harlow & Lane, supra).
  • Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol, 6:511, 1976). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, retroviruses, or other methods well known in the art. Colonies arising from single clones of immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al, Science, 246:1275, 1989.
  • Monoclonal antibodies or polyclonal sera are collected and titered against antigen in an immunoassay, for example, a solid phase immunoassay with the antigen immobilized on a solid support.
  • an immunoassay for example, a solid phase immunoassay with the antigen immobilized on a solid support.
  • polyclonal antisera with a titer of 10 4 or greater are selected and tested for their cross reactivity against non-sensory receptor proteins, or even other sensory receptor family members or other related proteins from other organisms, using a competitive binding immuno-assay.
  • Specific polyclonal antisera and monoclonal antibodies will usually bind with a Kd of at least about 0.1 mM, more usually at least about 1 pM, optionally at least about 0.1 p.M or better, and optionally 0.01 pM or better.
  • Sensory receptor proteins can be detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • immunological binding assays see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed., 1991).
  • Immunological binding assays typically use an antibody that specifically binds to a protein or antigen of choice (in this case a sensory receptor family member or an antigenic subsequence thereof).
  • the antibody e.g. , anti-sensory receptor
  • the antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
  • Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen.
  • the labeling agent may itself be one of the moieties comprising the antibody/antigen complex.
  • the labeling agent may be a labeled sensory receptor polypeptide or a labeled anti-sensory receptor antibody.
  • the labeling agent may be a third moiety, such a secondary antibody, that specifically binds to the antibody/sensory receptor complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived).
  • Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent.
  • the labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin.
  • detectable moieties are well known to those skilled in the art.
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, optionally from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C. a.
  • Immunoassays for detecting a sensory receptor protein in a sample may be either competi-tive or noncompetitive.
  • Noncompetitive immunoassays directly measure the amount of antigen.
  • the anti-sensory receptor antibodies are bound directly to a solid substrate on which they are immobilized. These immobilized antibodies then capture the sensory receptor protein present in the test sample.
  • the sensory receptor protein thus immobilized is then bound by a labeling agent, such as a second anti-sensory receptor antibody bearing a label.
  • the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second or third antibody is typically modified with a detectable moiety, such as biotin, to which another molecule specifically binds, e.g., streptavidin, to provide a detectable moiety.
  • a detectable moiety such as biotin
  • streptavidin another molecule specifically binds
  • the amount of sensory receptor protein present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) sensory receptor protein displaced (competed away) from an anti-sensory receptor antibody by the unknown sensory receptor protein present in a sample.
  • a known amount of sensory receptor protein is added to a sample and the sample is then contacted with an antibody that specifically binds to the sensory receptor.
  • the amount of exogenous sensory receptor protein bound to the antibody is inversely proportional to the concentration of sensory receptor protein present in the sample.
  • the antibody is immobilized on a solid substrate.
  • the amount of sensory receptor protein bound to the antibody may be determined either by measuring the amount of sensory receptor protein present in a sensory receptor/antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein.
  • the amount of sensory receptor protein may be detected by providing a labeled sensory receptor molecule.
  • a hapten inhibition assay is another preferred competitive assay.
  • the known sensory receptor protein is immobilized on a solid substrate.
  • a known amount of anti-sensory receptor antibody is added to the sample, and the sample is then contacted with the immobilized sensory receptor.
  • the amount of anti-sensory receptor antibody bound to the known immobilized sensory receptor protein is inversely proportional to the amount of sensory receptor protein present in the sample.
  • the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above. c. Cross-reactivity determinations
  • Immunoassays in the competitive binding format can also be used for cross- reactivity determinations.
  • a protein at least partially encoded by the nucleic acid sequences disclosed herein can be immobilized to a solid support.
  • Proteins are added to the assay that compete for binding of the antisera to the immobilized antigen.
  • the ability of the added proteins to compete for binding of the antisera to the immobi-lized protein is compared to the ability of the sensory receptor polypeptide encoded by the nucleic acid sequences disclosed herein to compete with itself.
  • the percentage cross- reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with each of the added proteins listed above are selected and pooled.
  • the cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the added considered proteins, e.g., distantly related homologs.
  • peptides comprising amino acid sequences representing conserved motifs that are used to identify members of the sensory receptor family can be used in cross-reactivity determinations.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immu-noassay as described above to compare a second protein, thought to be perhaps an allele or poly-morphic variant of a sensory receptor family member, to the immunogen protein (i.e., sensory receptor protein encoded by the nucleic acid sequences disclosed herein).
  • the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined.
  • the second protein required to inhibit 50% of binding is less than 10 times the amount of the protein encoded by nucleic acid sequences disclosed herein required to inhibit 50% of binding, then the second protein is said to specifically bind to the polyclonal antibodies generated to a sensory receptor immunogen.
  • Antibodies raised against sensory receptor conserved motifs can also be used to prepare antibodies that specifically bind only to GPCRs of the sensory receptor family, but not to GPCRs from other families.
  • Polyclonal antibodies that specifically bind to a particular member of the sensory receptor family can be make by subtracting out cross-reactive antibodies using other sensory receptor family members.
  • Species-specific polyclonal antibodies can be made in a similar way.
  • antibodies specific to human T2R01 can be made by, subtracting out antibodies that are cross-reactive with orthologous sequences, e.g., rat OLFR1 or mouse OLFR1. d.
  • Other assay formats e.g., rat OLFR1 or mouse OLFR1.
  • Western blot (immunoblot) analysis is used to detect and quantify the presence of sensory receptor protein in the sample.
  • the technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind the sensory receptor protein.
  • the anti-sensory receptor polypeptide antibodies specifically bind to the sensory receptor poly-peptide on the solid support.
  • These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti-sensory receptor antibodies.
  • LOA liposome immunoassays
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • Such detectable labels have been well- developed in the field of immuno-assays and, in general, most any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, bio-chemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g., DYNABEADSTM, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).
  • magnetic beads e.g., DYNABEADSTM, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like)
  • radiolabels e.g., 3 H, 125 1, 35 S, 14 C, or 32 P
  • enzymes e.g., horseradish peroxida
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels maybe used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to another molecules (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • the ligands and their targets can be used in any suitable combination with antibodies that recognize a sensory receptor protein, or secondary antibodies that recognize anti-sensory receptor.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
  • the label may be detected using a scintillation counter or with photographic film as in autoradiography.
  • the label may be detected by exciting the fluoro-chrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, with photographic film, or using electronic detectors such as charge coupled devices (CCDs) or photomultipliers.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • aggluti-nation assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
  • test compound specifically binds to a mammalian sensory, and more particularly, olfactory receptor of the invention, both in vitro and in vivo are described below, as are methods and compositions for determining whether a test compound is neurotoxic to an olfactory neuron expressing an olfactory transmembrane receptor polypeptide.
  • Any aspect of cell physiology can be monitored to assess the effect of sensant-binding to a naturally- occurring or chimeric olfactory receptor.
  • assays may be performed on intact cells expressing an olfactory receptor, on permeabilized cells or on membrane fractions produced by standard methods.
  • Olfactory receptors are normally located on the specialized cilia of olfactory neurons. These receptors bind odorants and initiate the transduction of chemical stimuli into electrical signals. An activated or inhibited G-protein will in turn alter the properties of target enzymes, channels, and other effector proteins.
  • the classic examples are the activation of cGMP phosphodiesterase by transducin in the visual system, adenylate cyclase by the stimulatory G-protein, phospholipase C by Gq and other cognate G-proteins, and modulation of diverse channels by Gi and other G- proteins. Downstream consequences can also be examined such as generation of diacyl glycerol and IP3 by phospholipase C, and in turn, for calcium mobilization by IP3.
  • the sensory receptor protein of the assay will typically be selected from a natural poly-peptide or conservatively modified variant thereof. Generally, the amino acid sequence identity will be at least 75%, 85%, 90%, 95%, or 99%.
  • the polypeptide of the assays can comprise a domain of a sensory receptor protein, such as an extracellular domain, transmembrane region, transmembrane domain, cytoplasmic domain, ligand binding domain, subunit association domain, active site, and the like. Either the sensory receptor protein or a domain thereof can be covalently linked to a heterologous protein to create a chimeric protein used in the assays described herein.
  • Modulators of sensory receptor activity can be tested using sensory receptor polypeptides as described above, either recombinant or naturally occurring. Protein can be isolated, expressed in a cell, expressed in a membrane derived from a cell, expressed in tissue or in an animal, either recombinant or naturally occurring.
  • Modulation can be tested using one of the in vitro or in vivo assays described herein. 1. In vitro binding assays
  • Sensory perception can also be examined in vitro with soluble or solid state reactions, using a full-length sensory receptor-GPCR or a chimeric molecule such as an extracellular domain or transmembrane region, or combination thereof, of a sensory receptor covalently linked to a heterologous signal transduction domain, or a heterologous extracellular domain and/or transmembrane region covalently linked to the transmembrane and/or cytoplasmic domain of a sensory receptor.
  • ligand-binding domains of the protein of interest can be used in vitro in soluble or solid state reactions to assay for ligand binding.
  • a chimeric receptor will be made that comprises all or part of a sensory receptor polypeptide, as well an additional sequence that facilitates the localization of the sensory receptor to the membrane, such as a rhodopsin, e.g., an N-terminal fragment of a rhodopsin protein.
  • Ligand binding to a sensory receptor protein, a domain, or chimeric protein can be tested in solution, in a bilayer membrane, attached to a solid phase, in a lipid monolayer, or in vesicles. Binding of a modulator can be tested using, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbence, refractive index) hydrodynamic (e.g., shape), chromatographic, or solubility properties. Sensory receptors with large (e.g., approximately 600 amino acid residues) extracellular N- terminal segments. These N-terminal segments are thought to form ligand-binding domains, and are therefore useful in biochemical assays to identify sensory receptor agonists and antagonists.
  • spectroscopic characteristics e.g., fluorescence, absorbence, refractive index
  • hydrodynamic e.g., shape
  • Sensory receptors with large (e.g., approximately 600 amino acid residues) extracellular N- terminal segments are thought to form ligand
  • Receptor-G-protein interactions can also be examined. For example, binding of the G-protein to the receptor or its release from the receptor can be examined. For example, in the absence of GTP, an activator will lead to the formation of a tight complex of a G protein (all three subunits) with the receptor. This complex can be detected in a variety of ways, as noted above. Such an assay can be modified to search for inhibitors, e.g., by adding an activator to the receptor and G protein in the absence of GTP, which form a tight complex, and then screen for inhibitors by looking at dissociation of the receptor-G protein complex. In the presence of GTP, release of the alpha subunit of the G protein from the other two G protein subunits serves as a criterion of activation.
  • G-protein An activated or inhibited G-protein will in turn alter the properties of target enzymes, channels, and other effector proteins.
  • the classic examples are the activation of cGMP phosphodiesterase by transducin in the visual system, adenylate cyclase by the stimulatory G-protein, phospholipase C by Gq and other cognate G proteins, and modulation of diverse channels by Gi and other G proteins.
  • Fluorescence Polarization (“FP") based assays may be used to detect and monitor sensant binding.
  • Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity.
  • Fluores-cence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polari- zation is rapid and does not destroy the sample.
  • this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
  • This section describes how fluorescence polarization can be used in a simple and quantitative way to measure the binding of odorants to the sensory receptors of the invention.
  • a fluorescently labeled molecule When a fluorescently labeled molecule is excited with plane polarized light, it emits light that has a degree of polarization that is inversely proportional to its molecular rotation. Large fluorescently labeled molecules remain relatively stationary during the excited state ( 4 nano-seconds in the case of fluorescein) and the polarization of the light remains relatively constant between excitation and emission. Small fluorescently labeled molecules rotate rapidly during the excited state and the polarization changes significantly between excitation and emission.
  • a single-stranded fluorescein-labeled oligonucleotide has a relatively low polarization value but when it is hybridized to a complementary strand, it has a higher polariza-tion value.
  • fluorescence-labeled sensants or auto-fluorescent sensants may be used.
  • the Beacon® and Beacon 2000TM System may be used in connection with these assays. Such systems typically express polarization in millipolarization units (1
  • the rotational relaxation time is small ( « 1 nanosecond) for small molecules
  • the invention provides soluble assays using molecules such as a domain such as ligand binding domain, an extracellular domain, a transmembrane domain (e.g., one comprising seven transmembrane regions and cytosolic loops), the transmembrane domain and a cytoplasmic domain, an active site, a subunit association region, etc.; a domain that is covalently linked to a heterologous protein to create a chimeric molecule; a sensory receptor protein; or a cell or tissue expressing a sensory receptor protein, either naturally occurring or recombinant.
  • molecules such as a domain such as ligand binding domain, an extracellular domain, a transmembrane domain (e.g., one comprising seven transmembrane regions and cytosolic loops), the transmembrane domain and a cytoplasmic domain, an active site, a subunit association region, etc.; a domain that is covalently linked to a heterologous protein to create a chimeric molecule
  • the invention provides solid phase based in vitro assays in a high throughput format, where the domain, chimeric molecule, sensory receptor protein, or cell or tissue expressing the sensory receptor is attached to a solid phase substrate.
  • the high throughput assays of the invention it is possible to screen up to several thousand different modulators or ligands in a single day.
  • each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentra-tion or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
  • a single standard microtiter plate can assay about 100 (e.g., 96) modulators.
  • the molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage, e.g., via a tag.
  • the tag can be any of a variety of compo-nents.
  • a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest (e.g., the taste transduction molecule of interest) is attached to the solid support by interaction of the tag and the tag binder.
  • tags and tag binders can be used, based upon known molecular interactions well described in the literature.
  • a tag has a natural binder, for example, biotin, protein A, or protein G
  • tag binders avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.
  • Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).
  • any haptenic or antigenic compound can be used in combination with an appro-priate antibody to form a tag/tag binder pair.
  • the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody.
  • receptor-ligand inter-actions are also appropriate as tag and tag-binder pairs.
  • agonists and antagonists of cell membrane receptors e.g., cell receptor-ligand interactions such as transfemn, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, describing arrays of biopolymers fixed to solid substrates).
  • Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UN radiation, and the like. 4.
  • Computer-based assays Yet another assay for compounds that modulate sensory receptor protein activity involves computer assisted drug design, in which a digital or analog processing system is used to generate a three-dimensional structure of a sensory receptor protein based on the structural information encoded by its amino acid sequence. The input amino acid sequence interacts directly and actively with a preestablished algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the protein. The models of the protein structure are then examined to identify regions of the structure that have the ability to bind, e.g., ligands. These regions are then used to identify ligands that bind to the protein.
  • the three-dimensional structural model of the protein is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a sensory receptor polypeptide into the computer system.
  • the nucleotide sequence encoding the polypeptide, or the amino acid sequence thereof can be any of those described herein, or fragments or variants thereof.
  • the amino acid sequence represents the primary sequence or subsequence of the protein, which encodes the structural information of the protein.
  • At least 10 residues of the amino acid sequence are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM.
  • the three-dimensional structural model of the protein is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art.
  • the amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary and quaternary structure of the protein of interest.
  • the software looks at certain parameters encoded by the primary sequence to generate the structural model.
  • energy terms primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding.
  • Secon-dary energy terms include van der Waals potentials.
  • Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secon-dary structural model.
  • the tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure.
  • the user at this point can enter addi-tional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure.
  • the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.
  • potential ligand binding regions are identified by the computer system.
  • Three-dimensional structures for potential ligands are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described above. The three-dimensional structure of the potential ligand is then compared to that of the sensory receptor protein to identify ligands that bind to the protein. Binding affinity between the protein and ligands is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.
  • Computer systems are also used to screen for mutations, polymorphic variants, alleles and interspecies homologs of sensory receptor genes. Such mutations can be associated with disease states or genetic traits. As described above, GeneChipTM and related technology can also be used to screen for mutations, polymorphic variants, alleles and interspecies homologs. Once the variants are identified, diagnostic assays can be used to identify patients having such mutated genes. Identification of the mutated sensory receptor genes involves receiving input of a first nucleic acid or amino acid sequence of a sensory receptor gene, or conservatively modified versions thereof. The sequence is entered into the computer system as described above. The first nucleic acid or amino acid sequence is then compared to a second nucleic acid or amino acid sequence that has substantial identity to the first sequence.
  • the second sequence is entered into the computer system in the manner described above. Once odorant dependent activation monitored. Control samples (untreated with activators or inhibitors) are assigned a relative sensory receptor activity value of 100. Inhibition of a sensory receptor protein is achieved when the sensory receptor activity value relative to the control is about 90%, optionally 50%, optionally 25-0%. Activation of a sensory receptor protein is achieved when the sensory receptor activity value relative to the control is 110%, optionally 150%, 200-500%, or 1000-2000%.
  • Changes in ion flux may be assessed by determining changes in polarization (i.e., electri-cal potential) of the cell or membrane expressing a sensory receptor protein.
  • polarization i.e., electri-cal potential
  • One means to deter-mine changes in cellular polarization is by measuring changes in current, and thereby measuring changes in polarization, with voltage-clamp and patch-clamp techniques, e.g., the "cell-attached" mode, the "inside-out” mode, and the "whole cell” mode (see, e.g., Ackerman et al, New Engl. JMed., 336:1575, 1997).
  • Whole cell currents are conveniently determined using the standard.
  • Other known assays include: assays to measure ion flux using radiolabeled or fluorescent probes such as voltage-sensitive dyes (see, e.g., Nestergarrd-Bogind et al, J.
  • the compounds to be tested are present in the range from 1 pM to 100 mM.
  • the effects of the test compounds upon the function of the polypeptides can be measured by examining any of the parameters described above. Any suitable physiological change that affects GPCR activity can be used to assess the influence of a test compound on the polypeptides of this invention.
  • the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as transmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca 2+ , DP3, cGMP, or cAMP.
  • effects such as transmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca 2+ , DP3, cGMP, or cAMP.
  • Prefened assays for G-protein coupled receptors include cells that are loaded with ion or voltage sensitive dyes to report receptor activity. Assays for determining activity of such receptors can also use known agonists and antagonists for other G-protein coupled receptors as negative or positive controls to assess activity of tested compounds. In assays for identifying modulatory compounds (e.g., agonists, antagonists), changes in the level of ions in the cytoplasm or membrane voltage will be monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively. Among the ion-sensitive indicators and voltage probes that may be employed are those disclosed in the Molecular Probes 1997 Catalog.
  • promiscuous G-proteins such as G ⁇ l 5 and G ⁇ l 6 can be used in the assay of choice (Wilkie et al, Proc. Natl. Acad. Sci., 88:10049, 1991). Such promiscuous G-proteins allow coupling of a wide range of receptors.
  • Receptor activation typically initiates subsequent intracellular events, e.g., increases in second messengers such as IP3, which releases intracellular stores of calcium ions.
  • second messengers such as IP3
  • Activation of some G-protein coupled receptors stimulates the formation of inositol triphosphate (EP3) through phospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge & Irvine, Nature 312:315, 1984).
  • EP3 in turn stimulates the release of intracellular calcium ion stores.
  • a change in cytoplasmic calcium ion levels, or a change in second messenger levels such as IP3 can be used to assess G-protein coupled receptor function.
  • Cells expressing such G-protein coupled receptors may exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it may be desirable although not necessary to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such as EGTA, to distinguish fluorescence response resulting from calcium release from internal stores.
  • Other assays can involve determining the activity of receptors which, when activated, result in a change in the level of intracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymes such as adenylate cyclase.
  • cyclic nucleotide-gated ion channels e.g., rod photoreceptor cell channels and olfactory neuron channels that are permeable to cations upon activation by binding of cAMP or cGMP (see, e.g., Altenhofen et al, Proc. Natl. Acad. Sci., 88:9868, 1991 and Dhallan et al, Nature 347:184, 1990).
  • cyclic nucleotide levels it may be preferable to expose the cells to agents that increase intracellular cyclic nucleotide levels, e.g., forskolin, prior to adding a receptor-activating compound to the cells in the assay.
  • agents that increase intracellular cyclic nucleotide levels e.g., forskolin
  • Cells for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-crated ion channel, GPCR phosphatase and DNA encoding a receptor (e.g., certain glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like), which, when activated, causes a change in cyclic nucleotide levels in the cytoplasm.
  • a receptor e.g., certain glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like
  • sensory receptor protein activity is measured by expressing a sensory receptor gene in a heterologous cell with a promiscuous G-protein that links the receptor to a phospholipase C signal transduction pathway (see Offermanns & Simon, J Biol. Chem., 270:15175, 1995).
  • the cell line is HEK-293 (which does not naturally express sensory receptor genes) and the promiscuous G-protein is Gal 5 (Offermanns & Simon, supra).
  • Modu-lation of olfactory transduction is assayed by measuring changes in intracellular Ca 2+ levels, which change in response to modulation of the sensory receptor signal transduction pathway via administration of a molecule that associates with a sensory receptor protein. Changes in Ca 2+ levels are optionally measured using fluorescent Ca 2+ indicator dyes and fluorometric imaging.
  • the changes in intracellular cAMP or cGMP can be measured using immunoassays.
  • the method described in Felley-Bosco et al, Am. J. Resp. Cell and Mol Biol, 11:159, 1994, may be used to determine the level of cGMP.
  • an assay kit for measuring cAMP and/or cGMP is described in U.S. Patent No. 4,115,538, herein incorporated by reference.
  • phosphatidyl inositol (PI) hydrolysis can be analyzed according to U.S. Patent No. 5,436,128, herein incorporated by reference. Briefly, the assay involves labeling of cells with 3H-myoinositol for 48 or more hrs. The labeled cells are treated with a test compound for one hour. The treated cells are lysed and extracted in chloroform-methanol-water after which the inositol phosphates were separated by ion exchange chromatography and quantified by scintillation counting. Fold stimulation is determined by calculating the ratio of cpm in the presence of agonist, to cpm in the presence of buffer control. Likewise, fold inhibition is determined by calculating the ratio of cpm in the presence of antagonist, to cpm in the presence of buffer control (which may or may not contain an agonist).
  • transcription levels can be measured to assess the effects of a test compound on signal transduction.
  • a host cell containing a sensory receptor protein of interest is contacted with a test compound for a sufficient time to effect any interactions, and then the level of gene expression is measured.
  • the amount of time to effect such interactions may be empiri-cally determined, such as by running a time course and measuring the level of transcription as a function of time.
  • the amount of transcription may be measured by using any method known to those of skill in the art to be suitable. For example, mRNA expression of the protein of interest may be detected using northern blots or their polypeptide products may be identified using immunoassays. Alternatively, transcription based assays using reporter gene maybe used as described in U.S. Patent No.
  • the reporter genes can be, e.g., chloramphenicol acetyltransferase, luciferase, '3-galactosidase and alkaline phosphatase.
  • the protein of interest can be used as an indirect reporter via attachment to a second reporter such as green fluorescent protein (see, e.g., Mistili & Spector, Nature Biotech. 15:961, 1997).
  • the amount of transcription is then compared to the amount of transcription in either the same cell in the absence of the test compound, or it may be compared with the amount of transcription in a substantially identical cell that lacks the sensory receptor protein of interest.
  • a substantially identical cell maybe derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. Any difference in the amount of transcription indicates that the test compound has in some manner altered the activity of the sensory receptor protein of interest.
  • Non-human animals expressing one or more sensory receptor sequences of the invention can also be used for receptor assays.
  • Such expression can be used to determine whether a test compound specifically binds to a mammalian olfactory transmembrane receptor polypeptide in vivo by contacting a non-human animal stably or transiently transfected with a nucleic acid encoding an olfactory receptor or ligand binding region thereof with a test compound and determining whether the animal reacts to the test compound by specifically binding to the receptor polypeptide.
  • translocation domains in the fusion polypeptides generates a cell expressing high levels of olfactory receptor.
  • Animals transfected or infected with the vectors of the invention are particularly useful for assays to identify and characterize odorants/ligands that can bind to a specific or sets of receptors.
  • Such vector-infected animals expressing libraries of human olfactory sequences can be used for in vivo screening of odorants and their effect on, e.g., cell physiology (e.g., on olfactory neurons), on the CNS (e.g., olfactory bulb activity), or behavior.
  • Means to infect/express the nucleic acids and vectors, either individually or as libraries, are well known in the art.
  • a variety of individual cell, organ or whole animal parameters can be measured by a variety of means.
  • recording of stimulant-induced waves (bulbar responses) from the main olfactory bulb or accessory olfactory bulb is a useful tool for measuring quantitative stable olfactory responses.
  • electrodes When electrodes are located on the olfactory bulb surface it is possible to record stable responses over a period of several days (see, e.g., Kashiwayanagi, Brain Res. Protoc. 1:287, 1997).
  • electroolfactogram recordings were made with a four- electrode assembly from the olfactory epithelium overlying the endoturbinate bones facing the nasal septum.
  • Extracellular potassium activity (aK) measurements can also be carried out in in vivo. An increase in aK can be measured in the mucus and the proximal part of the nasal epithelium (see, e.g., Khayari, Brain Res. 539:1, 1991).
  • the sensory receptor sequences of the invention can be for example expressed in animal nasal epithelium by delivery with an infecting agent, e.g., adenovirus expression vector.
  • an infecting agent e.g., adenovirus expression vector.
  • Recom-binant adeno virus-mediated expression of a recombinant gene in olfactory epithelium using green fluorescent protein as a marker is described by, e.g., Touhara, Proc. Natl. Acad. Sci. USA 96:4040, 1999.
  • the endogenous olfactory receptor genes can remain functional and wild-type (native) activity can still be present. In other situations, where it is desirable that all olfactory receptor activity is by the introduced exogenous hybrid receptor, use of a knockout line is preferred.
  • Methods for the construction of non-human transgenic animals, particularly transgenic mice, and the selection and preparation of recombinant constructs for generating transformed cells are well known in the art.
  • Construction of a "knockout” cell and animal is based on the premise that the level of expression of a particular gene in a mammalian cell can be decreased or . completely abrogated by introducing into the genome a new DNA sequence that serves to interrupt some portion of the DNA sequence of the gene to be suppressed.
  • "gene trap insertion” can be used to disrupt a host gene
  • mouse embryonic stem (ES) cells can be used to produce knockout transgenic animals (see, e.g., Holzschu, Transgenic Res 6:97, 1997).
  • the insertion of the exogenous is typically by homologous recombination between complementary nucleic acid sequences.
  • the exogenous sequence is some portion of the target gene to be modified, such as exonic, intronic or transcriptional regulatory sequences, or any genomic sequence which is able to affect the level of the target gene's expression; or a combination thereof.
  • Gene targeting via homologous recombi-nation in pluripotential embryonic stem (ES) cells allows one to modify precisely the genomic sequence of interest. Any technique can be used to create, screen for, propagate, a knockout animal, e.g., see Bijvoet, Hum. Mol. Genet. 7:53, 1998); Moreadith, J. Mol. Med. 75:208, 1997; Tojo, Cytotechnology 19:161, 1995; Mudgett, Methods Mol Biol 48:167, 1995; Longo, Transgenic Res.
  • nucleic acid libraries can also be used as reagents to produce "knockout” human cells and their progeny.
  • the compounds tested as modulators of a sensory receptor family member can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Alternatively, modulators can be genetically altered versions of a sensory receptor gene. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds-can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
  • the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.
  • high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds).
  • potential modulator or ligand compounds potential modulator compounds
  • Such "combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired charac-teristic activity.
  • the compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e. , the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing, of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37:487, 1991; and Houghton et al, Nature 354:84, 1991).
  • peptide libraries see, e.g., U.S. Patent No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37:487, 1991; and Houghton et al, Nature 354:84, 1991.
  • Other chemistries for generating chemical diversity libraries can also be used.
  • Such chemistries include, but are not limited to: peptoids (e.g., WO 91/19735), encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., U.S. Patent No. 5,288,514), diversomers such as hydantoins, benzodiaze-pines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sci. 90:6909, 1993), vinylogous polypep-tides (Hagihara et ⁇ /., J 4rn r. Chem. Soc.
  • a method for simulating a fragrance or flavor sensed by a mammal comprising: for each of a plurality of sensory receptors, or fragments or variants thereof, ascertaining the extent to which the sensory receptor interacts with the fragrance and or flavor; and combining a plurality of compounds, each having a previously-determined interaction with one or more of the sensory receptors, in amounts that together provide a stimulation profile that mimics the profile for the fragrance and or flavor. Interaction of a fragrance and/or flavor with a sensory receptor can be determined using any of the binding or reporter assays described herein.
  • the interactions can be aggregated or a profile generated using known signal processing techniques (e.g., a neural network) as described below.
  • the sensory receptor, or fragments or variants thereof e.g., fusion proteins with reporters, chimeric proteins
  • the plurality of compounds may then be combined to form a mixture. If desired, one or more of the plurality of the compounds can be combined covalently.
  • the combined compounds substantially stimulate at least 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99% or all of the receptors that are substantially stimulated by the fragrance or flavor.
  • a method in which a plurality of standard compounds are tested against a plurality of sensory receptors, or fragments or variants thereof, to ascertain the extent to which the sensory receptors each interact with each standard compound, thereby generating a receptor stimulation profile for each standard compound.
  • These receptor stimulation profiles may then be stored in a relational database on data storage medium.
  • the method may further comprise providing a desired receptor-stimulation profile for an odor and/or taste; comparing the desired receptor stimulation profile to the relational database; and ascertaining one or more combinations of standard compounds that most closely match the desired receptor-stimulation profile.
  • the method may further comprise combining standard compounds in one or more of the ascertained combinations to simulate the odor and/or taste.
  • a further aspect of the invention is to provide a method for representing sensory percep-tion of a particular odor and/or taste in a mammal (e.g., human), comprising: providing values X ⁇ to X n representative of the quantitative stimulation of each of n sensory receptors of the mammal; where n is greater than or equal to 5, n is greater than or equal to 10, n is greater than or equal to 20, n is greater than or equal to 50, n is greater than or equal to 75, n is greater than or equal to 100, n is greater than or equal to 125, n is greater than or equal to 150, n is greater than or equal to 175, n is greater than or equal to 200, n is greater than or equal to 225, n is greater than or equal to 250, n is greater than or equal to 275, n is greater than or equal to 300, n is greater than or equal to 325, or n is greater than or equal to 350; and generating from the values a quantitative representation of sensory perception.
  • the sensory receptors may be a receptor disclosed herein, or fragments or variants thereof.
  • the representation may constitute a point or a volume in n-dimensional space, may constitute a graph or a spectrum, or may constitutes a matrix of quantita-tive representations.
  • the providing step may comprise contacting a plurality of recombi-nantly-produced sensory receptors, or fragments or variants thereof, with a composition and quantitatively measuring the interaction of the composition with the receptors.
  • the maximum number of taste receptors that are needed to mimic the native repertoire e.g. , about 50
  • the maximum number of olfactory receptors that are needed e.g., about 350. But the number of sensory receptors that need to be represented in an assay to provide useful results may be much less.
  • the sensory receptors used in this method may include a receptor disclosed herein, or fragment or variant thereof.
  • the maximum number of taste receptors that are needed to mimic the native repertoire e.g., about 50
  • the maximum number of olfactory receptors that are needed e.g. , about 350. But the number of sensory receptors that need to be represented in an assay to provide useful results may be much less.
  • Assaying for ligand-receptor binding in a large set of sensory receptors is envisioned to mimic the function of the nose or tongue in sensory perception of chemical sensants.
  • the resultant signal is a summa-tion of the interactions of all sensory receptors in the set: none, one, or a few of which have ligand-binding domains that are a perfect fit for the sensant; some of which have ligand-binding domains that are only an imperfect fit for the sensant; and most of which do not significantly bind to the sensant.
  • the individual sensory receptor's binding affinity for sensant varies accordingly.
  • a suitably large set of ligand-binding domains from sensory receptors will be analogous to any ability of the native repertoire of olfactory or taste receptors to tolerate varying degrees of mismatch between ligand and ligand-binding domain.
  • the complication of desensiti-zation of sensory receptor signaling will also be avoided by using the invention instead of prepa-rations of nasal or tongue epithelium containing native sensory receptors.
  • the event of binding between ligand and receptor can be separated from more complicated downstream signaling events in the epithelial cells. But the latter events can be reconstituted in the invention by transferring the sensory receptor of interest into appropriate host cells containing an intact and functional signaling pathway.
  • Sensory modulators can be administered directly to a mammal (e.g., human) for modu-lation of sensory perception in vivo. Administration is by any of the routes normally used for introducing a modulator compound into ultimate contact with the tissue to be treated (e.g., nose or tongue).
  • the olfactory modulators are administered in any suitable manner, optionally with acceptable carriers. Suitable methods of administering such modulators are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Acceptable carriers are determined at least in part by the particular components of the composition to be administered (e.g., stabilizing the sensants), as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington 's Pharmaceutical Sciences, 17th ed. 1985).
  • the sensory modulators can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichloro-difluoromethane, propane, nitrogen, and the like that may or may not contribute to sensory perception.
  • pressurized acceptable propellants such as dichloro-difluoromethane, propane, nitrogen, and the like that may or may not contribute to sensory perception.
  • Other possible formulation include dry or liquid forms, powders or tablets, solutions of polar (e.g., water) or nonpolar (e.g., alcohol) solvents, emulsions or suspensions, creams, gels, lotions, and syrups.
  • Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants; buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered, for example, by orally, topically, intravenously, intraperitoneally, intravesically, or intrathecally.
  • the compositions are administered orally or nasally.
  • the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
  • Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the modu-lators can also be administered as part a of prepared drug, food, or cosmetic.
  • an un-pleasant odor or taste e.g., sulfur or bitter, respectively
  • a pleasant odor or taste can be mimicked or enhanced.
  • Primary sensants are prefened because the subset of activated cells is kept small and the effects limited to projection into a specific region of the brain.
  • novel olfactants or combinations thereof that bind only a few olfactory receptors would also be useful.
  • the dose administered to a mammal should be sufficient to effect a bene-ficial response in the subject over time.
  • the dose will be determined by the efficacy of the parti-cular sensory modulators employed and the condition of the subject, as well as the body weight or surface area of the area to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular subject.
  • the effective amount of the modulator to be administered in a physician may evaluate circulating plasma levels of the sensory modulator, modulator toxicities,, and the production of anti-modulator antibodies.
  • the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for the typical mammal.
  • sensory modulators can be administered at a rate determined by the ED 50 of the modulator, and the side-effects of the inhibitor at various concentrations, as applied to the mass and overall health of the mammal. Administration can be accomplished via single or divided doses.
  • Sensory receptor genes are useful tools for identifying cells expressing sensory receptors, for forensics and paternity determinations, and for examining signal transduction in isolated cells.
  • Sensory receptor family member-specific reagents that specifically hybridize to sensory receptor nucleic acids, such AOFLF1 probes and primers, and sensory receptor specific reagents that specifically bind to a sensory receptor protein, e.g., anti-sensory receptor antibodies are used to examine expression in cells and regulation of signal trans-duction.
  • one or more family member-specific reagents may be used to detect poly-morphisms that are linked to genetic anosmia or to detect allelic exclusion.
  • Nucleic acid assays for the presence of DNA and RNA for a sensory receptor family member in a sample include numerous techniques are known to those skilled in the art, such as Southern analysis, Northern analysis, dot blots, RNase protection, SI analysis, amplification techniques such as PCR, and in situ hybridization.
  • in situ hybridization for example, the target nucleic acid is liberated from its cellular sunoundings in such as to be available for hybrid-ization within the cell while preserving the cellular morphology for subsequent interpretation and analysis.
  • a sensory receptor protein can be detected with the various immunoassay techniques described above.
  • the test sample is typically compared to both a positive control (e.g., a sample expressing a recombinant sensory receptor protein) and a negative control.
  • kits for screening for novel modulators of sensory receptor family members can be prepared from readily available materials and reagents, as well as any of the aforementioned products.
  • kits can comprise any one or more of the following materials: sensory receptor nucleic acids or proteins, reaction tubes, and instructions for testing sensory receptor activity.
  • the kit contains a biologically active sensory receptor.
  • AOLFRl sequences can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user. Examples AOLFRl sequences:
  • AOLFR12 sequences MERNHNPDNCNVLNFFFADKXNK-RRNFGQIVSDVGRICYSVSLSLGEPTTMGRNNLTRPSEFIL LGLSSRPEDQKPLFAVFLPIY ITVIGNLLIILAIRSDTRLQTPMYFFLSILSFVDICYVTVIIPKMLV NFLSETKTISYGECLTQMYFFLAFGNTDSYLLAAMAIDRYVAICNPFHYITIMSHRCCVLLLVLS FCIPHFHSLLHILLTNQLIFCASNVIHHFFCDDQPVLKLSCSSHFVKEITVMTEGLAVIMTPFSCIII SYLRILITVLKIPSAAGK-1-U AFSTCGSHLTVVTLFYGSISYVYFQPLSNYTVKDQIATIIYTVLTP MLNPF-CYSL-RNKD KQGLA LMHRMKCQ (SEQ ID NO: 23) ATGGAAAGAAACCACAATCCAGATAATTGTAATGTTTTAAATTTTCTTTGCTGATAAGA
  • AOLFR16 sequences MRRNCTLVTEFILLGLTSRRELQILLFTLFLAIYMVTVAGNLGMIVLIQANAWLHMPMYFFLSH LSFVDLCFSSNVTPKMLEIFLSEKJ SISYPACLVQCYLFIALVHVEIYILAVMAFDRYMAICNPLL YGSRMSKSVCSFLITVPYVYGALTGLMETMWTYNLAFCGPNEINHFYCADPPLIKLACSDTYN KELSMFIVAGWNLSFSLFIICISYLYIFPAILKIRSTEGRQKAFSTCGSHLTAVTIFYATLFFMYLR PPSKESVEQG-O ⁇ VAVFYTTVIPMLNLIIYSLRNK-NV- ALIK ⁇ LSMKIYFS (SEQ ID NO: 31)
  • AOLFR26 sequences MAAKNSSVTEFILEGLTHQPGLRIPLFFLFLGFYTVTVVGNLGLITLIGLNSHLHTPMYFFLFNLS LIDFCFSTTITPKMLMSFVSRKNIISFTGCMTQLFFFCFFVVSESFILSAMAYDRYVAICNPLLYT VTMSCQVCLLLLLGAYGMGFAGAMAHTGSIMNLTFCADNLVNHFMCDILPLLELSCNSSYMN ELVVFIVVAVDVGMPIVTVFISYALILSSILHNSSTEGRSKAFSTCSSHIIWSLFFGSGAFMYLKP LSILPLEQGKVSSLFYTIIVPVLNPLIYSLRNKDVKVALRRTLGRKIFS (SEQ ID NO: 49)
  • AOLFR37 sequences MEKANETSPVMGFVLLRLSAHPELEKTFFVLILLMYLVILLGNGVLILVTILDSRLHTPMYFFLG NLSFLDICFTTSSVPLVLDSFLTPQETISFSACAVQMALSFAMAGTECLLLSMMAFDRYVAICNP LRYSVIMSKAAYMPMAASSWAIGGAASVVHTSLAIQLPFCGDNVINHFTCEILAVL LACADIS INVISMEVTNVIFLGVPVLFISFSY IITTILRIPSAEGRKKVFSTCSAHLTVVIVFYGTLFFMYG KPKSKDSMGADKEDLSDKLIPLFYGVVTPMLNPIIYSLPJ> «I)VKAAVRRLLRPKGFTQ (SEQ ID NO: 69)
  • AOLFR38 sequences MYLVTVLRNLLIILAVSSDSHLHTPMCFFLSNLC ADIGFTSAMVPKMIVDMQSHSRVISYAGC LTQMSFFVLFACIEDMLLTVMAYDRFVAICHPLHYPVIiSWPHLGVFLVLVSFFLSLLDSQLHSW IVLQFTFFKNVEISNFVCDPSQLLNLACSDSVINSIFIYLDSIMFGFLPISGILLSYANNVPSILRISS SDRKSKAFSTCGSHLAWCLFYGTGIGVYLTSAVSPPPRNGVVASVMYAVVTPMLNPFIYSLR NRDIQSALWRLRSRTVESHDLLSQDLLHPFSCVGE GQPH (SEQ ID NO: 71)
  • AOLFR60 sequences MFLPNDTQFHPSSFLLLGIPGLETLHIWIGFPFCAVYMIALIGNFTILLVIKTDSSLHQPMFYFLA MLATTDVGLSTATIP- ⁇ LGIFWINLRGIIFEACLTQMFFIHNFTLMESAVLVAMAYDSYVAICN PLQYSAILTNKVVSVIGLGVFVRALIFVIPSILLILRLPFCGNHVIPHTYCEHMGLAHLSCASIKINI IYGLCAICNLVFDITVIALSYVHILCAVFRLPTHEPRLKSLSTCGSHVCVILAFYTPALFSFMTHC FGRNVPRYIHILLANLYVVWPMLNPVIYGVRTKQIYKCVK- LLQEQGMEKEEYLIHTRF (SEQ ID NO: 109)
  • AOLFR66 sequences MSFLNGTSLTPASFILNGIPGLED VHLWISFPLCTMYSIAITGNFGLMYLIYCDEALHRPMYVFL ALLSFTDVLMCTSTLPNTLFIL TNLKEroFKACLAQMFFVHTFTGMESGVLMLMALDHCVAI CFPLRYATILTNSVLAKAGFLTFLRGVMLVIPSTFLTKRLPYCKGNVIPHTYCDHMSVAKISCGN VRVNAIYGLIVALLIGGFDILCITISYTMILQAWSLSSADARQKAFSTCTAHFCAIVLTYVPAFF TFFTHHFGGHTIPLHII]V-ANLYLLMPPTMNPIVYGVT TRQVRESVIRFFLKGKDNSHNF (SEQ ID NO: 121)
  • AOLFR72 sequences MAPENFTRVTEFILTGVSSCPELQIPLFL LVLYGLTMAGNLGIITLTSVDSRLQTPMYFFLQHL AL- ⁇ STLGNSTVIAPKMLINFLVK- TTSFYECATQLGGFLFFIVSEVIMLALJN ⁇ YMVWSRRLCLLLVSLTYLYGFSTAIWSSYVFSVSYCSSNIINHFYCDNVPLLALSCSDTYLPE TVWISAATNVNGSLIIVLVSYFNIVLSILKICSSEGRK AFSTCASHMMAVTIFYGTLLF- ⁇ RSNHSLDTDDKMASWYTLVIPMLNPLIYSLRNKDVKTALQRFMTNLCYSFKTM (SEQ ID NO: 133)
  • AOLFR89 sequences MLDPSISSHTLYLHSLFPQGLRKGTMWQKNQTSLADFILEGLFDDSLTHLFLFSLTMWFLLAVS GNTLTILLIC-TOPQLHTPMYFLLSQLSLMDLMHVSTTILKMATNYLSGKKSISFVGCATQHFLYL CLGGAECFLLAVMSYDRYVAICHPLRYAVL- -D «-KVGLMMAVMSWLGASVNSLIHMAILMHF PFCGPRKVYIffYCEFPAVVKLVCGDITVYETTVYISSILLLLPIFLISTSYVFILQSVIQMRSSGSK RNAFATCGSHLTVVSLWFGACIFSYMRPRSQCTLLQNKVGSVFYSIITPTLNSLIYTLR-NKDVA KALRRVLRRDVITQCIQRLQLWLPRV (SEQ ID NO: 165)
  • AOLFR94 sequences METWVNQSYTDGFFLLGIFSHSTADLVLFSVVMAVFTVALCGNVLLIFLIYMDPHLHTPMYFF LSQLSLMDLMLVCTNVPKMAANFLSGRKSISFVGCGIQIGLFVCLVGSEGLLLGLMAYDRYVA ISHPLHYPILMNQRVCLQITGSSWAFGIIDGLIQMVV ⁇ MNFPYCGLRKVNHFFCEMLSLLKLAC VDTSLFEKVIFACCVFMLLFPFSIIVASYAHILGTVLQMHSAQAWKKALATCSSHLTAVTLFYG AAMFIYLRPRHYRAPSHDKVASIFYTVLTPMLNPLIYSLRNREVMGALRKGLDRCRIGSQH (SEQ ID NO: 175)
  • AOLFR95 sequences MLGSKPRVHLYILPCASQQVSTMGDRGTSNHSEMTDFILAGFRVRPELHILLFLLFLFVYAMILL GNVGMMTIIMTDPRLNTPMYFFLGNLSFIDLFYSSVIEPKAMINFWSENKSISFAGCVAQLFLFA LLIVTEGFLLAAMAYDRFIAICNPLLYSVQMSTRLCTQLVAGSYFCGCISSVIQTSMTFTLSFCAS -I vT)HFYCDSRPLQRLSCSDLFIHRMISFSLSCIIILPTIIVIIVSYMYIVSTVLKIHSTEGHKKAFST CSSHLGVVSVLYGA FMYLTPDRFPELSKVASLCYSLVTPMLNPLIYSLRNKDVQEALKKFLE KKNIIL (SEQ ID NO: 177)
  • AOLFR96 sequences MICENHTRVTEFILLGFTNNPEMQVSLFIFFLAIYTVTLLGNFLIVTVTSVDLALQTPMYFFLQN LSLLEVCFTLVMVPKMLVDLVSPRKIISFVGCGTQMYFFFFFGSSECFLLSMMAYDRFVAICNP LHYSV-OS -NRSLCLWMAIGSWMSGV VSMLQTAWMMALPFCGPNAVDHFFCDGPPVLKLVTV DTTMYEMQALASTLLFIMFPFCLILVSYTRIIITILRMSSATGRQKAFSTCSSHLIWSLFYGTASL TYLRPKSNQSPESKKLVSLSYTVITPMLNPIIYGLRNNEVKGAVKRTITQKVLQKLDVF (SEQ ID NO: 179)
  • AOLFR97 sequences MTEFHLQSQMPSIRLIFRRLSLGRIKPSQSPRCSTSFMVVPSFSIAEHWRRMKGANLSQGMEFEL LGLTTDPQLQRLLFWFLGMYTATLLGNLVMFLLIHVSATLHTPMYSLLKSLSFLDFCYSSTVV PQTLVNFLAK-RKVISYFGCMTQMFFYAGFATSECYLIAAMAYDRYAAICNPLLYSTIMSPEVC ASLIVGSYSAGFLNSLIHTGCIFSLKFCGAHWTHFFCDGPPILSLSCVDTSLCEILLFIFAGFNLLS CTLTILISYFLILNTILKMSSAQGRFKAFSTCASHLTAICLFFGTTLFMYLRPRSSYSLTQDRTVA VIYTVVIPVLNPLMYSLPJ IKDVKKALIKV GRKTME (SEQ ID NO: 181)
  • AOLFR99 sequences MERVNETVV-REVIFLGFSSLARLQQLLFVIFLLLYLFTLGTNAIIISTIVLDRALHIPMYFFLAILSC SEICYTFIIVPKMLVDLLSQKKTISFLGCAIQMFSFLFLGCSHSFLLAVMGYDRYLAICNPLRYSV LMGHGVCMGLVA--AACACGFTVAQIITSLVFHLPFYSSNQLHHFFCDIAPVLKLASHHNHFSQIV IFMLCTLVLAIPLLLILVSYVHILSAILQFPSTLGRCKAFSTCVSHLIIVTVHYGCASFIYLRPQSNY SSSQDALISVSYTIITPLFNPMIYSLRNKEFKSALCKIVRRTISLL (SEQ ID NO: 185)
  • HYPTLMTPTLCAEI GCWLGGLAGPVVEISLISRLPFCGPNRIQHVFCDFPPVLSLACTDTSINV LVDFVINSCKILATFLLILCSYVQIICTVLRIPSAAGKRKAISTCASHFTVVLIFYGSILSMYVQLK KSYSLDYDQALAVVYSVLTPFLNPFr ⁇ SLRNKEIKEAVRRQLKRIGILA (SEQ ID NO: 187)
  • AOLFR105 sequences MQGLNHTSVSEFILVGFSAFPHLQLMLFLLFLLMYLFTLLGNLLIMATVWSERSLHMPMYLFLC ALSITEILYTVAIIPRMLADLLSTQRSIAFLACASQMFFSFSFGFTHSFLLTVMGYDRYVAICHPL RYNVLMSLRGCTCRVGCSWAGGLVMGMWTSAIFHLAFCGHKEIHHFFCHVPPLLKLACGDD VLVVAKGVGLVCITALLGCFLLILLSYAFrVAAILKIPSAEGRNl- A FSTCASHLTVVVNHYGFAS VIYLKPKGPQSPEGDTLMGITYTVLTPFLSPIIFSLR-NKELKVAMKKTCFTKLFPQNC (SEQ ID NO: 193)
  • AOLFR106 sequences METANYTKVTEFVLTGLSQTPEVQLVLFVIFLSFYLFILPGNILIICTISLDPHLTSPMYFLLANLA FLDIWYSSITAPEMLIDFFVERKIISFDGCIAQLFFLHFAGASEMFLLTVMAFDLYTAICRPLHYA TIMNQRLCCILVALSWRGGFms ⁇ QVALIVRLPFCGPNELDSYFCDITQVVRIACANTFPEELVM ICSSGLISVVCLIALLMSYAFLLALFKKLSGSGENTNRAMSTCYSHITIWLMFGPSIYIYARPFD SFSLDKVYSVFNTLIFPLRNPIIYTLPJ ⁇ JKEVKAAMRKLVTKYILCKEK (SEQ ID NO: 195)
  • AOLFR114 sequences MERINHTSSVSEFILLGLSSRPEDQKTLFVLFLIVYLVTITGNLLIILAIRFNPHLQTPMYFFLSFLS LTDICFTTSVW-IO lLMNFLSEK-KTISYAGCLTQMYFLYALGNSDSCLLAVMAFDRYVAVCDPF HYVTTMSHHHCVLLVAFSCSFPHLHSLLHTLLLNRLTFCDSNVIHHFLCDLSPVLKLSCSSIFVN EIVQMTEAPIVLVT-RFLCL FSYIRILTTVLOPSTSGKRKAFSTCGFYLTVVTLFYGSIFCVYLQP PSTYAVKDHVATIVYTVLSSMLNPFIYSLRNKDLKQGLRKLMSKP.S (SEQ ID NO: 209) ATGGAAAGAATCAACCACACCAGCAGTGTCTCCGAGTTTATCCTCCTGGGACTCTCCTCCC GGCCTGAGGACCAAAAGACACTCTTTGTTCTCTTCCTCATCGTGT
  • AOLFR116 sequences MDEANHSVVSEFVFLGLSDSRKIQLLLFLFFSVFYVSSLMGNLLIVLTVTSDPRLQSPMYFLLAN LSIINLVFCSSTAJPKMIYDLFR-KHKTISFGGCVVQffFIED VGGTEMVLLIAMAFDRYVAICKPLH YLTIMNPQRCILFLVISWIIGIIHSVIQLAFWDLLFCGPNELDSFFCDLPRFIKLACIETYTLGFMV TANSGFISLASFLILIISYIFILVTVQKKSSGGIFKAFSMLSAHVIVWLVFGPLIFFYIFPFPTSHLD KFLAIFDAVITPVLNPVIYTFRNKEMMVAMRRRCSQFVNYSKIF (SEQ ID NO: 213)
  • AOLFR118 sequences MNHMSASLKISNSSKFQVSEFILLGFPGIHSWQHWLSLPLALLYLSALAANTLILIIIWQNPSLQQ PMYIFLGILCMVDMGLATTIIPKILAIFWFDAKVISLPECFAQIYAIHFFVGMESGILLCMAFDRY VAICHPLRYPSIVTSSLILKATLFMVLRNGLFVTPVPVLAAQRDYCSKNEIEHCLCSNLGVTSLA CDDRRPNSICQLVLAWLGMGSDLSLIILSYILILYSVLRLNSAEAAAKALSTCSSHLTLILFFYTIV VVISVTHLTEMKATLIPVLLNVLHNIIPPSLNPTVYALQTKELRAAFQKVLFALTKEIRS (SEQ ID NO: 217)
  • AOLFR120 sequences MQP YTKNWTQVTEF VMMGFAGIHEAHLLFFILFLTMYLFTLVENLAIILWGLDHRLRRPMYF FLTHLSCLEIWYTSVTVPKMLAGFIGVDGGKNISYAGCLSQLFIFTFLGATECFLLAAMAYDRY VAICMPLHYGAFVSWGTCIRLAAACWLVGFLTPILPIYLLSQLTFCGPNVIDHFSCDASPLLALS CSDVTWKETVDFLVSLAVLLASSMVIAVSYGNIVWTLLHIRSAAERWKAFSTCAAHLTWSLF YGTLFFMYVQTKVTSSIr ⁇ NKVVSWYSVVTPMLNPLIYSLRNKEVKGALGRVFSLNFWKGQ (SEQ ID NO: 221)
  • AOLFR121 sequences MKRKNFTEVSEFIFLGFSSFGKHQITLFWFLTVYILTLVANIIIVTIICIDHHLHTPMYFFLSMLA SSETVYTLVIVPRMLLSLIFHNQPISLAGCATQMFFFVILATNNCFLLTAMGYDRYVAICRPLRY TVIMSKGLCAQLVCGSFGIGLTMAVLHVTAMFNLPFCGTVVDHFFCDIYPVMKLSCIDTTINEII NYGVSSFVIFVPIGLIFISYVLVISSILQLASAEGRKKTFATCVSHLTVV ⁇ VHCGCASIAYLKPKSES SIEKDLVLSVTYTIITPLLNPVVYSLRNKEVKDALCRVVGRNIS (SEQ ID NO: 223)
  • AOLFR124 sequences MNHSVVTEFIILGLTK-KPELQGIIFLFFLIVYLVAFLGNMLIIIA- YNNTLHTPMYVFLLTLAVV DIICTTSIIPKMLGTMLTSENTISYAGCMSQLFLFTWSLGAEMVLFTTMAYDRYVAICFPLHYST VM- raHMCVALLSMVMAIAVTNSWVHTALIMRLTFCGPNTIDHFFCEIPPLLALSCSPVPJNEV MVYVADITLAIGDFILTCISYGFIIVAILRIRTVEGKRKAFSTCSSHLTVVTLYYSPVIYTYIRPASS YTFERDKWAALYTLVTPTLNPMVYSFQNREMQAGIRKVFAFLKH (SEQ ID NO: 229)
  • AOLFR127 sequences MSNEDMEQDNTTLLTEFVLTGLTYQPE KMPLFLVFLVIYLITIV NLGLLALIWNDPQLHIPM YFFLGSLAEVDAWISSTVTP-KMLVNFLAK-NRMISLSECMIQFFSFAFGGTTECFLLATMAYDRY VAICKPLLYPVIMNNSLCIRLLAFSFLGGFLHALIHEVLIFRLTFCNSNIIHHFYCDIIPLFMISCTD PSINELMWILSGSIQVFTIVTVLNSYTFALFTILKKKSVRGVRKAFSTCGAHLLSVSLYYGPLIF MYLI ⁇ ASPQADDQDMIDSVFYT ⁇ iPLLNPIIYSLRNKQVIDSFTKMVKRNV (SEQ ID NO: 235)
  • AOLFR133 sequences MTEFIFLVLSPNQEVQRVCFVIFLFLYTAIVLGNFLIVLTVMTSRSLGSPMYFFLSYLSFMEICYS SATAPKX,ISDLLAERKVISWWGCMAQLFFLHFFGGTEIFLLTVMAYDHYVAICKPLSYTTIMN WQVCTVLVGIAWVGGFMHSFAQILLIFHLLFCGPNVINHYFCDLVPLLKLACSDTFLIGLLIVAN GGTLSVISFGVLLASYMVILLHLRTWSSEGWCKALSTCGSHFAWILFFGPCVFNSLRPSTTLPI DKMVAWYTVITAILNPVIYSLRNAEMRKAMKRLWIRTLRLNEK (SEQ ID NO: 245)
  • AOLFR136 sequences MTMENYSMAAQFVLDGLTQQAELQLPLFLLFLGIYWTVVGNLGMILLIAVSPLLHTPMYYFL SSLSFVDFCYSSVITPKMLVNFLGKKNTILYSECMVQLFFFWFVVAEGYLLTAMAYDRYVAIC SPLLYNAIMSSWVCSLLVLAAFFLGFLSALTHTSAMMKLSFCKSHIINHYFCDVLPLLNLSCSNT HLNELLLFIIAGFNTLVPTLAVAVSYAFILYSILHIRSSEGRSKAFGTCSSHLMAVVIFFGSITFMY FK PSSNSLDQEKVSSVFYTTVIPMLNPLIYSLRNKDVKKALRKVLVGK (SEQ ID NO: 251)
  • AOLFR140 sequences MLTLNKTDLIPASFILNGVPGLEDTQLWISFPFCSMYVVAMVGNCGLLYLIHYEDALHKPMYY FLAMLSFTDLVMCSSTIPKALCIFWFHLKDIGFDECLVQMFFIHTFTGMESGVLMLMALDRYV AICYPLRYSTILTNPVIAKVGTATFLRGVLLIIPFTFLTKRLPYCRGNILPHTYCDHMSVAKLSCG NVKVNAIYGLMVALLIGGFDILCITISYTMILRAWSLSSADARQKAFNTCTAHICAIVFSYTPAF FSFFSHRFGEHIIPPSCHIIVANIYLLLPPTMNPIVYGVKTKQIRDCVIRILSGSKDTKSYSM (SEQ ID NO: 259)
  • AOLFR141 sequences MSSTLGHNMESPNHTDVDPSVFFLLGIPGLEQFHLWLSLPVCGLGTATIVGNITILVWATEPVL HKPVYLFLCMLSTIDLAASVSTVPKLLAIFWCGAGHISASACLAQMFFIHAFCMMESTVLLAM AFDRYVAICHPLRYATILTDTIIAHIGVAAWRGSLLMLPCPFLIGRLNFCQSHVILHTYCEHMA VVKLACGDTRPNRVYGLTAALLVIGVDLFCIGLSYALSAQAVLRLSSHEARSKALGTCGSHVC VILISYTPALFSFFTHRFGHHVPVHIHILLANVYLLLPPALNPVVYGVKTKQIRKRVVRVFQSGQ GMGIKASE (SEQ ID NO: 261)
  • AOLFR150 sequences MELGNVTRVKEFIFLGLTQSQDQSLVLFLFLCLVYMTTLLGNLLIMVTVTCESRLHTPMYFLLR NLAILDICFSSTTAPKVLLDLLSKKKTISYTSCMTQIFLFHLLGGADIFSLSVMAFDCYMAISKPL HYVTIMSRGQCTALISASWMGGFVHSIVQISLLLPLPFCGPNVLDTFYCDVPQVLKLTCTDTFA LEFLMISNNGLVTTLWFIFLLVSYTVILMTLRSQAGGGRRKAISTCTSPHHCGDPAFCALHLCLC PALHCPPHRKGHLCHLHCHLPSAEPFDLHSEEPGNEVSHEKTEEKTRAF (SEQ ID NO: 277)
  • AOLFR159 sequences MGPRNQTAVSEFLLMKVTEDPELKLIPFSLFLSMYLVTILGNLLILLAVISDSHLHTPMYFLLFN LSFTDICLTTTTVPKILVNIQAQNQSITYTGCLTQICLVLVFAGLESCFLAVMAYDRYVAICHPL RYTVLMNVHFWGLLILLSMFMSTMDALVQSLMVLQLSFCKNVEIPLFFCEWQVIKLACSDTL INNILIYFASSVFGAIPLSGIIFSYSQIVTSVLRMPSARGKYKAFSTCGCHLSVFSLFYGTAFGVYIS SAVAESSRITAVASVMYTVVPQMMNPFIYSLRNKEMK1 LRKLIGRLFPF (SEQ ID NO: 291)
  • AOLFR161 sequences MEPRNQTSASQFILLGLSEKPEQETLLFSLFFCMYLVMWGNLLIILAISIDSHLHTPMYFFLANL SLVDFCLATNTIPKMLVSLQTGSKAISYPCCLIQMYFFHFFGIVDSVIIAMMAYDRFVAICHPLH YAKIMSLRLCRLLVGALWAFSCFISLTHILLMARLVFCGSHEVPHYFCDLTPILRLSCTDTSVNR IFILIVAGMVIATPFVCILASYARILVAIMKVPSAGGRKKAFSTCSSHLSWALFYGTTIGVYLCP SSVLTTVKEKASAVMYTAVTPMLNPFIYSLRNRDLKGALRKLVNRKITSSS (SEQ ID NO: 295)
  • AOLFR170 sequences MSFTSLIPSLCFSLTLPFLFCYLSLLPFLSAFLFITRWLLAFLSLFSVSVPVSSVSSSMVLCLYLSVS ASPSVFCFSCMQGPILWIMANLSQPSEFVLLGFSSFGELQALLYGPFLMLYLLAFMGNTIIIVMVI ADTHLHTPMYFFLGNFSLLEILVTMTAVPRMLSDLLVPHKVITFTGCMVQFYFHFSLGSTSFLIL TDMALD-RFVAICHPLRYGTLMSRAMCVQLAGAAWAAPFLAMVPTVLSRAHLDYCHGDVINH FFCDNEPLLQLSCSDTRLLEF DFLMALTFVLSSFLVTLISYGYIVTTVLRIPSASSCQKAFSTCG SHLTLVFIGYSSTIFLYVRPGKAHSVQVRKVVALVTSVLTPFLNPFILTFCNQTVKTVLQGQMQ RLKGLCKAQ (SEQ ID NO: 313)
  • AOLFR171 sequences MVGNLLIWVTTIGSPSLGSLMYFFLAYLSLMDAIYSTAMSPKLMIDLLCDKIAISLSACMGQLFI EHLLGGAEWLLVVMAYDRYVAISKPLHYLNIMNRLVCILLLVVAMIGGFVHSVVQIVFLYSLP ICGPNVIDHSVCDMYPLLELLCLDTYFIGLTWANGGIICMVIFTFLLISCGVILNFLKTYSQEER HKALPTCISHIIVVALWWCIFMYVRPVSNFPFDKLMTVFYSIITLMLNPLIYSLRQSEM-O ⁇ AM KNLWCEKLSIVRKRVSPTLNIFIPSSKATNRR (SEQ ID NO: 315)
  • AOLFR180 sequences MTNKMYAIYIKNLNYFSFLIVQCLQPTMAIFNNTTSSSSNFLLTAFPGLECAHVWISIPVCCLYTI ALLGNSMIFLVIITK-RRLH-KPMYYFLSMLAAVDLCLTITTLPTVLGVLWFHAREISFKACFIQMF FVHAFSLLESSVLVAMAFDRFVAICNPLNYATILTDRMVLVIGLVICIRPAVFLLPLLVAINTVSF HGGHELSHPFCYHPEVIKYTYSKPWISSFWGLFLQLYLNGTDVLFILFSYVLILRTVLGIVARKK QQKALSTCVCHICAVTIFYVPLISLSLAHRLFHSTPRVLCSTLANIYLLLPPVLNPIIYSLKTKTIR QAMFQLLQSKGSWGFNVRGLRGRWD (SEQ ID NO: 331)
  • AOLFR182 sequences MTLGSLGNSSSSVSATFLLSGIPGLERMHIWISIPLCFMYLVSIPGNCTILFIIKTERSLHEPMYLFL SMLALIDLGLSLCTLPTVLGIFWVGAREISHDACFAQLFFIHCFSFLESSVLLSMAFDRFVAICHP LHYVSILTNTVIGRIGLVSLGRSVALIFPLPFMLKRFPYCGSPVLSHSYCLHQEVMKLACADMK ANSIYGMFVIVSTVGIDSLLILFSYALILRTVLSIASRAERFKALNTCVSHICAVLLFYTPMIGLSV IHRFGKQAPHLVQVVMGFMYLLFPPVMNPIVYSVKTKQIRDRVTHAFCY (SEQ ID NO: 335)
  • AOLFR196 sequences MLESNYTMPTEFLFVGFTDYLPLRVTLFLVFLLVYTLTMVGNILLIILVNINSSLQIPMYYFLSNL SFLDISCSTAITPKMLANFLASRKSISPYGCALQMFFFASFADAECLILAAMAYDRYAAICNPLL YTTLMSRRVCVCFIVLAYFSGSTTSLVHVCLTFRLSFCGSNIVNHFFCDIPPLLALSCTDTQINQL LLFALCSFIQTSTFWIFISYFCILITVLSIKSSGGRSKTFSTCASHLIAVTLFYGALLFMYLQPTTS YSLDTDKVVA YTVWPMFNPIIYSFRN ⁇ VKN ⁇ ID NO: 363)
  • AOLFR199 sequences MDTGNKTLPQDFLLLGFPGSQTLQLSLFMLFLVMYILTVSGNVAILMLVSTSHQLHTPMYFFLS NLSFLEIWYTTAAVPKALAILLGRSQTISFTSCLLQMYFVFSLGCTEYFLLAAMAYDRCLAICYP LHYGAIMSSLLSAQLALGSWVCGFVAIAVPTALISGLSFCGPRAINHFFCDIAP IALACTNTQA VELVAFVIAVWILSSCLITFVSYVYIISTILRIPSASGRSKAFSTCSSHLTWLIWYGSTVFLHVR TSIKDALDLIKAVHVLNTVVTPVLNPFIYTLRNKEVRETLLKKWKGK (SEQ ID NO: 369)
  • AOLFR212 sequences MAGNNFTEVTVFILSGFANHPELQVSLFLMFLFIYLFTVLGNLGLITLIRMDSQLHTPMYFFLSN LAFIDIFYSSTVTPKALVNFQSNRRSISFVGCFVQMYFFVGLVCCECFLLGSMAYNRYIAICNPL LYSVVMSQKVSNWLGVMPYVIGFTSSLISVWVISSLAFCDSSINHFFCDTTALLALSCVDTFGT EMVSFVLAGFTLLSSLLIITVTYIIIISAILRIQSAAGRQKAFSTCASHLMAVTIFYGSLIFTYLQPD NTSSLTQAQVASWYTIVIPMLNPLIYSLR--SIKDVKNALLRVIHRKLFP (SEQ ID NO: 395)
  • AOLFR214 sequences MDKSNSSWSEFVLLGLCSSQKLQLFYFCFFSVLYTVIVLGNLLIILTVTSDTSLHSPMYFLLGN LSFVDICQASFATPKMIADFLSAHETISFSGCIAQIFFIHLFTGGEMVLLVSMAYDRYVAICKPLY YVVIMSRRTCTVLVMISWAVSLVHTLSQLSFTVNLPFCGPNVVDSFFCDLPRVTKLACLDSYIIE ILIVVNSGILSLSTFSLLVSSY ⁇ iLVTVWLKSSAAMAKAFSTLASHIAVVILFFGPCIFIYVWPFTIS PLD-mAIFYTVFTPVLNPIIYTLRM ⁇ MKAAV ⁇ (SEQ ID NO: 399)
  • AOLFR221 sequences MRNLSGGHVEEFVLVGFPTTPPLQLLLFVLFFAIYLLTLLENALIVFTIWLAPSLHRPMYFFLGH LSFLELWYINVTIPRLLAAFLTQDGRVSYVGCMTQLYFFIALACTECVLLAVMAYDRYLAICGP LLYPSLMPSSLATRLAAASWGSGFFSSMMKLLFISQLSYCGPNIINHFFCDISPLLNLTCSDKEQA ELVDFLLALVMILLPLLAWSSYTAIIAAILRIPTSRGRHKAFSTCAAHLAWVIYYSSTLFTYAR PRAMYTFNIM ISVLYTIIWFFNPAIYCLRNKEVKEAFRKTVMGRCHYPRDVQD (SEQ ID NO: 413).
  • AOLFR222 sequences MGQTNVTSWRDFVFLGFSSSGELQLLLFALFLSLYLVTLTSNVFIIIAIRLDSHLHTPMYLFLSFL SFSETCYTLGIIPRMLSGLAGGDQAISYVGCAAQMFFSASWACTNCFLLAAMGFDRYVAICAPL HYASHMNPTLCAQLVITSFLTGYLFGLGMTLVIFHLSFCSSHEIQHFFCDTPPVLSLACGDTGPS ELRIFILSLLVLLVSFFFITISYAYILAAILRIPSAEGQKKAFSTCASHLTWIIHYGCASFVYLRPK ASYSLERDQLIAMTYTWTPLLNPIVYSLRTRAIQTALRNAFRGRLLGKG (SEQ ID NO: 415).
  • AOLFR241 sequences MPQILIFTYLNMFYFFPPLQILAENLTMVTEFLLLGFSSLGEIQLALFWFLFLYLVILSGNVTIIS VIHLDKSLHTPMYFFLGILSTSETFYTFVILPKMLINLLSVARTISFNCCALQMFFFLGFAITNCLL LGVMGYDRYAAICHPLHYPTLMSWQVCGKLAAACAIGGFLASLTWNLVFSLPFCSANKVNH YFCDISAVILLACTNTDVNEFVIFICGVLVLVVPFLFICVSYLCILRTILKIPSAEGRRKAFSTCAS HLSVVIVHYGCASFIYLRPTANYVSNKDRLVTVTYTIVTPLLNPMVYSLR KDVQLAIRKVLG KKGSLKLYN (SEQ ID NO: 451).
  • AOLFR242 sequences MNTTLFHPYSFLLLGIPGLESMHLWVGFPFFAVFLTAVLGNITILFVIQTDSSLHHPMFYFLAILS SIDPGLSTSTIPKMLGTFWFTLREISFEGCLTQMFFIHLCTGMESAVLVAMAYDCYVAICDPLCY TLVLTNKWSVMALAIFLRPLVFVIPFVLFILRLPFCGHQIIPHTYGEHMGIARLSCASIRVNIIYG LCAISILVFDIIAIVISYVQILCAVFLLSSHDARLKAFSTCGSHVCVMLTFYMPAFFSFMTHRFGR NIPHFIHILLANFYWIPPALNSVIYGVRTKQIRAQVLKMFFNK (SEQ ID NO: 453).
  • AOLFR245 sequences MDLKNGSLVTEFILLGFFGRWELQIFFFVTFSLIYGATVMGNILIMVTVTCRSTLHSPLYFLLGN LSFLDMCLSTATTPKMIIDLLTDHKTISVWGCVTQMFFMHFFGGAEMTLLIIMAFDRYVAICKP LHYRTIMSHKLLKGFAILSWIIGFLHSISQIVLTMNLPFCGHNVINNIFCDLPLVIKLACIETYTLE LFVIADSGLLSFTCFILLLVSYIVILVSVPKKSSHGLSKALSTLSAHIIWTLFFGPCIFIYVWPFSSL ASNKTLAVFYTVITPLLNPSIYTLRNKKMQEAIRKLRFQYVSSAQNF (SEQ ID NO: 459).
  • AOLFR254 sequences MTNTSSSDFTLLGLLVNSEAAGIVFTVILAVFLGAVTANLVMIFLIQVDSRLHTPMYFLLSQLSI MDTLFICTTVPKLLADMVSKEKIISFVACGIQIFLYLTMIGSEFFLLGLMAYDCYVAVCNPLRYP VLMNRKKCLLLAAGAWFGGSLDGFLLTPITMNVPYCGSRSINHFFCEIPAVLKLACADTSLYET LMYICCVLMLLIPISIISTSYSLILLTIHRMPSAEGRKKAFTTCSSHLTVVSIFYGAAFYTYVLPQS FHTPEQDKVVSAFYTIVTPMLNPLIYSLRNKDVIGAFKKVFACCSSAQKVATSDA (SEQ ID NO: 477).
  • AOLFR130B sequences MEGKNQTAPSEFIILGFDHLNELQYLLFTIFFLTYICTLGGNVFIIWTIADSHLHTPMYYFLGNL ALIDICYTTTNVPQMMVHLLSEKKIISYGGCVTQLFAFIFFVGSECLLLAAMAYDRYIAICKPLR YSFIMNKALCSWLAASCWTCGFLNSVLHTVLTFHLPFCGNNQINYFFCDIPPLLILSCGDTSLNE LALLSIGILISWTPFLCIILSYLYIISTILRIRSSEGRHKAFSTCASHLLIVILYYGSAIFTYVRPISSYS LEKD-RLISVLYSVVTPMLNPVIYTLRNKDIKEAVKAIGRKWQPPVFSSDI (SEQ ID NO: 493)

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Abstract

Cette invention se rapporte à l'utilisation de récepteurs couplés aux protéines G sensorielles, qui reconnaissent des agents sensoriels chimiques, en particulier ceux impliquant des récepteurs olfactifs et gustatifs; à des fragments polypeptidiques et des mutants de ceux-ci; à des classes de ces récepteurs; à des polynucléotides codant ces récepteurs, des fragments et des mutants de ceux-ci et des représentants de classes de récepteurs; à des vecteurs génétiques contenant ces polynucléotides; et à des cellules et des organismes non humains génétiquement modifiés pour exprimer ces complexes de récepteurs, des fragments et des mutants d'un récepteur olfactif ou gustatif, et des représentants de classes de récepteurs, en vue de simuler la perception sensorielle d'agents odorants et d'agents gustatifs. Cette invention concerne également l'utilisation de ces produits comme biocapteurs ou comme constituants de biocapteurs pour détecter, identifier, mesurer ou traiter de quelque façon que ce soit la liaison entre le récepteur et son ligand apparenté (c'est-à-dire agent sensoriel chimique). Cette invention trouve une application notamment dans la mise au point et la formulation de compositions d'agents odorants et d'agents gustatifs.
PCT/US2001/020122 2000-06-22 2001-06-22 Cartographie de recepteurs, perception sensorielle et biocapteurs d'agents sensoriels chimiques WO2001098526A2 (fr)

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AU2001270121A AU2001270121A1 (en) 2000-06-22 2001-06-22 Receptor fingerprinting, sensory perception, and biosensors of chemical sensants

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US21381200P 2000-06-22 2000-06-22
US60/213,812 2000-06-22
US09/804,291 US20030088059A1 (en) 2000-03-13 2001-03-13 Human olfactory receptors and genes encoding same
US09/804,291 2001-03-13

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008289A2 (fr) * 2000-07-26 2002-01-31 Curagen Corporation Nouvelles proteines et acides nucleiques codant ces proteines
WO2002029061A2 (fr) * 2000-10-06 2002-04-11 Inctye Genomics, Inc. Recepteurs couples aux proteines g
WO2002046229A2 (fr) * 2000-12-05 2002-06-13 Curagen Corporation Nouvelles proteines et acides nucleiques codant celles-ci
WO2002050275A2 (fr) * 2000-12-18 2002-06-27 Curagen Corporation Nouvelles proteines et acides nucleiques codant ces dernieres
EP1270724A2 (fr) * 2001-06-18 2003-01-02 National Institute of Advanced Industrial Science and Technology Récepteurs couplés aux protéines de liaison guanosin triphosphate
EP1551972A2 (fr) * 2001-08-09 2005-07-13 Curagen Corporation Acides nucleiques, polypeptides, polymorphismes nucleotidiques simples et techniques d'utilisation de ceux-ci
US7341842B2 (en) 2000-04-17 2008-03-11 The Mount Sinai School Of Medicine TRP8, a transient receptor potential channel expressed in taste receptor cells
US7351583B2 (en) 1999-10-05 2008-04-01 Agensys, Inc. Antibodies to G protein-coupled receptor
US7361338B2 (en) 1999-10-05 2008-04-22 Agensys, Inc. Methods to inhibit growth of prostate cancer cells
EP2087358A2 (fr) * 2006-11-15 2009-08-12 Redpoint Bio Corporation Technologie à base de matrice d'épices pour l'identification de composés
US7803982B2 (en) 2001-04-20 2010-09-28 The Mount Sinai School Of Medicine Of New York University T1R3 transgenic animals, cells and related methods
EP2806031A3 (fr) * 2010-09-03 2015-07-29 Kao Corporation Procédé de recherche d'un agent d'élimination des mauvaises odeurs, agents d'élimination des mauvaises odeurs et procédé d'élimination des mauvaises odeurs
WO2024126820A1 (fr) * 2022-12-15 2024-06-20 Givaudan Sa Récepteurs olfactifs modifiés

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7351583B2 (en) 1999-10-05 2008-04-01 Agensys, Inc. Antibodies to G protein-coupled receptor
US8236510B2 (en) 1999-10-05 2012-08-07 Agensys, Inc. Protein showing enhanced expression in cancer cells
US7795391B2 (en) 1999-10-05 2010-09-14 Agensys, Inc. Protein showing enhanced expression in cancer cells
US7361338B2 (en) 1999-10-05 2008-04-22 Agensys, Inc. Methods to inhibit growth of prostate cancer cells
US7960128B2 (en) 2000-04-17 2011-06-14 The Mount Sinai School Of Medicine TRP8, a transient receptor potential channel expressed in taste receptor cells
US7341842B2 (en) 2000-04-17 2008-03-11 The Mount Sinai School Of Medicine TRP8, a transient receptor potential channel expressed in taste receptor cells
US7960127B2 (en) 2000-04-17 2011-06-14 The Mount Sinai School Of Medicine TRP8, a transient receptor potential channel expressed in taste receptor cells
US7364867B2 (en) 2000-04-17 2008-04-29 The Mount Sinai School Of Medicine Method of identifying bitter compounds by employing TRP8, a transient receptor potential channel expressed in taste receptor cells
WO2002008289A3 (fr) * 2000-07-26 2003-07-03 Curagen Corp Nouvelles proteines et acides nucleiques codant ces proteines
WO2002008289A2 (fr) * 2000-07-26 2002-01-31 Curagen Corporation Nouvelles proteines et acides nucleiques codant ces proteines
WO2002029061A2 (fr) * 2000-10-06 2002-04-11 Inctye Genomics, Inc. Recepteurs couples aux proteines g
WO2002029061A3 (fr) * 2000-10-06 2002-10-17 Inctye Genomics Inc Recepteurs couples aux proteines g
WO2002046229A3 (fr) * 2000-12-05 2003-07-10 Curagen Corp Nouvelles proteines et acides nucleiques codant celles-ci
WO2002046229A2 (fr) * 2000-12-05 2002-06-13 Curagen Corporation Nouvelles proteines et acides nucleiques codant celles-ci
WO2002050276A2 (fr) * 2000-12-18 2002-06-27 Curagen Corporation Nouvelle proteine et acides nucleiques codant pour celle-ci
WO2002050275A2 (fr) * 2000-12-18 2002-06-27 Curagen Corporation Nouvelles proteines et acides nucleiques codant ces dernieres
WO2002050275A3 (fr) * 2000-12-18 2003-07-24 Curagen Corp Nouvelles proteines et acides nucleiques codant ces dernieres
WO2002050276A3 (fr) * 2000-12-18 2003-07-24 Curagen Corp Nouvelle proteine et acides nucleiques codant pour celle-ci
US7803982B2 (en) 2001-04-20 2010-09-28 The Mount Sinai School Of Medicine Of New York University T1R3 transgenic animals, cells and related methods
EP1270724A2 (fr) * 2001-06-18 2003-01-02 National Institute of Advanced Industrial Science and Technology Récepteurs couplés aux protéines de liaison guanosin triphosphate
EP1270724A3 (fr) * 2001-06-18 2003-05-21 National Institute of Advanced Industrial Science and Technology Récepteurs couplés aux protéines de liaison guanosin triphosphate
EP1551972A2 (fr) * 2001-08-09 2005-07-13 Curagen Corporation Acides nucleiques, polypeptides, polymorphismes nucleotidiques simples et techniques d'utilisation de ceux-ci
EP1551972A4 (fr) * 2001-08-09 2005-08-31 Curagen Corp Acides nucleiques, polypeptides, polymorphismes nucleotidiques simples et techniques d'utilisation de ceux-ci
EP2087358A2 (fr) * 2006-11-15 2009-08-12 Redpoint Bio Corporation Technologie à base de matrice d'épices pour l'identification de composés
EP2806031A3 (fr) * 2010-09-03 2015-07-29 Kao Corporation Procédé de recherche d'un agent d'élimination des mauvaises odeurs, agents d'élimination des mauvaises odeurs et procédé d'élimination des mauvaises odeurs
US9233082B2 (en) 2010-09-03 2016-01-12 Kao Corporation Method for searching for malodor control agent, malodor control agent, and malodor control method
US9526680B2 (en) 2010-09-03 2016-12-27 Kao Corporation Method for searching for malodor control agent, malodor control agent, and malodor control method
WO2024126820A1 (fr) * 2022-12-15 2024-06-20 Givaudan Sa Récepteurs olfactifs modifiés

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