HK1129701A - T2r taste receptors and genes encoding same - Google Patents

Description

T2R taste receptor and genes encoding same

This application is a divisional application of the same-name application having application number 01809145.8 and filed on 4.4.2001.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No.60/195,532, filed on 7/4/2000 and U.S. Ser. No.60/247,014, filed on 13/11/2000, both of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to newly identified mammalian chemosensory G protein-coupled receptors, families of these receptors, and genes and cDNAs encoding these receptors. The invention relates, inter alia, to newly identified mammalian chemosensory G protein-coupled receptors that are active in taste signaling, to families of such receptors, to genes and cDNAs encoding such receptors, and to methods of using such receptors, genes and cDNAs in the analysis and discovery of taste modulators.

Background

The taste system provides sensory information about the external chemical components. Taste transduction is one of the most complex forms of chemically triggered perception in animals, and is ubiquitous throughout the animal kingdom, from metazoans to the most complex vertebrates. Mammals are generally considered to have five basic taste forms: sweet, bitter, sour, salty and fresh (umami, taste of monosodium glutamate).

Each distinct taste form is believed to be mediated by distinct transduction pathways. These pathways are thought to be mediated by receptors, such as metabolic tropism (metabotropic) receptors or inotropic (inotropic) receptors, which are expressed in taste receptor cell subsets (subsets). For example, some receptors are thought to be mediated by G protein-coupled receptors, while other Taste sensations are thought to be mediated by channel proteins (see, e.g., Kawamura et al, Umami: A Basic Taste (1987); Kinnamon et al, Ann. Rev. physiol.), 54: 715-31 (1992); Lindemann, physiologically, (physio. 1.Rev.), 76: 718-66 (1996); Stewart et al, journal of physiology in the United states (am. J. physiol.), 272: 1-26 (1997)).

In mammals, taste receptor cells are assembled into taste buds that are widely distributed in different papillae of tongue epithelial cells. The circumvallate papillae, which is located at the rearmost part of the tongue, contains hundreds of taste buds. In contrast, the foliated papillae located on the posterior side of the tongue contain tens to hundreds of taste buds. Furthermore, the fungiform papillae, which is located in the front of the tongue, has only one or a few taste buds.

According to the species, each taste bud contains 50-150 cells, including precursor cells, supporting cells and taste receptor cells. See, for example, Lindemann, physiological review (physiol. rev.), 76: 718-66(1996). The receptor basal cell is filled with afferent nerve endings that transmit information to the taste centers of the cerebral cortex through synapses in the brainstem and thalamus. Elucidation of the mechanisms of taste cell signaling and information processing is important for understanding taste function, regulation and perception.

A number of animal physiological studies have shown that taste receptor cells may respond selectively to different chemical stimuli (see, e.g., Akabas et al, Science, 242: 1047-50(1988), gilbertson et al, J.Gen.Physiol., 100: 803-24(1992), Bernhardt et al, J.Physiol., 490: 325-36(1996), Cummings et al, J.Neuropysiol., 75: 1256-63 (1996)). In particular, cells expressing these receptors, upon exposure to specific chemical stimuli, elicit taste sensation by depolarizing to generate an action potential. Action potentials are generally thought to trigger neurotransmitter release at taste afferent neuronal synapses, initiating signaling on neural pathways, thereby mediating taste perception (see, e.g., Roper, annual review of neuroscience (ann. rev. neurosci.), 12: 329-53 (1989)). Nonetheless, little is currently known about the manner in which taste sensation is elicited (see, e.g., Margolske, BioEssays, 15: 645-50 (1993); Avenet et al J. Membrane biol., 112: 1-8 (1989)).

As described above, taste receptors specifically recognize molecules that elicit a particular taste sensation. These molecules are also referred to herein as "tastants". Many taste receptors belong to the 7-transmembrane receptor superfamily (Hoon et al, Cell (Cell) 96: 451 (1999); Adler et al, Cell 100: 693(2000)), which are also known as G-protein coupled receptors (GPCRs). G protein-coupled receptors control many physiological functions such as endocrine function, exocrine function, heart rate, lipolysis, carbohydrate metabolism and transmembrane signaling. Biochemical analysis and molecular cloning of many of these receptors has revealed many rationales for the function of these receptors.

For example, U.S. Pat. No.5,691,188 describes how upon binding of a ligand (ligand) to a GPCR, this receptor may undergo a conformational change leading to G-protein activation. The G protein comprises three subunits: an alpha subunit, a beta subunit, and a gamma subunit bound to an amidino nucleotide. The G protein circulates in two forms depending on whether GDP and GTP are bound to the alpha subunit. After GDP binding, the G protein exists as a heterotrimer (hetetomerimer): i.e., the G α β γ complex. When GTP is bound, the α subunit dissociates from the heterotrimer, leaving the G β γ complex. When the G α β γ complex binds to an activated G protein-coupled receptor on the cell membrane, the rate of GTP exchange binding to GDP is increased, and the rate of dissociation of the bound G α subunit from the G α β γ complex is also increased. The free G α subunit and G β γ complex are therefore capable of transmitting a signal to downstream elements of a variety of signaling pathways. These events form the basis for a diversity of different cellular signaling phenomena, including, for example, signaling phenomena where taste and/or smell are considered neurological sensory perception.

The sequence of all or part of numerous human and other eukaryotic chemosensory receptors is now clear. (see, e.g., Pilpel, Y and Lancet, D, Protein Science, 8: 969-977 (1999); Mombaerts, P., annual review of neuroscience, 22: 487-50 (1999); EP0867508A2, US 5874243, WO 92/17585, WO 95/18140, WO 97/17444, WO 99/67282). Although much is known about the psychophysics and physiology of taste cell function, there is a lack of understanding about the molecules and pathways that mediate sensory signaling responses. The identification and isolation of novel taste receptors and taste signaling molecules can lead to novel methods for chemically and genetically modulating taste transduction pathways. For example, if receptors and channel proteins are present, high affinity agonists, antagonists, inverse agonists and modulators of taste activity can be screened. These taste modulating compounds are useful in the pharmaceutical and food industries to improve the taste of a variety of consumer products or to block some unpleasant tastes, such as the bitter taste of certain products.

Summary of The Invention

The present invention addresses, in part, the need for a better understanding of the interaction between chemosensory receptors and chemical stimuli. Thus, the present invention provides novel taste receptors and methods of using these receptors, as well as genes and cdnas and the like encoding such receptors, to identify molecules that can be used to modulate taste transduction.

The invention relates, inter alia, to a family of recently discovered G protein-coupled receptors, and to the genes and cDNAs encoding these receptors. These receptors are thought to be primarily involved in bitter taste transduction, but may also be involved in signal transduction from other taste forms.

The invention provides methods for describing and/or predicting taste perception in mammals, including humans. Preferably, these methods can be accomplished by using these receptors and using genes encoding the receptors described herein.

To this end, it is an object of the present invention to provide a novel family of mammalian G protein receptors, referred to herein as T2R, T2R, which are active in taste perception. It is another object of the invention to provide such fragments and variants of T2R (variants) that retain gustatory binding activity.

It is another object of the present invention to provide nucleic acid sequences or molecules encoding such T2R and fragments or variants thereof.

It is still another object of the present invention to provide expression vectors comprising a nucleic acid sequence encoding such T2R or fragments or variants thereof operably linked to at least one regulatory sequence such as a promoter, enhancer or other sequence involved in the transcription and/or translation of a positive or negative gene.

It is still another object of the present invention to provide human or non-human cells functionally expressing at least one T2R or a fragment or variant thereof.

It is still another object of the present invention to provide a T2R fusion protein or polypeptide comprising at least one fragment of at least one such T2R.

It is another object of the invention to provide an isolated nucleic acid molecule encoding a T2R polypeptide, the molecule comprising a nucleic acid sequence that is at least 50%, preferably 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19 and 23 and conservatively modified variant sequences thereof.

It is another object of the invention to provide an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence that is at least 35% to 50%, preferably 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 24 and conservatively modified variant sequences thereof, wherein the fragment is at least 20, preferably 40, 60, 80, 100, 150, 200 or 250 amino acids in length. Optionally, the fragment may be an antigenic fragment capable of binding to an anti-T2R antibody.

It is a further object of the invention to provide an isolated polypeptide comprising a variant of said fragment wherein there is a variation of up to 10, preferably 5,4, 3,2 or 1 amino acid residues.

It is another object of the invention to provide agonist antagonists of these T2R, or fragments or variants thereof.

It is another object of the invention to provide methods for demonstrating and/or predicting taste perception in mammals, including humans. Preferably, these methods can be accomplished by using the T2R receptor, or a fragment or variant thereof, described herein, and using the gene encoding the T2R, or a fragment or variant thereof.

It is another object of the present invention to provide novel molecules or combinations of molecules capable of eliciting a specific taste perception in a mammal. These molecules or combinations can be obtained by the following methods: determining the taste perception value in a mammal for a known molecule or combination of molecules; determining a taste perception value for one or more unknown molecules or combinations of molecules in a mammal; comparing the taste perception values of the one or more unknown compositions and the one or more known compositions in the mammal; selecting a molecule or combination of molecules capable of eliciting a particular taste perception in a mammal; and combining two or more unknown molecules or combinations of molecules to form a molecule or combination of molecules that is capable of eliciting a particular mammalian taste perception. The combining step results in individual molecules or combinations of molecules that are capable of eliciting a particular mammalian taste perception.

It is another object of the present invention to provide a method of screening one or more compounds to identify the presence or absence of a detectable taste in a mammal, comprising: a step of contacting one or more of the compounds referred to above with at least one of the disclosed T2R, fragments or variants thereof, wherein preferably the mammal is a human.

It is another object of the present invention to provide a method of stimulating taste sensation comprising the steps of: determining the extent of interaction of T2R with a tastant for each of the plurality of T2R, or fragments thereof, described herein, preferably human T2R; combining a plurality of compounds, wherein each compound has a predetermined interaction with one or more T2R, such that together they provide a receptor-stimulation pattern that mimics a taste pattern. The effect of tastants on T2R can be determined by any of the binding or reporting assays described herein. A plurality of compounds may then be combined to form a mixture. If desired, one or more of the plurality of compounds may be covalently bound. The bound compound substantially stimulates at least 50%, 60%, 70%, 75%, 80%, or 90% or all of the receptors that may be substantially stimulated by tastants.

Another aspect of the invention is to provide a method wherein a plurality of standard compounds are tested against a plurality of T2R, or fragments or variants thereof, to determine the extent of interaction of each T2R with each standard compound, thereby generating a receptor stimulation pattern for each standard compound. These receptor stimulation patterns may be stored in a relational database on a data storage medium. The method may further comprise providing a desired receptor stimulation pattern for a taste sensation; comparing the desired receptor stimulation pattern to a relational database; and identifying one or more standard compound combinations that most closely match the desired receptor stimulation pattern. The method may further comprise combining the standard compounds into one or more compositions that have been identified to stimulate taste.

It is another object of the present invention to provide a method for demonstrating taste perception of a particular substance in a mammal, comprising the steps of: providing X₁To X_nA value representing the amount of stimulation of each of n T2R in the vertebrate, wherein n is greater than or equal to 4 and n is greater than or equal to 12; n is greater than or equal to 24, or n is greater than or equal to 40; from this value, a quantitative representation of taste perception is generated. T2R may be a taste receptor disclosed herein, or a fragment or variant thereof, and the representation may comprise a dot or volume of n-dimensional space, may comprise a graph or a spectrum, or may comprise a quantitative representation matrix. Likewise, the step of providing may comprise contacting a plurality of recombinantly produced T2R, or fragments or variants thereof, with a test composition and quantitatively measuring the effect of the composition on the receptor.

It is a related object of the invention to provide a method for predicting mammalian tasteA method of taste perception (which perception is the production of an unknown taste perception in a mammal by one or more molecules or combinations of molecules) comprising the steps of: providing X₁To X_nA value representing the amount of stimulation of each of n T2R in the vertebrate, wherein n is greater than or equal to 4 and n is greater than or equal to 12; n is greater than or equal to 24, or n is greater than or equal to 40; producing a known taste perception in a mammal for one or more molecules or combinations of molecules; and generating a quantitative representation of the taste perception of the mammal from the value for one or more molecules or combinations of molecules that produce a known taste perception in the mammal, providing X₁To X_nA value representing the amount of stimulation of each of n T2R in the vertebrate, wherein n is greater than or equal to 4 and n is greater than or equal to 12; n is greater than or equal to 24, or n is greater than or equal to 40; producing an unknown taste perception in the mammal for one or more molecules or combinations of molecules; generating a quantitative representation of the taste perception of the mammal from the values for one or more molecules or combinations of molecules that produce an unknown taste perception in the mammal, predicting the taste perception of the mammal from the unknown taste perception produced by the one or more molecules or combinations of molecules by comparing the quantitative representation of the unknown taste perception produced by the mammal for the one or more molecules or combinations of molecules to the quantitative representation of the known taste perception produced by the mammal for the one or more molecules or combinations of molecules. T2R for use in the present method may include a taste receptor disclosed herein, or a fragment or variant thereof.

Detailed Description

The present invention thus provides isolated nucleic acid molecules encoding taste cell-specific G protein-coupled receptors ("GPCR"), and the polypeptides they encode. These nucleic acid molecules and the polypeptides they encode are members of the taste cell-specific GPCR T2R family. Further, the recently identified T2R gene families (database accession numbers AF227129-AF227149 and AF240765-AF240768) encoding candidate bitter taste receptors facilitate the search and identification of related genes in public nucleotide sequence databases. The present invention relates to the recently identified members of this family. For more detailed information on the T2R family, see Adler et al, Cell, 100: 693-702(2000) and Chandrashekar et al, Cell, 100: 703-11(2000), both of which are incorporated herein by reference in their entirety.

Nucleic acids encoding T2R proteins and polypeptides of the invention can be isolated from a variety of sources by genetic engineering, amplification, synthesis, and/or recombinant expression methods according to the methods disclosed in WO 00/35374, which is incorporated herein by reference in its entirety.

The invention provides, inter alia, nucleic acids encoding a novel family of taste cell-specific G protein-coupled receptors. These nucleic acids and the encoded receptors are referred to herein as "T2R" family members of taste cell-specific G protein-coupled receptors ("GPCRs"). Taste cell-specific GPCRs are generally considered to be components of taste transduction pathways, particularly bitter taste transduction pathways, and are involved in taste detection of substances such as bitter tastants, 6-n-propylthiouracil (PROP), sucrose octaacetate (soa), melitriose undecanoate (rua), denatonium, copper glycinate (copper glycinate) and quinine. However, T2R may also be associated with other taste modalities.

Furthermore, it is generally believed that T2R family members may act in conjunction with other T2R family members, other taste cell-specific GPCRs, or combinations thereof, thereby affecting chemosensory taste transduction. For example, it is believed that members of the T2R family may be co-expressed in the same taste receptor cell type, and that the co-expressed receptors may physically interact to form heterodimeric (heterodimeric) taste receptors. Alternatively, co-expressed receptors may each bind to the same type of ligand alone, and their combined binding may result in a specific perceived taste sensation.

The invention also provides methods of screening for modulators of these taste cell-specific GPCRs, such as activators (activators), inhibitors (inhibitors), stimulators (stimulants), enhancers, agonists, antagonists, and the like. These taste transduction modulators are useful for pharmacological and genetic modulation of taste signaling pathways, and the like. These screening methods can be used to identify high affinity agonists and antagonists of taste cell activity. These modulating compounds can then be used in the food and pharmaceutical industries to tailor the taste, e.g., to reduce or mask the bitter taste of foods and pharmaceuticals.

Thus, the present invention provides assays for taste modulation in which a member of the T2R family acts as a direct or indirect reporter of the effect of modulators on taste transduction. GPCRs, for example, are useful in assays that measure changes in ligand binding, ionic concentration, membrane potential, current, ionic flux, transcription, signaling, receptor-ligand interactions, second messenger concentrations, and the like, e.g., in vivo, in vitro, and ex vivo (ex vivo). In one embodiment, a T2R family member can act as an indirect reporter (reporter) by binding to a second reporter molecule such as green fluorescent protein (see, e.g., misili&Spector, Nature Biotechnology, 15: 961-964(1997)). In another embodiment, a T2R family member is recombinantly expressed in a cell, and Ca can be measured²⁺Changes in levels and other intracellular information such as cAMP, cGMP or IP3 to assay for modulation of taste transduction by GPCR activity.

In a preferred embodiment, the T2R polypeptide is expressed in eukaryotic cells as a chimeric receptor, along with heterologous sequences that facilitate plasma membrane transport (trafficking) or maturation and are directed through the secretory pathway. In a preferred embodiment, the heterologous sequence is a rhodopsin sequence, for example an N-terminal fragment of rhodopsin. This chimeric T2R receptor can be expressed in any eukaryotic cell, such as HEK-293 cells. Preferably, these cells comprise a functional G protein, such as G α 15, which is capable of coupling the chimeric receptor to an intracellular signaling pathway or to a signaling protein such as phospholipase C. Activation of the chimeric receptor in these cells can be detected by any standard method, for example, by detecting changes in intracellular calcium by measuring FURA-2 dependent fluorescence in the cells. If the host cells do not express the appropriate G protein, they may be transfected with a gene encoding a promiscuous G protein (e.g., as described in U.S. patent application No. US 60/243,770). This patent application is incorporated herein by reference in its entirety.

Methods of analyzing modulators of taste transduction include in vitroA ligand binding assay in which: a T2R polypeptide, a portion thereof, such as an extracellular domain, a transmembrane region, or a combination thereof, or a chimeric protein comprising one or more T2R family member domains of a T2R family member; oocytes, primary cells, or tissue culture cells express the T2R gene, or express a T2R fragment or fusion protein, such as rhodopsin fusion protein; phosphorylation and dephosphorylation of a T2R family member; a G protein that binds to a GPCR; captin (arrestin) binding; internalization (internalization); a ligand binding assay; voltage, membrane potential and resistance changes; carrying out an ion flow test; changes in intracellular second messengers such as cGMP, cAMP, and inositol triphosphate; intracellular Ca²⁺A change in level; neurotransmitter release.

In addition, the present invention provides methods for detecting T2R nucleic acid and protein expression for studies of taste transduction modulation and the specific identification of taste receptor cells. Members of the T2R family also provide nucleic acid probes useful for paternity and forensic identification. The T2R gene also serves as a useful nucleic acid probe for identifying taste receptor cells, such as foliate, fungiform, circumvallate, geschmackstreifen and epiglottis taste receptor cells, particularly bitter taste-sensing gustducin (gustducin) expressing cells. Furthermore, the nucleic acids and polypeptides encoded by them can be used as probes for the careful study of taste-induced behavior.

T2R polypeptides can also be used to prepare monoclonal and polyclonal antibodies for identifying taste receptor cells. Identification of taste receptor cells can be performed using techniques such as reverse transcription and amplification of mRNA, extraction of total or poly A + RNA, Nothern blotting, dot blotting, in situ hybridization, RNase protection, S1 digestion, probing DNA microchip arrays, Western blotting, and the like.

The T2R gene and the polypeptides encoded thereby include a family of related taste cell-specific G protein-coupled receptors. Within the genome, these genes exist in individual form or are located within one of several gene clusters (cluster). One gene cluster located in the human genome region 12p13 contains at least 24 genes, while another gene cluster located at 7q33 contains at least 7 genes. In summary, 60 distinct T2R family members were co-identified from within different organisms, including several putative pseudogenes (pseudogenes). It is estimated that the human genome may include about 40 different T2R genes, encoding about 30 functional human receptors.

Several T2R genes have been associated with previously located sites involved in bitter taste control. For example, human T2R1 is located in the human 5p15 compartment, where a locus has been precisely located that can affect the ability to taste a PROP substance (see Reed et al, am. J. hum. Gene., 64: 1478-80 (1999)). In addition, the gene cluster located in the human genomic region 12p13, which is associated with a region of mouse chromosome 6, has been shown to contain a number of bitter taste genes that are generally thought to affect taste perception or perception, such as sucrose octaacetate, melitriose undecanoate, actinone, and quinine (see, e.g., Lush et al, Genet. Res., 6: 167-74 (1995)). These associations show that the T2R gene is involved in taste detection of various substances, particularly bitter substances. In addition, mouse T2R5 was specifically sensitive to actinone, and therefore it was hypothesized that mutations in the mT2R5 gene could produce an actinone-non-sensitive phenotype. Similarly, human T2R4 and mouse T2R8 were able to specifically react to denatonium and PROP.

Functionally, the T2R gene comprises a family of 7 related transmembrane G protein-coupled receptors that are thought to be involved in taste transduction, possibly interacting with G proteins to mediate taste signaling (see, e.g., Fong, Cell Signal, 8: 217 (1996); Baldwin, Curr. Opin. CellBiol., 6: 180 (1994)). In particular, it is believed that T2R interacts with the G protein gustducin in a ligand-specific manner.

Structurally, the T2R family member nucleotide sequence may encode a family of related polypeptides comprising an extracellular domain, 7 transmembrane domains and a cytoplasmic domain. Related T2R family genes from other species are related to SEQ ID NOS: 1, 3, 5,7, 9, 11, 13, 15, 17, 19 and 23, or conservatively modified variants thereof, are at least 20-30% identical in nucleotide sequence over a region at least 50 nucleotides in length, optionally 100, 200, 500 or more nucleotides in length; or the encoded polypeptide has the sequence shown in SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, and 24, or conservatively modified variants thereof, is at least about 30-40% identical in amino acid sequence over a region of at least about 25 amino acids, optionally 50 to 100 amino acids. The T2R gene is known to be selectively expressed in the tongue, palate epithelium, papilla phyllifolia, geschmackstreifen and epiglottis taste receptor cell subsets. In contrast, studies have shown that the T2R gene is less expressed in the fungiform papillae. Furthermore, it is known that the T2R gene is selectively expressed in gustducin-positive cells.

Several consensus amino acid sequences or domains have been identified that are characteristic of the T2R family members. In particular, it has been found that T2R family members generally comprise sequences at least about 50%, optionally 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more identical to T2R transmembrane region I, T2R transmembrane region II, T2R transmembrane region III, T2R transmembrane region IV, T2R transmembrane region V, and T2R transmembrane region VII. These conserved domains can thus be used to identify members of the T2R family by identity, specific hybridization or amplification, or specific binding to antibodies raised against the domains. These T2R transmembrane regions have the following amino acid sequence:

T2R family consensus sequence 1:

E(F/A)(I/V/L)(V/L)G(I/V)(L/V)GN(G/T)FI(V/A)LVNC(IM)DW(SEQ ID NO：25)

T2R family consensus sequence 2:

(D/G) (F/L) (I/L) L (T/I) (G/A/S) LAISRI (C/G/F) L (SEQ ID NO: 26) T2R family consensus sequence 3:

NH(L/F)(S/T/N)(L/I/V)W(F/L)(A/T)T(C/S/N)L(S/N/G)(I/V)(SEQ ID NO：27)

T2R family consensus 4:

FY(F/C)LKIA(N/S)FS(H/N)(P/S)(L/I/V)FL(W/Y)LK(SEQ ID NO：28)

T2R family consensus sequence 5:

LLI(I/F/V)SLW(K/R)H(S/T)(K/R)(Q/K)(M/I)(Q/K)(SEQ ID NO：29)

T2R family consensus sequence 6:

HS(F/L)(I/V)LI(L/M)(G/S/T)N(P/S/N)KL(K/R)(Q/R)(SEQ IDNO：30)

specific regions of the nucleotide and amino acid sequence of human T2R can be used to identify polymorphic variants or alleles or homologues (homologies) of humans or other species. Such identification can be performed in vitro, for example, under stringent hybridization conditions or PCR (e.g., using primers encoding the consensus sequence of T2R, described above) and sequencing, or by using such sequence information in a computer system for comparison to other nucleotide sequences. In general, polymorphic variants or alleles of T2R family members can be identified by comparing sequences of about 25 amino acids or more, such as 50-100 amino acids. Amino acid identity of about 30-40%, alternatively 50-60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95-99% or more generally indicates that the protein is a polymorphic variant, or allele, of a member of the T2R family, or is a homolog or ortholog (ortholog). Sequence identity can be calculated by the methods disclosed below. If there is additionally at least one conserved sequence that is completely identical or at least 75% identical to the previously identified T2R family consensus sequence, it is almost certain that the further positive T2R gene belongs to the T2R family. Sequence comparison is achieved by any of the following sequence comparison algorithms. Antibodies that specifically bind to the T2R polypeptide or a conserved region thereof may also be used to identify alleles, interspecies homologs, and polymorphic variants of the T2R protein.

Polymorphic variants, or alleles, and related T2R homologues or orthologs can be confirmed by examining taste cell-specific expression of putative T2R polypeptides. Generally, a T2R polypeptide having the amino acid sequence disclosed herein can serve as a positive control for comparison to a putative T2R polypeptide to demonstrate the identification of alleles or homologues of the T2R family member. Such homologues or alleles likewise have 7 transmembrane structures of the G protein-coupled receptor.

Nucleotide and amino acid sequence information for T2R family members can also be used to construct models of taste cell-specific polypeptides in a computer system. These models can then be used to identify compounds that activate or inhibit the T2R receptor protein. These compounds that modulate the activity of T2R family members are useful for studying the role of T2R genes and polypeptides in taste transduction.

Isolation of T2R family members provides a means for analyzing taste transduction inhibitors and activators. Using measurements such as ligand binding; phosphorylation and dephosphorylation; g protein binding; g protein activation; modulating molecular binding; voltage, membrane potential and conductance changes; ion flux; intracellular second messengers such as cAMP, cGMP and IP 3; and intracellular calcium levels, and the like, can be tested as inhibitors and activators of T2R of taste transmitters (transducers), particularly bitter taste transmitters, using the biologically active T2R protein. These activators and inhibitors identified using the T2R family members are useful for further study of taste transduction and for identification of specific taste agonists and antagonists. These activators and inhibitors are useful as pharmaceutical and food flavoring agents, for example, to reduce bitter taste in food or pharmaceutical products.

The invention also provides assays, particularly high throughput assays, for identifying molecules that interact with a T2R polypeptide and/or modulate a T2R polypeptide. Among the many methods, unique portions of the T2R family members, such as extracellular, transmembrane, or intracellular domains or regions, can be used. In various embodiments, the extracellular domain, transmembrane region, or combination thereof can be bound to a solid matrix and used, for example, to isolate a ligand, agonist, inverse agonist, antagonist, or any other molecule capable of binding to a T2R polypeptide and/or modulating the activity of the extracellular domain or transmembrane region of a T2R polypeptide.

In a particular embodiment, a region of T2R polypeptide, e.g., an extracellular, transmembrane, or intracellular region, is fused to a heterologous polypeptide, thereby forming a chimeric polypeptide, e.g., a chimeric polypeptide having GPCR activity. These chimeric polypeptides are useful, for example, in assays for identifying ligands, agonists, inverse agonists, antagonists or other modulators of the T2R polypeptide. In addition, these chimeric polypeptides can also be used to generate novel taste receptors with novel ligand binding specificities, modulation patterns, signaling pathways or other such properties, or to generate novel taste receptors with novel combinations of ligand binding specificities, modulation patterns, signaling pathways, and the like. Also, expression of T2R nucleic acids and T2R polypeptides can be used to generate tongue topology maps that can potentially be used to study the relationship between tongue taste receptor cells and brain taste sensory neurons. In particular, the method of detecting T2R could potentially be used to identify taste cells that are sensitive to bitter substances. Chromosomal mapping of the gene encoding human T2R could also potentially be used to identify diseases, mutations and traits caused by members of the T2R family or associated with members of the T2R family.

In general, the present invention provides isolated T2R family member nucleic acid molecules and their encoded taste receptors. The invention also encompasses not only nucleic acids and polypeptide sequences having a particular amino acid sequence, but also fragments, in particular, fragments of, for example, 40, 60, 80, 100, 150, 200, or 250 nucleotides, or more, and protein fragments of, for example, 10, 20, 30, 50, 70, 100, or 150 amino acids, or more.

Various conservative mutations and substitutions are contemplated within the scope of the present invention. For example, amino acid substitutions using known techniques including PCR, gene cloning, cDNA mapping, host cell transfection and in vitro transcription of recombinant genes are well within the level of skill in the art. Variants with functional activity can thus be screened.

More particularly, specific regions of the nucleic acid sequences disclosed herein, and the polypeptides they encode, may be used to identify polymorphic variants, interspecies homologs, and alleles of the sequences. The identification can be in vitro, such as under stringent hybridization conditions, PCR and sequencing, or using sequence information in a computer system to compare with other nucleic acid sequences. Different alleles of the T2R gene within a single species population are also useful for determining whether differences in allelic sequence between members of the population are correlated with differences in taste perception.

The nucleic acid molecules of the invention are typically of the intron-free (intron) type and encode a putative T2R protein, typically about 300 residues long, which, as predicted by hydrophobic mapping analysis, contains 7 transmembrane regions, suggesting that they belong to the G protein-coupled receptor (7TM) superfamily. In addition to overall structural similarity, each of the T2R identified herein has a sequence signature characteristic of a T2R family member. In particular, all sequences contained a very close match to the T2R family consensus sequence identified above. In combination with all the above-mentioned structural features of the identified genes and encoded proteins, it is clear that they represent a new member of the T2R receptor family.

It is also speculated that the T2R receptors and their genes may be used alone or in combination with other types of taste receptors, for developing detection systems and assay methods, for chemically identifying different types of molecules specifically recognized by these receptors, and for diagnostic and research purposes.

The nucleic acid sequences of the present invention and other nucleic acids used in the practice of the present invention, whether RNA, cDNA, genomic DNA, vectors, viruses, or hybrids thereof (hybrids), may be isolated from a variety of sources, genetically engineered, amplified, and/or recombinantly expressed. Any recombinant expression system may be used, including mammalian cells, and also including, for example, bacterial, yeast, insect or plant systems.

Identification of T2R Polypeptides

The amino acid sequences of the T2R proteins and polypeptides of the invention can be identified by putative translation of the encoding nucleic acid sequence. These diverse amino acid sequences and encoding nucleic acid sequences can be compared to each other or to other sequences in a number of ways. In a particular embodiment, the pseudogenes disclosed herein can be used to identify functional alleles or related genes in genomic databases known in the art.

For example, in sequence comparison, one sequence is typically used as a reference sequence and compared to additional test sequences. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, as described below with respect to the BLASTN and BLASTP programs, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

As used herein, a "comparison window" includes a reference to any contiguous number of positions, (selected from 20 to 600, typically about 50 to about 200, and more often about 100 to about 150), wherein a sequence can be compared to a reference sequence of the same number in a contiguous position after optimal alignment of the two sequences. Methods of aligning sequences for comparison are well known in the art. The method may be performed, for example, by using Smith & Waterman, adv.appl.math.2: 482(1981) by Needleman & Wunsch, J mol. biol.48: 443(1970) using Pearson & Lipman, proc.natl.acad.sci.usa 85: 2444(1988) methods of similarity search, optimized alignment of comparison sequences by computerized embodiments of these algorithms GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics software Package, Genetics Computer Group, 575 Science, Dr., Madison, Wis.), or methods using manual alignment and visual inspection (see, e.g., Current Protocols in molecular Biology (Ausubel et al, eds.1995 suppl.).

Preferred examples of algorithms suitable for determining sequence identity and percent sequence similarity are the BLAST and BLAST2.0 algorithms, respectively, identified by Altschul et al, Nuc.acids Res.25: 3389-: 403-. Software for performing BLAST analyses has been available from the national center for bioengineering information (http://www.ncbi.nlm.nih.gov/) Are disclosed to be obtained. The algorithm involves first determining high-scoring sequence pairs (HSPs) by determining short words in the length of the query sequence W, when compared to words of the same length as a sequence in a databaseWhen paired, they either match or meet some positive threshold score T. T here refers to the neighborhood word score threshold (Altschul et al, Nuc. acids Res.25: 3389-. These initial neighborhood word values (word hits) serve as seeds to initiate searches for longer HSPs containing them. The word value is extended to both sides along the sequence until the cumulative alignment score can be increased. For nucleotide sequences, the parameter M (reward score for a pair of matching residues; invariably>0) And N (penalty score for mismatched residues; always is<0) To calculate a cumulative score. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word value in each direction stops when the following situation occurs: the cumulative alignment score X amount decreases from its maximum obtainable value; (ii) the cumulative score becomes 0 or less due to the one or more negative score residue alignments; or reached either end of the sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The default value used by the BLASTN program (for nucleotide sequences) is a word size (W) of 11, the expected value (E) is 10, M-5, N-4 and two strand comparisons. For amino acid sequences, the BLASTP program uses a word size of 3 as a default value, an expectation value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff)&Henikoff, PNAS, 89: 10915(1989)), 50 for alignment (B), 10 for expected value (E), 5 for M, 4 for N and a two strand comparison.

Another advantageous embodiment of the algorithm is PILEUP. PILEUP produces multiple sequence alignments from a set of related sequences, using progressive, pairwise alignments to show relationship and percent sequence identity. It can also draw a so-called "tree" or "dendrogram" to show the clustering relationships used to generate the alignment (see, e.g., FIG. 2). PILEUP uses a simplified Feng & Doolittle progressive alignment method, J mol. evol.35: 351-360(1987). The method is similar to Higgins & Sharp, CABIOS 5: 151-153(1989) are similar. The program can align up to 300 sequences, each with a maximum allowable length of 5000 nucleotides or amino acids. The multiple alignment program begins with a pairwise alignment of the two most similar sequences, resulting in a cluster (cluster) of the two aligned sequences. The cluster is then aligned with the next most related sequence or cluster of aligned sequences. Two cluster sequences are aligned by a simple extension of the pairwise alignment of two separate sequences. The final comparison is accomplished by a series of progressive, pairwise comparisons. The program is run after specifying the specific sequences of the regions of sequence comparison and their amino acid or nucleotide coordinates and program parameters. Using PILEUP, the reference sequence was compared to other test sequences, and the following parameters were used to determine percent sequence identity: default gap weight (3.00), default gap length weight (0.10), and end gap weight. PILEUP can be obtained from the GCG sequence analysis software package, for example version 7.0(Devereaux et al, Nuc. acids Res. 12: 387-. Comparison of these protein sequences with all known proteins in public databases using the BLASTP algorithm revealed high homology to T2R family members, each T2R family sequence having at least about 50%, and preferably at least 55%, at least 60%, at least 65%, and most preferably 70% amino acid identity to at least one known member of that family.

B. Definition of

The following terms as used herein, unless otherwise indicated, each have the meaning ascribed to them.

"taste cells" include neuroepithelial cells which, when grouped, form the taste buds of the tongue, e.g., foliate, fungiform and circumvallate cells (see, e.g., Roper et al, Ann. Rev. neurosci.12: 329-353 (1989)). Taste cells are also found in the palate and other tissues, such as the esophagus and stomach.

"T2R" refers to one or more members of the family of G protein-coupled receptors that are selectively expressed in taste cells of the tongue and palate epithelium, such as foliate, geschmackstreifen, epiglottis, fungiform and circumvallate cells, and in esophagus and stomach cells (see, e.g., Adler et al, Cell, 100: 693-. This family is also referred to as the "SF family" (see, e.g., PCT/US00/24821, which is incorporated herein by reference in its entirety). These taste cells can be identified because they express specific molecules such as taste (transducin), taste cell specific G proteins, or other taste specific molecules (McLaughin et al, Nature, 357: 563-69 (1992)). Taste receptor cells can also be identified by morphology (see, e.g., Roper, supra). Members of the T2R family have the ability to act as taste transduction receptors. The T2R family members are also referred to as the "GR" family, for taste (gustatory) receptors, or the "SF" family.

The "T2R" nucleic acid encodes a GPCR family possessing 7 transmembrane domains with "G protein-coupled receptor activity", e.g., upon stimulation by enzymes such as phospholipase C and adenylate cyclase, which can bind to G proteins in response to external stimuli and promote the production of second messengers such as IP3, cAMP, cGMP and Ca²⁺(for a description of GPCR structure and function, see, e.g., Fong, supra, and Baldwin, supra). These nucleic acids encode proteins that are expressed in taste cells, particularly taste cells expressing taste (transducin) that are reactive with bitter tastants. A single taste cell may comprise multiple distinct T2R polypeptides.

The term "T2R" family thus includes polymorphic variants, alleles, mutants, and homologues having the following characteristics: (1) and SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, and 24 have about 30-40% amino acid sequence identity, more specifically about 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% amino acid sequence identity, over a window of about 25 amino acids, alternatively 50-100 amino acids; (2) specifically binding to an antibody raised against an immunogen comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, and 24, and conservatively modified variants thereof; (3) under stringent conditions specifically binds to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5,7, 9, 11, 13, 15, 17, 19 and 23, and conservatively modified variants thereof (at least about 100, alternatively at least about 500 and 1000 nucleotides in size); (4) comprising a nucleotide sequence substantially identical to a sequence selected from SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, and 24, or a sequence having at least about 40% identity to the amino acid sequence of said polypeptide; or (5) can be amplified by primers that hybridize under stringent hybridization conditions to primers encoding the nucleic acid sequences of SEQ ID NOS: 25-30, and hybridizing.

As mentioned above, while the T2R gene shows substantial sequence differences at the protein and DNA levels, all of the T2R genes isolated to date contain certain consensus sequences, particularly regions that are identical or have or at least 70-75% identity to the previously identified T2R consensus sequence (SEQ ID NOS: 25-30).

Topologically, some chemosensory GPCRs have an "N-terminal domain", "extracellular domain", a "transmembrane domain" comprising 7 transmembrane regions and associated cytoplasmic and extracellular loops, a "cytoplasmic region", and a "C-terminal domain" (see, e.g., Hoon et al, Cell, 96: 541-&Axel, Cell, 65: 175-187(1991)). These regions can be structurally identified using methods known to those skilled in the art, such as sequence analysis programs to identify hydrophilic and hydrophobic domains (see, e.g., Stryer, Biochemistry, (3)^rded.1988); see also any internet-based sequence analysis program, such as can be found on dot.imgen.bcm.tmc.edu). These regions are useful for making chimeric proteins and in vitro assays of the invention, such as ligand binding assays.

"extracellular domain" thus refers to the domain of the T2R polypeptide that protrudes outward from the cell membrane and is exposed to the outside of the cell. These regions generally include the "N-terminal domain" exposed to the extracellular side, as well as the extracellular loops of the transmembrane domain exposed to the extracellular side, i.e., the extracellular loops between transmembrane regions 2 and 3, between transmembrane regions 4 and 5, and between transmembrane regions 6 and 7. The "N-terminal domain" region begins at the N-terminus and extends to a position near the start of the transmembrane region. These extracellular regions are useful for in vitro soluble and solid phase ligand binding assays. In addition, the transmembrane regions described below may also be involved in ligand binding in combination with the extracellular domain or alone, and thus are useful for in vitro ligand binding assays.

The "transmembrane domain" includes 7 "transmembrane regions" and refers to the domain of the T2R polypeptide that is located within the cytoplasmic membrane and may likewise include the relevant cytoplasmic (intracellular) and extracellular loops, also referred to as transmembrane "regions". The following reactions were used, as described by Kyte & Doolittle, j. mol. biol., 157: 105-32(1982), or Stryer (supra), can identify the 7 transmembrane regions and extracellular and cytoplasmic loops.

By "cytoplasmic domain" is meant the domain of T2R that faces the interior of the cell, e.g., the "C-terminal domain" and the intracellular loops of the transmembrane domain, e.g., the intracellular loops between transmembrane regions 1 and 2, between transmembrane regions 3 and 4, and between transmembrane regions 5 and 6. "C-terminal domain" refers to the region spanning the end of the last transmembrane domain and the C-terminus of the protein, which is normally located within the cytoplasm of the cell.

The term "7-transmembrane receptor" refers to a polypeptide belonging to the superfamily of transmembrane proteins, which possesses 7 domains and traverses the plasma membrane 7 times (hence, these 7 regions are referred to as "transmembrane" or "TM" domains TM I to TM VII). The olfactory family and certain taste receptors belong to this superfamily. 7-transmembrane receptor polypeptides have similar and characteristic primary, secondary and tertiary structures, as described in more detail below.

The term "ligand binding region" refers to a sequence derived from a chemosensory receptor, or a sequence derived from a taste receptor, which incorporates essentially transmembrane domains II through VII (TMII through VII). This region may bind ligands, particularly taste receptors.

The term "cytoplasmic membrane translocation domain" or simply "translocation domain" refers to a polypeptide domain that is functionally equivalent to the exemplary translocation domain (5' -MNGTEGPNFYVPFSNKTGVV; SEQ ID NO: 31). These polypeptide domains, when incorporated into the amino terminus of a polypeptide coding sequence, are effective in "associating" or "translocating" a hybrid ("fusion") protein to the cytoplasmic membrane. This particular "translocation domain" was originally derived from the amino terminus of the human rhodopsin receptor polypeptide, a 7-transmembrane receptor. Another translocation domain is derived from bovine rhodopsin and is useful for facilitating translocation. The rhodopsin-derived sequence is very efficient in translocating the 7-transmembrane fusion protein to the cytoplasmic membrane.

By "functionally equivalent" is meant that the ability and efficiency of the domain to translocate a newly translated protein to the plasma membrane under similar conditions is comparable to the exemplary sequence SEQ ID NO: 31 are equally effective; relative efficiencies (in a quantitative manner) can be measured and compared using the methods described herein. Domains falling within the scope of the invention can be identified by routinely screening their efficiency in translocating a newly synthesized polypeptide to the plasma membrane in cells (mammals, toads, etc.) versus the 20 amino acid long translocation domain of SEQ ID NO: 31 are identified in the same manner.

In assays testing compounds capable of modulating taste transduction mediated by a T2R family member, the phrase "functional effect" includes determining any one of parameters, such as functional, physical and chemical effects, that are indirectly or directly affected by the receptor. It includes ligand binding, ion flux, membrane potential, current flow, transcription, G protein binding, GPCR phosphorylation or dephosphorylation, signaling, receptor ligand action, second messenger concentrations (e.g., cAMP, cGMP, IP3, or intracellular Ca) in vivo, in vitro, and ex vivo (ex vivo)²⁺) Etc., and also other physiological effects such as an increase or decrease in neurotransmitter or hormone release, etc.

By "determining functional effects" is meant testing compounds that increase or decrease parameters that are indirectly or directly affected by a T2R family member, such as functional, physical and chemical effects. These functional effects can be measured by any means known to those skilled in the art, such as spectroscopic characteristics (e.g., fluorescence, absorption, refractive index), fluid dynamics (e.g., shape), chromatographic or solubility, patch clamp assays (patch clamping), voltage sensitive staining, whole cell current, radioisotope efflux, inducible markers, changes in oocyte T2R gene expression; T2R expression by tissue culture cells; transcriptional activation of the T2R gene; a ligand binding assay; voltage, membrane potential and conductance changes; carrying out an ion flow test; changes in intracellular second messengers such as cAMP, cGMP and inositol triphosphate (IP 3); changes in intracellular calcium levels and neurotransmitter release, among others.

"inhibitors," "activators," and "modulators" of the T2R gene or protein are used interchangeably and refer to inhibitory, activating, and regulatory molecules, such as ligands, agonists, antagonists, and their homologues and mimetics (mimetics), identified using in vivo and in vitro taste transduction assays. For example, an inhibitor refers to a compound, such as an antagonist, that, upon binding, is capable of partially or completely blocking stimulation, reducing, preventing, delaying activation, negatively regulating signaling, or inactivating, desensitizing. An activator refers to a compound, such as an agonist, that is capable of stimulating, increasing, opening, activating, promoting, enhancing activation, positively modulating signaling, or sensitizing to, for example, binding. Modulators include, for example, compounds that alter the interaction of a receptor with: extracellular proteins capable of binding activators or inhibitors (e.g., ebnerin and other hydrophobic carrier family members); a G protein; kinases (e.g., rhodopsin kinase and β adrenergic receptor kinase analogs involved in receptor deactivation and desensitization); and a captin (arrestin) which is also capable of deactivating and desensitizing the receptor. Modulators include genetically modified T2R family members, e.g., members with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small molecule compounds, and the like.

Assays for such inhibitors and activators include, for example, expression of T2R family members in cells or cell membranes, use of putative modulator compounds with or without tastants such as bitter tastants, and then determination of functional effects on taste transduction as described above. Samples or assays comprising a member of the T2R family are treated with a potential activator, repressor, or modulator to test the extent of modulation as compared to control samples without the repressor, activator, or modulator. Control samples (untreated with modulators) were assigned a relative T2R activity value of 100%. Inhibition of T2R is judged to be achieved when the T2R activity value is about 80% activity relative to the control, alternatively 50% or 25-0%. Activation of T2R was judged to be achieved when the T2R activity value was 110% activity relative to the control, alternatively 150%, alternatively 200-500% or 1000-3000%.

The terms "purified", "substantially purified" and "isolated" as used herein refer to the state in which it is free of other, different compounds with which the compounds of the invention are naturally associated. Preferably, "purified", "substantially purified", and "isolated" mean that the composition comprises 0.5%, 1%, 5%, 10%, or 20%, most preferably at least 50% or 75% of the mass of a sample, by weight of a given sample. In a preferred embodiment, these terms are used to refer to a compound of the invention that is at least 95% by weight of the mass of a given sample. As used herein, the terms "purified", "substantially purified" and "isolated" nucleic acids and proteins, when referring to nucleic acids or proteins, refer to a state or concentration of purification that is different from the state or concentration of purification that is naturally found in the mammalian body, particularly the human body. Any degree of purification or concentration greater than the state of purification or concentration naturally occurring in a mammalian body, particularly a human, including (1) purification from other related structures or compounds or (2) association with structures and compounds not normally associated with the mammalian body, particularly a human, is within the definition of "isolated". The nucleic acids or proteins or classes of nucleic acids or proteins described herein can be isolated according to a variety of methods and procedures known to those skilled in the art, or linked to structures and compounds not naturally associated therewith.

The term "isolated" as used herein when referring to a nucleic acid or polypeptide refers to a state or concentration of purification which is different from the state or concentration naturally occurring in the mammalian body, particularly the human body. Any degree of purification or concentration greater than that naturally occurring in the mammalian body, particularly human, including (1) purification from other naturally occurring related structures or compounds, or (2) association with structures and compounds not normally associated with the body, is within the definition of "isolated" as used herein. The nucleic acids or polypeptides described herein can be isolated according to a variety of methods and procedures known to those skilled in the art, or combined with structures and compounds not normally associated in nature.

The term "amplification" as used herein refers to the use of any suitable amplification methodology for the production or detection of recombinants or naturally expressed nucleic acids as detailed below. For example, the invention provides methods and reagents (e.g., specific 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 (e.g., gustatory binding sequences of the invention) of the invention in vivo or in vitro.

The term "expression vector" refers to any recombinant expression system used to express a nucleic acid sequence of the present invention in any cell (including prokaryotic, yeast, fungal, plant, insect or mammalian cells), in vivo or in vitro, constitutively or inducibly. The term includes linear or circular expression systems. The term includes expression systems that are episomal or have integrated into the genome of the host cell. The expression system may or may not have the ability to self-replicate (i.e., to induce transient expression in the cell). The term includes recombinant expression "cassettes which contain only the minimal elements required for transcription of the recombinant nucleic acid. "

The term "library" refers to a mixed preparation containing different nucleic acid or polypeptide molecules, such as a recombinantly produced library of receptor regions, particularly libraries of taste receptor ligand binding regions produced by amplifying nucleic acids with degenerate primer pairs, or an isolated collection of vectors containing amplified ligand binding regions, or a mixture of cells, each cell randomly transfected with at least one vector encoding a taste receptor.

The term "nucleic acid" or "nucleic acid sequence" refers to a deoxyribonucleic acid or ribonucleic acid oligonucleotide, either in single-stranded or double-stranded form. The term includes nucleic acids, i.e., oligonucleotides containing known analogs of natural nucleotides. The term also includes nucleic acid-like structures having synthetic backbones.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more pending codons is replaced by mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res., 19: 5081 (1991); Ohtsuka et al, J. biol. chem., 260: 2605-2608 (1985); Rossolini et al, mol. cell. Probes, 8: 91-98 (1994)). The term nucleic acid may be used interchangeably with gene, cDNA, mRNA, oligonucleotide and polynucleotide (polynucleotide).

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues in the sequence is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The "translocation domain", "ligand binding domain", and chimeric receptor components described herein also include "analogs" or "conservative variants" and "mimetics" (peptide mimetics) having a structure and activity substantially identical to the exemplified sequences. Thus, the term "conservative variant" or "analog" or "mimetic" refers to a polypeptide having an amino acid sequence that has been modified such that the modification does not substantially affect the structure and/or activity of the polypeptide (of the conservative variant) described herein. This includes conservatively modified variations of an amino acid sequence, i.e., substitutions, additions or deletions of amino acid residues that are not critical to the activity of the protein, or amino acid substitutions (e.g., acidic, basic, positive, negative, polar or non-polar, etc.) using residues of the same nature, such that the substitution of a critical amino acid also does not substantially affect structure and/or activity.

More specifically, "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to sequences which are essentially identical thereto. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acid molecules encode any particular protein.

For example, the codons GCA, GCC, GCG and GCU all encode alanine. Thus, at every position of alanine that is determined by a codon, the codon can be changed to any of the codons described that do not change the encoded polypeptide.

These nucleic acid variations are "silent variations," which are conservatively modified variations. Each nucleic acid sequence herein that encodes a polypeptide also describes each and every possible silent variation of that nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

Conservative substitution tables of amino acids that provide functional similarity are well known in the art. For example, one exemplary guideline for selecting conservative substitutions includes (original residue followed by exemplary substitutions): ala/gly or ser; arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gln; ile/leu or val; leu/ile or val; lys/arg or gln or glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu. Another exemplary guideline uses the following six groups, each group containing conservatively mutually substituted amino acids: 1) alanine (a), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (I); 5) isoleucine (I), leucine (L), methionine (M), valine (V), and 6) phenylalanine (F), tyrosine (Y), tryptophan (W); (see, e.g., Creighton, Proteins, W.H.Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Vrlag (1979)). One skilled in the art will appreciate that the substitutions identified above are not the only possible conservative substitutions. For example, for some reason, one might substitute all charged amino acids as conservative substitutions for one another, whether they are positive or negative. In addition, individual substitutions, deletions or additions in the coding sequence which result in a change, addition or deletion of a single amino acid or a small number of amino acids are likewise 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 a polypeptide of the invention, such as a translocation domain, a ligand binding region, or a chimeric receptor. The mimetic can be either entirely composed of chemically synthesized analogs of unnatural amino acids, or a chimeric molecule of partially natural peptide amino acids and partially unnatural amino acid analogs. The mimetic can likewise have any number of conservative substitutions of the natural amino acids, provided such substitutions also do not substantially alter the structure and/or activity of the analog.

As with polypeptides of the invention that are conservative variants, routine experimentation can be used to determine whether a mimetic is within the scope of the invention, i.e., its structure and/or activity is not substantially altered. Peptidomimetic compositions can include any combination of non-natural structural components, which are typically derived from three structural groups: a) a residue linking group other than a natural amide bond ("peptide bond"); b) non-natural residue substitutions of naturally occurring amino acid residues; or c) residues which can induce secondary structure mimicry, i.e.residues which are capable of inducing or stabilizing secondary structures such as beta turns, gamma turns, beta sheet and alpha helix conformations, etc. A polypeptide can be identified as a mimetic when all or some of its residues are added by chemical means rather than by natural peptide bonds. The individual peptidomimetic residues can be linked by peptide bonds, other chemical bonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N '-Dicyclohexylcarbodiimide (DCC) or N, N' -Diisopropylcarbodiimide (DIC). Alternative linking groups which may become conventional amide bonds ("peptide bonds") include, for example, ketomethylene (e.g., -C (═ O) -CH)₂-substitution by-C (═ O) -NH-), aminomethyl (CH)₂-NH), vinyl, alkenyl (CH ═ CH), ether (C)H₂-O), thioether (CH)₂-S), tetrazoles, thiazoles, retroamides, thioamides or esters (see, e.g., Spatola, Chemistry and biochemistry of amino acids, Peptides and Proteins, Vol. 7, pp267-357, Marcell Dekker, Peptide Back boron Modifications, NY (1983)). Polypeptides comprising all or a portion of the non-natural residues in place of the naturally occurring residues can also be identified as mimetics; non-natural residues are well described in the scientific and patent literature.

A "label" or "detectable moiety" is a component that can be detected spectroscopically, photochemically, biochemically, immunochemically, or chemically. For example useful indicia include³²P, fluorescein, electron dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, or haptens and proteins that can be detected (e.g., by incorporating a radiolabel into the peptide) or used to detect antibodies that can specifically react with the peptide.

"labeled nucleic acid probe or oligonucleotide" refers to a nucleic acid or oligonucleotide that is covalently bound, either through a linker or a chemical bond, or non-covalently bound, such as by ionic, van der Waals, electrostatic, or hydrogen bonding, to a label, such that detection of the label bound to the probe detects the presence of the probe.

As used herein, a "nucleic acid probe or oligonucleotide" is defined as a nucleic acid that binds to a target nucleic acid of complementary sequence through one or more forms of chemical bonding (typically through complementary base pairing via hydrogen bonding). Probes as referred to herein may include natural (i.e., A, G, C or T) or modified bases (7-deazaguanylic acid, hypoxanthine, etc.). In addition, the bases in the probe may be linked by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, for example, a probe may be a peptide nucleic acid whose constituent bases are linked by peptide bonds rather than phosphodiester bonds. It will be appreciated by those skilled in the art that probes may bind target sequences that are not fully complementary thereto, depending on the stringency of the hybridization conditions. The probe is optionally labeled directly with an isotope, chromogenic substance, fluorescent substance, chromogen, or indirectly with biotin, which can be bound by a streptavidin complex, for example. By analyzing the presence or absence of the probe, one can detect the presence or absence of the selected sequence or subsequence.

When the term "heterologous" is used with respect to a portion of a nucleic acid, it is meant that the nucleic acid comprises two or more subsequences that are not in the same relationship to each other in nature. For example, nucleic acids are typically recombinantly produced, with two or more unrelated gene sequences recombined together to form a new functional nucleic acid molecule, e.g., a promoter from one source and a coding region from another source. Likewise, a heterologous protein means that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

A "promoter" is an array of nucleic acid sequences that is capable of directing transcription of a nucleic acid. Promoters, as used herein, include necessary nucleic acid sequences near the start of transcription, such as a TATA element in the case of a polymerase II type promoter. Promoters optionally include distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the transcription start site. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, or an arrangement of transcription factor binding sites) and another nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

"recombinant" as used herein refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., "recombinant polynucleotide"), as well as 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. "recombinant means" also include the linkage of nucleic acids of different origin, having different coding regions or domains or promoter sequences, into an expression cassette or vector for expression, e.g., inducible or constitutive expression of a fusion protein comprising a translocation domain of the invention and a nucleic acid sequence amplified using primers of the invention.

The phrase "selectively (or specifically) hybridizes to … …" refers to the binding, formation of a duplex, or hybridization of a molecule under stringent hybridization conditions only to a particular nucleotide sequence present in a complex mixture (e.g., total cell or library DNA or RNA).

The phrase "stringent hybridization conditions" refers to conditions under which a probe hybridizes only to a target sequence normally present in a complex mixture of nucleic acids, and not to other sequences. Stringent conditions are sequence dependent and will be different in different circumstances. Long sequences have hybridization specificity only at high temperatures. A more detailed guide to Nucleic Acid Hybridization is found in Ti jssen, Techniques in biochemistry and Molecular Biology-Hybridization with Nucleic acids, "Overview of Principles of Hybridization and the strategy of Nucleic Acid Assays" (1993). Generally, stringent conditions are selected to be about 5-10 ℃ lower than the thermal melting point (Tm) for the specific sequence at a specific ionic strength pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence after equilibrium is reached (the target sequence is in excess, at Tm, 50% of the probes are saturated at equilibrium). Stringent conditions mean a salt concentration of less than about 1.0M sodium ion, typically about 0.01 to 1.0M sodium ion (or other salt) concentration (pH7.0 to 8.3), at least about 30 ℃ for short probes (e.g., 10 to 50 nucleotides) and at least about 60 ℃ for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be established by the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal is at least twice, optionally 10 times, the background value. Exemplary stringency conditions are as follows: 50% formamide, 5 XSSC and 1% SDS, at 42 ℃ or 5 XSSC and 1% SDS, at 65 ℃ and rinsed with 0.2 XSSC and 0.1% SDS at 65 ℃. Such hybridization and rinsing steps can be performed, for example, for 1, 2, 5, 10, 15, 30, 60 minutes or more.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially related if the polypeptides encoded by the nucleic acids are substantially related. This occurs, for example, when copies of a nucleic acid are produced using the maximum codon degeneracy permitted by the genetic code. In these cases, the nucleic acid typically hybridizes under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include hybridization in a buffer with 40% formamide, 1M NaCl, 1% SDS at 37 deg.C, rinsed at 45 deg.C using 1 XSSC. Such hybridization and rinsing steps can be performed, for example, for 1, 2, 5, 10, 15, 30, 60 minutes or more. Positive hybridization was at least twice background. One of ordinary skill in the art will readily recognize that other hybridization and wash conditions may be used to provide conditions of similar stringency.

"antibody" refers to a polypeptide comprising the framework regions of an immunoglobulin gene or fragment thereof, which specifically binds to and recognizes an antigen. Known immunoglobulin genes include K, λ, α, γ, Δ, ε, and μ constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either K or λ. Heavy chains are classified as gamma, mu, alpha, delta and epsilon, which therefore define the immunoglobulin classes, i.e., IgG, IgM, IgA, IgD and IgE, respectively.

Exemplary immunoglobulin (antibody) structural units include tetramers (tetramers). Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

"chimeric antibody" refers to an antibody molecule in which (a) the constant regions or portions thereof are altered, replaced, or exchanged such that the antigen binding site (variable region) is linked to a constant region that is different or altered in class, effector function, and/or species, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that confers new properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, replaced or exchanged for a variable region having a different or altered antigen specificity.

An "anti-T2R antibody" is an antibody or antibody fragment that specifically binds to a polypeptide encoded by the T2R gene, cDNA, or subsequence thereof.

The term "immunoassay" is an assay that uses an antibody to specifically bind an antigen. Immunoassays are characterized by the use of the specific binding properties of a particular antibody to isolate, target, and/or quantify an antigen.

When referring to proteins or peptides, the phrase "specifically (or selectively) binds to" an antibody or "specifically (or selectively) immunoreacts with … …" refers to a binding reaction that can determine the presence of a protein of interest in a heterologous protein or other biological population. Thus, under the specified immunoassay conditions, a given antibody binds to a particular protein at least twice the background value and does not substantially bind significantly to other proteins present in the sample. Specific binding to an antibody under these conditions may require an antibody selected for its specificity for a particular protein.

For example, polyclonal antibodies raised against a particular species, such as rat, mouse, or human T2R family members, can be selected to obtain those polyclonal antibodies that are specifically immunoreactive only with the T2R polypeptide or immunogenic component thereof, and not with other proteins (with the exception of T2R polypeptide orthologs or polymorphic variants and alleles). This selection process can be accomplished by removing antibodies that cross-react with other species of T2R molecules or other T2R molecules. Antibodies may also be selected that recognize only T2R GPCR family members but not other GPCR families. There are a variety of immunoassay formats that can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are commonly used to select Antibodies specifically immunoreactive with a particular protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, (1988), among others, for a description of immunoassay formats and conditions that can be used to determine a specific immune response). Typically, the specific or selective reaction is at least twice background or noise, more preferably 10 to 100 times greater than background.

The phrase "selectively binds to … …" refers to the ability of a nucleic acid to "selectively hybridize" to other nucleic acids described above, or the ability of an antibody to "selectively bind" to a protein described above.

The term "expression vector" refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention constitutively or inducibly in vivo or in vitro in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cells. The term includes linear or circular expression systems. The term includes expression systems that are episomal or have integrated into the genome of the host cell. Expression systems have the ability to self-replicate or not, i.e., to induce transient expression within the cell. The term includes recombinant expression "cassettes" which include only the minimal elements required for transcription of a recombinant nucleic acid.

"host cell" refers to a cell that comprises an expression vector and supports replication or expression of the expression vector. The host cell may be a prokaryotic cell such as E.coli, or a eukaryotic cell such as yeast, insect, amphibian, or mammalian cells, e.g., CHO, Hela, and HEK-293, and others, e.g., cultured cells, explants (explants), and in vivo cells, and the like.

C. Isolation and expression of T2R

Isolation and expression of T2R of the invention or fragments or variants thereof can be carried out by known cloning methods using probes or primers constructed based on the T2R nucleic acid sequences disclosed herein. Related T2R sequences can also be identified from human or other species genomic databases using the sequences disclosed herein and known computer-based search techniques, such as BLAST sequence search techniques. In particular embodiments, the pseudogenes disclosed herein can be used to identify functional alleles or related genes.

The expression vector may then be used to infect or transfect host cells for functional expression of these sequences. These genes and vectors can be prepared and expressed in vivo or in vitro. One skilled in the art will appreciate that by modulating the expression or activity of genes and nucleic acids (e.g., promoters, enhancers, etc.) within the vectors of the invention, the desired phenotype of altering and controlling the expression of the nucleic acid can be achieved. Any known method described as useful for increasing or decreasing expression or activity may be used. The practice of the present invention may be combined with any method or protocol known in the art, which is well described in the scientific and patent literature.

Alternatively, these nucleic acid molecules can be synthesized in vitro using known chemical synthesis methods. For example, the method described below, carrothers, 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, Free radic.biol.med.19: 373-380 (1995); blomers, 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 can then be obtained either by synthesizing the complementary strand and annealing it under suitable conditions, or by adding the complementary strand with a suitable primer sequence and with a DNA polymerase.

Nucleic acid manipulation techniques, such as those that generate sequence mutations, subcloning, probe labeling, 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.), Vols.1-3, Cold spring Harbor Laboratory (1989); ausubel, ed., Current protocols Molecular Biology, Jon Wiley & Sons, Inc., New York (1997); ti jssen, ed., Laboratory Techniques in Biochemistry and molecular biology: hybridization with Nucleic Acid Probes, Part I, Theoryand Nucleic Acid Preparation, Elsevier, N.Y. (1993).

Nucleic acids, vectors, capsids, polypeptides, etc. can be analyzed and quantified by any of the methods well known to those skilled in the art. These include, for example, analytical biochemical methods such as Nuclear Magnetic Resonance (NMR), spectroscopy, radiation, electrophoresis, capillary electrophoresis, High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), ultrafiltration chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion, immunoelectrophoresis, Radioimmunoassay (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescence assays, Southern analysis, Northern analysis, dot blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acids or target substances or signal amplification methods, radiolabels, liquid scintillation counting, and affinity chromatography, and the like.

Oligonucleotide probes can be used to amplify nucleic acid fragments encoding the ligand binding region of T2R. The nucleic acids described herein can also be cloned or quantitatively measured using amplification techniques. Amplification Methods are well known in the art and include, for example, polymerase chain reaction, PCR (Innis ed., PCRProtocols, a Guide to Methods and Applications, Academic Press, N.Y. (1990)), Innis ed., PCR Strategies, Academic Press, Inc., N.Y. (1995), Ligase Chain Reaction (LCR) see, for example, (Wu, Genomics 4: 560 (1989)), Landegren, Science 241, (1988); Barringer, Gene 89: 117(1990)), transcriptional amplification (Kwoh, PNAS, 86: 1173 (1989)), self-sustained sequence replication (Guatelli, PANS, 87: 1874 (1990)), Q-beta replicase amplification (Smith, J.in. Microbiol. 35: 1477 (1997), beta. replicase), and other RNA-mediated amplification Methods (Bessel 257, Bessel et al) such as RNA polymerase enzyme amplification Methods (Bergey) and RNA polymerase (1996, bee) 3: 1989), methods enzymol.152: 307-16 (1987); sambrook; ausubel; U.S. patent nos.4, 683,195 and 4,683,202; soknanan, Biotechnology 13: 563-64(1995).

Once amplified, the nucleic acid molecules are cloned as desired, either alone or in a library format, into any one of the vectors, using conventional molecular biology methods, according to methods known in the art; cloning methods for in vitro amplification of nucleic acids are described, for example, in U.S. Pat. No.5,426,039. To aid in cloning of the amplified sequences, restriction sites can be "built-in" (build intro) to the PCR primer pair. For example, the Pst I and Bsp E1 sites were designed into exemplary primer pairs of the present invention. The sequence of these unique restriction sites allows for in-frame alignment of the ligation relative to the 7-membrane receptor "donor" coding sequence to which they are spliced (the ligand binding region coding sequence is located within the 7-membrane polypeptide, so in-frame results should be avoided if one wishes to translate the construct (construct) downstream of the restriction sites; this is not necessary if the inserted ligand binding region contains substantially most of the transmembrane V II region). Primers can be designed to retain the original sequence of the "donor" 7-membrane acceptor. Alternatively, the primer may encode amino acid residues that are conservatively substituted (e.g., hydrophobic residues are replaced with hydrophobic residues, see discussion below) or substituted functionally unaffected (e.g., do not interfere with cytoplasmic membrane insertion, do not cause peptidase-induced cleavage, do not cause abnormal folding of the receptor, etc.).

The primer pair may be designed to selectively amplify a ligand binding region of the T2R protein. These regions may vary widely for different ligands. Thus, the binding region that may be minimal for one ligand may be very limited for another potential ligand. Therefore, there is a need to amplify ligand binding regions of different sizes, containing different domain structures; for example, Transmembrane (TM) domains II to VII, III to VI, or II to VI of 7-transmembrane T2R, or variants thereof (e.g., subsequences of only specific domains, order of mixed domains, etc.).

Since the domain structures and sequences of many 7-membrane T2R proteins are known, one skilled in the art can readily select domain flanking and internal domain sequences as model sequences to design degenerate amplification primer pairs. For example, PCR amplification using primer pairs can produce nucleic acid sequences encoding domain regions II through VII. To amplify a nucleic acid sequence comprising a transmembrane domain i (tm i) sequence, degenerate primers may be designed from nucleic acids encoding the amino acid sequence of T2R family consensus sequence 1 described above. Such degenerate primers can be used to generate binding regions incorporating TM I to TM III, TM I to TM IV, TM I to TM V, TM I to TM VI, or TMI to TM VII. Additional degenerate primers can be designed based on other consensus sequences of the T2R family provided herein. Such degenerate primers can be used to generate binding regions that incorporate TM III to TM IV, TM III to TM V, TM III to TM VI, or TM III to TM VII

Examples of the design of degenerate primers are well known in the art. For example, a cosense-degenerate hybrid oligonucleotide primer (CODEHOP) strategy computer program can be generated fromhttp://blocks.fhcrc.org/codehop.htmlWhere obtained, the primers can be directly ligated to Blockmaker multiple sequence alignment addresses for hybrid primer prediction, starting with a set of related protein sequences, such as the known taste receptor ligand binding region (see, e.g., Rose, nucleic acids Res. 26: 1628-.

Means for synthesizing oligonucleotide primer pairs are well known in the art. "natural" base pairs or synthetic base pairs may be used. For example, the use of artificial nucleotide bases can provide a diverse way to manipulate primer sequences, resulting in more complex amplification product complex mixtures. Multiple families of artificial nucleotide bases, rotated through internal bonds, are able to adopt multiple hydrogen bond orientations, thus providing a means of degenerate molecular recognition. Incorporation of these analogs into a single site of a PCR primer will result in the generation of a complex library of amplified products. See, for example, Hoops, Nucleic Acids res. 25: 4866-4871(1997). Nonpolar molecules can also be used to mimic the shape of natural DNA bases. Non-hydrogen bond-shaped mimetics of adenine can replicate efficiently and selectively as non-polar mimetics of thymine (see, e.g., Morales, nat. struct. Bio. o. 5: 950-. For example, the two degenerate bases may be the pyrimidine base 6H, 8H-3, 4-dihydroxypyrimido [4, 5-c ] [1, 2] oxazin-7-one or the purine base N6-methoxy-2, 6-diaminopurine (see, e.g., Hill, Proc. Natl. Acad. Sci, USA 95: 4258-. Examples of degenerate primers of the present invention are the incorporation of the nucleobase analog 5 ' -dimethoxytrityl-N-benzoyl-2 ' -deoxycytidine and 3 ' - [ (2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite (see "P" in the sequences described below). Such pyrimidine analogs form hydrogen bonds with purines, including A and G residues.

Taste receptors disclosed herein are essentially equivalent polymorphic variants, alleles and interspecies homologs that can be isolated using the nucleic acid probes described below. Alternatively, expression libraries can be used to clone the T2R polypeptide and polymorphic variants, alleles and interspecies homologs thereof by detecting expressed homologues that immunoreact with antisera or purified antibodies raised against the T2R polypeptide, which expressed homologues can likewise recognize and selectively bind to the T2R homologue.

Nucleic acids encoding the ligand binding region of taste receptors can be obtained by amplifying (e.g., PCR) the appropriate nucleic acid sequence using an appropriate primer pair (matching or degenerate primers). The amplified nucleic acid can be genomic DNA from any cell or tissue or mRNA or cDNA derived from taste receptor-expressing cells.

In one example, a hybrid protein coding sequence can be constructed that includes a nucleic acid encoding T2R fused to a translocation sequence. Hybrid T2R comprising translocation motifs (motifs) and taste motif binding domains of other chemosensory receptors, particularly the taste receptor family, can also be provided. These nucleic acid sequences may be operably linked to transcriptional or translational control elements such as transcriptional and translational initiation sequences, promoters and enhancers, transcriptional and translational terminators, polyadenylation sequences, and other sequences useful for the transcription of DNA into RNA, and the like. In the construction of recombinant expression cassettes, vectors and transgenic animals, the promoter fragment can be used to direct expression of the nucleic acid of interest in all cells or tissues of interest.

In another embodiment, the fusion protein may include a C-terminal or N-terminal translocation sequence. In addition, the fusion protein may include additional elements, such as elements for protein detection, purification, or other applications. Domains that facilitate detection and purification include, for example, metal chelating peptides such as polyhistidine strands, histidine-tryptophan modules or other domains that can be purified on immobilized metals; a maltose binding protein; protein a domains that can be purified on immobilized immunoglobulins; or a domain used in the FLAGS extension/affinity purification system (Immunex Corp, SeattleWA).

Between the translocation domain (for efficient cytoplasmic membrane expression) and the remainder of the newly translated polypeptide, it may be advantageous to include a cleavable linker sequence such as factor Xa (see, e.g., Ottavi, Biochimie 80: 289-293 (1998)), subtilisin (subtilisin) protease recognition motif (see, e.g., Polyak, Protein Eng. 10: 615-619(1997)), enterokinase (Invitrogen, San Diego, Calif.), etc., to facilitate purification. For example, a construct may comprise a polypeptide-encoding nucleic acid sequence (thioredoxin linked to 6 histidine residues followed by an enterokinase cleavage site, (see, e.g., Williams, Biochemistry 34: 1787. 1797 (1995)), and a C-terminal translocation domain histidine residues to facilitate detection and purification, while the enterokinase cleavage site provides a means of purifying the protein of interest from the remaining fusion protein.

Expression vectors containing the ligand binding region coding sequences (whether individual expression vectors or libraries of expression vectors) can be introduced into the genome or cytoplasm or nucleus of a cell to express the sequence of interest by a variety of conventional methods described in the scientific and patent literature. See, e.g., Roberts, Nature 328: 731 (1987); berger is as before; schneider, Protein expr. purif.6435: 10 (1995); sambrook; tijssen; ausubel. Product information manuals provided by the manufacturers of biological reagents and test equipment will also provide information about biological methods. Vectors may be isolated from natural sources, obtained from libraries such as ATCC or GenBank, or prepared by synthetic or recombinant means.

The nucleic acid may be expressed in an expression cassette, vector or virus that is stably or transiently expressed in the cell (e.g., an episomal expression system). Selectable markers can be incorporated into expression cassettes and vectors to confer selectable phenotypes on transformed cells and sequences. For example, a selectable marker may encode episomal maintenance and replication, and thus does not have to be integrated into the host genome. For example, the marker may encode antibiotic resistance (e.g.chloramphenicol, kanamycin, G418, bleomycin, hygromycin) or herbicide resistance (e.g.chlorosulfuron or Basta) to select for cells transformed with the DNA sequence of interest (see, e.g.Blindelet-Rouuult, Gene 190: 315-. Selectable marker genes that confer resistance to substrates such as neomycin or hygromycin can only be used in tissue culture, while chemoresistance genes can serve as selectable markers in vivo or in vitro.

The chimeric nucleic acid sequence may encode a ligand binding region of T2R within any 7 transmembrane polypeptide. Because 7 transmembrane receptor polypeptides have similar primary sequences and secondary and tertiary structures, domains (e.g., extracellular domain, TM domain, cytoplasmic domain, etc.) can be readily identified by sequence analysis. For example, homology modeling, fourier analysis and helix cycle detection can be used to identify 7 domains with 7 transmembrane receptor sequences. A Fast Fourier Transform (FFT) algorithm can be used to evaluate the dominant period representing the hydrophobicity and variability profile of the analyzed sequence. The periodic detection enhancement and alpha helix periodic index can be measured by methods such as Donnelly, Protein sci. 2: 55-70 (1993). Other algorithms and modeling algorithms are well known in the art (see, e.g., Peitsch, Receptors, Channels 4; 161- & 164 (1996); kyte& Doolittle，J. Md. Bio.，157：105-132 (1982)；Cronet，Protein Eng. 6：59-64(1993)；http://bioinfo.weizman.ac.il/)。

The invention encompasses not only nucleic acid molecules and polypeptides having the specified nucleic acid and amino acid sequences, but also fragments thereof, in particular fragments of e.g. 40, 60, 80, 100, 150, 200 or 250 nucleotides or more, and polypeptide fragments of e.g. 10, 20, 30, 50, 70, 100 or 150 amino acids or more. Alternatively, the nucleic acid fragment may encode an antigenic polypeptide capable of binding to an antibody raised against a T2R family member. Furthermore, a protein fragment of the invention is optionally an antigenic fragment capable of binding to an antibody raised against a T2R family member.

The invention additionally encompasses chimeric proteins comprising at least a 10, 20, 30, 50, 70, 100 or 150 amino acid or more sequence of at least one T2R polypeptide disclosed herein, coupled to an additional amino acid sequence (which represents all or part of another GPCR, preferably a 7 transmembrane superfamily member). These chimeras can be obtained directly from the receptor of the invention and another GPCR, or in combination of two or more receptors of the invention. In one embodiment, a portion of the chimera corresponds to or is derived from the transmembrane domain of a T2R polypeptide of the invention. In another embodiment, a portion of the chimera corresponds to or is derived from one or more of the transmembrane regions of a T2R polypeptide described herein, and the remainder of the chimera is from another GPCR. Chimeric receptors are well known in the art, as are techniques for making chimeric receptors and the selection and definition of G protein-coupled receptor domains and fragments for incorporation. Thus, the knowledge of the skilled person can be readily used for the preparation of such chimeric receptors. Use of such chimeric receptors can provide, for example, taste selective characteristics of one of the receptors specifically described herein, coupled with signaling characteristics of another receptor, such as one known in prior art experimental systems.

For example, a region such as a ligand binding region, extracellular domain, transmembrane domain, cytoplasmic domain, N-terminal domain, C-terminal domain, or any combination thereof, can be covalently linked to a heterologous protein. For example, the T2R transmembrane region may be linked to the heterologous GPCR transmembrane domain, or the heterologous GPCR extracellular domain may be linked to the T2R transmembrane region. Other alternative heterologous proteins include, for example, green fluorescent protein, β -gal, glutamate receptor, and the N-terminus of rhodopsin.

Host cells expressing T2R, fragments or variants of the invention are also within the scope of the invention. To obtain high levels of expression of a cloned gene or nucleic acid (e.g., a cDNA encoding T2R, a fragment, or a variant of the invention), one skilled in the art typically subclones the nucleic acid sequence of interest into an expression vector containing a strong promoter to direct transcription, a transcription/translation terminator, and, for nucleic acid molecules encoding proteins, a ribosome binding site for translation initiation. Suitable bacterial promoters are well known in the art, for example, as described in Sambrook et al. However, either bacterial or eukaryotic expression systems may be used.

Any known method for introducing an exogenous nucleotide sequence into a host cell may be used. These methods include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, cytoplasmic vectors, viral vectors, and any other known method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, e.g., Sambrook et al). Provided that the particular genetic engineering procedure is warranted to successfully introduce at least one nucleic acid molecule into a host cell capable of expressing T2R, a fragment or a variant of interest.

After introduction of the expression vector into the cells, the transfected cells are cultured under conditions suitable for expression of the receptor, fragment or variant of interest, and then recovered from the culture using standard techniques. Embodiments of such techniques are well known in the art. See, for example, WO 00/06593, which is incorporated herein by reference in its entirety in accordance with the present disclosure.

D. Immunoassay for T2R

In addition to using nucleic acid hybridization techniques to detect the T2R gene and gene expression, immunoassays can also be used to detect T2R, such as identifying variants of taste receptor cells and T2R family members. Immunoassays can be used to qualitatively or quantitatively analyze T2R. A general overview of the application of this technique is found in Harlow & Lane, Antibodies: a Laboratory Manual (1988).

1. Antibodies to T2R family members

Methods for generating Monoclonal and polyclonal Antibodies that specifically react with T2R family members are methods known to those of skill in the art (see, e.g., Coligan, Current protocols in Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature, 256: 495-. Such techniques include the preparation of corresponding antibodies by selecting antibodies from recombinant antibody libraries of phage or similar vectors, and the preparation of monoclonal and polyclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al, Science, 246: 1275-.

A number of immunogens comprising T2R can be used to generate antibodies specifically reactive with members of the T2R family. For example, recombinant T2R protein, or an antigenic fragment thereof, can be isolated using the methods described herein. Suitable antigenic regions include, for example, the above-disclosed consensus sequences for identifying members of the T2R family. Recombinant proteins can be expressed in eukaryotic or prokaryotic cells using the methods described above, and purified using methods known in the art. Recombinant proteins are preferred immunogens for the production of monoclonal or polyclonal antibodies. Alternatively, synthetic peptides derived from the sequences disclosed herein can be used as immunogens after conjugation to a carrier protein. Naturally occurring proteins can also be used in pure or non-pure form. The product is then injected into an animal capable of producing antibodies. Monoclonal or polyclonal antibodies are available for subsequent immunoassays to measure proteins.

More specifically, methods for producing polyclonal antibodies are well known to those skilled in the art. For example, inbred mice (e.g., BALB/C mice) or rabbits are immunized with the T2R polypeptide using standard adjuvants, such as freund's adjuvant, and standard immunization procedures. The immune response of the animal to the immunogen preparation can then be monitored by collecting blood and determining the titer of the response to T2R. When a suitably high titer of antibodies to the immunogen is obtained, animal blood can be collected and antisera prepared. If desired, antibodies reactive with the T2R polypeptide can be enriched by further fractionation of the antisera (see Harlow & Lane, supra).

Monoclonal antibodies can be obtained by a variety of techniques well known to those skilled in the art. Briefly, spleen cells of an animal immunized with an antigen of interest are immortalized, usually by fusion with a myeloma (see Kohler & Milstein, Eur.J.Immunol., 6: 511-519 (1976)). Another method of immortalization involves the use of E-B virus (Epstein Barr Virus), oncogenes, retroviral transformation, or other methods known in the art. Clones formed from a single immortalized cell are screened to select clones that are capable of producing antibodies with the desired specificity and affinity for the antigen. The yield of monoclonal antibody produced by these cells can be increased by a variety of techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, the ratio of the two components is determined according to Huse et al, Science, 246: 1275-1281(1989) by screening a nucleic acid library of human B cells, nucleic acid sequences encoding monoclonal antibodies or binding fragments thereof can be isolated.

Monoclonal antibodies and polyclonal sera are typically collected and titrated with the immunogenic protein in an immunoassay, such as a solid phase immunoassay, in which the immunogen is immobilized on a solid support. Typically, polyclonal antisera with titers of 104 or higher are selected and tested for their cross-reactivity with non-T2R protein, or even other T2R family members, or other related proteins of other organisms, using competitive binding immunoassays. Specific polyclonal antisera and monoclonal antibodies will generally bind with a Kd of at least about 0.1mM, more often at least about 1pM, alternatively at least about 0.1pM or better, and alternatively 0.01pM or better.

Once antibodies specific for the T2R family members are obtained, each T2R protein can be detected using a series of immunoassay methods. For a review of immunological and immunoassay procedures, see Stits& Terreds.，Basic and Clinical Immunology(7^thed.1991). Furthermore, the immunoassay of the present invention mayThis is accomplished in several forms, such as Maggio, ed., Enzymemmunoassay (1980) and Harlow&Lane (as detailed above).

2. Immunological binding assays

T2R protein can be detected and/or quantified using any of the known immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of general immunoassays, see methods in cell biology: the use of Antibodies in Cell Biology (Methods in Cell Biology: Antibodies in Cell Biology, volume 37, Asai, ed.1993); basic and clinical immunology (Stiters, Basic and clinical immunology)& Terr，eds.，7^thed.1991). Immunological binding assays (or immunoassays) typically use antibodies that are capable of specifically binding to a selected protein or antigen, which in this application is a member of the T2R family or antigenic subsequence thereof. Antibodies (e.g., anti-T2R) can be produced using a variety of means known to those of skill in the art and various methods as described above.

Immunoassays also often use labels that specifically bind and label antigen-antibody complexes. The label itself may be a part of the antibody/antigen complex. Thus, the label may be a labeled T2R polypeptide or a labeled anti-T2R antibody. Alternatively, the label may be a third moiety, such as a second antibody, which is capable of specifically binding to the antibody/T2R complex (the second antibody typically specifically binds to an antibody of the species producing the first antibody). Other proteins capable of specifically binding to immunoglobulin constant regions, such as protein A or protein G, may also be used as labels. These proteins exhibit strong non-immunogenic reactivity with immunoglobulin constant regions from many species (see, e.g., Kronval et al, J.Immunol., 111: 1401-1406 (1973); Akerstrom et al, J.Immunol., 135: 2589-2542 (1985)). The label, such as biotin, can be modified with a detectable moiety that can specifically bind to another molecule, such as streptavidin. A variety of detectable moieties are well known to those of skill in the art.

Throughout the assay, incubation and/or rinsing steps are required for each binding of one reagent. The incubation step is from 5 seconds to several hours, alternatively from about 5 minutes to about 24 hours. However, the incubation time will depend on the assay format, antigen, solution volume, concentration, etc. Typically, the tests are conducted at room temperature, although they may be conducted over a range of temperatures, such as 10 ℃ to 40 ℃.

a. Non-competitive assay format

Immunoassays for the detection of T2R protein in a sample can be either competitive or noncompetitive. Noncompetitive immunoassays are those in which the amount of antigen is determined directly. For example, in a preferred "sandwich" (sandwich) assay, anti-T2R antibody can be bound directly to its immobilized solid support. These immobilized antibodies can then capture any T2R protein in the test sample. The T2R protein is thus immobilized and then bound to a label, such as a second T2R antibody carrying a label. Alternatively, the second antibody may be unlabeled, but it may bind a labeled third antibody that is specific for an antibody 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, which can specifically bind to another molecule, such as streptavidin, to provide a detectable moiety.

b. Competitive assay format

In a competitive assay, the amount of T2R protein present in a sample is measured indirectly by measuring the amount of known added (foreign) T2R protein that is removed (competitively removed) from the anti-T2R antibody with the unknown T2R protein present in the sample. In a competitive assay, a known amount of T2R protein is added to a sample, which is then contacted with an antibody that specifically binds T2R. The amount of exogenous T2R protein bound to the antibody is inversely proportional to the concentration of T2R protein in the sample. In a particularly preferred embodiment, the antibody is immobilized on a solid phase substrate. The amount of T2R protein bound to the antibody can be determined by measuring the amount of T2R protein in the T2R/antibody complex, or by measuring the amount of protein remaining uncomplexed. The amount of T2R protein can be detected by providing a labeled T2R molecule.

Hapten inhibition assays are another preferred competitive assay. In this assay it is known that the T2R protein is immobilized on a solid substrate. A known amount of anti-T2R antibody was added to the sample, which was then contacted with immobilized T2R. The amount of anti-T2R antibody bound to the known immobilized T2R protein is inversely proportional to the amount of T2R protein present in the sample. Also, the amount of immobilized antibody can be detected by detecting the antibody-immobilized portion or the remaining antibody portion in the solution. The antibody may be detected directly when labelled or indirectly by the subsequent addition of a labelled moiety which specifically binds the antibody as described above.

c. Cross reaction assay

Immunoassays in competitive binding formats can also be used to determine cross-reactivity. For example, a protein encoded at least in part by a nucleic acid sequence disclosed herein can be immobilized on a solid support. Proteins (e.g., T2R polypeptide and homologs thereof) are added to the assay system and compete with the immobilized antigen for binding to antisera. The ability of the added protein to compete with the immobilized protein for binding to antisera was compared to the ability of the T2R polypeptide encoded by the nucleic acid sequence disclosed herein to compete with itself. The percent cross-reactivity of the above proteins was calculated using standard calculations. Antisera that cross-reacted less than 10% with each of the above added proteins were selected and pooled. Cross-reactive antibodies can optionally be removed from pooled antisera by immunoabsorption with the addition of specific proteins, such as an allogenic homolog. In addition, peptides containing amino acid sequences representing consensus sequences useful for identifying T2R family members can be used to determine cross-reactivity.

The immunoabsorbed and pooled antisera are then used to perform a competitive binding immunoassay as described above for comparison of a second protein (which is believed to be possibly an allelic or polymorphic variant of a T2R family member) with the immunogenic protein (i.e., the T2R polypeptide encoded by the nucleic acid sequences disclosed herein). To achieve this comparison, both proteins were tested over a wide range of concentrations, and the amount of each protein required to inhibit 50% binding of the antisera to the immobilized protein was determined. A second protein that is encoded by a nucleic acid sequence disclosed herein is considered to be capable of specifically binding to the polyclonal antibody raised by the T2R immunogen if the amount of the second protein required to inhibit 50% binding is less than 10 times the amount required for 50% binding inhibition by the protein encoded by the protein.

Antibodies directed against the T2R consensus sequence may also be used to prepare antibodies that specifically bind only to GPCRs of the T2R family, but not to GPCRs of other families. For example, polyclonal antibodies that specifically bind to a particular T2R family member can be prepared by subtracting cross-reactive antibodies using other T2R family members. Species-specific polyclonal antibodies can be obtained by similar routes. For example, the production of antibodies specific for human T2R1 can be achieved by subtracting out antibodies that cross-react with, for example, rat T2R1 or mouse T2R1 ortholog sequences.

d. Other test formats

Western blot (immunoblot) analysis can be used to detect and quantify T2R protein present in a sample. The technique generally involves separating the proteins of the sample by molecular weight-based gel electrophoresis, transferring the separated proteins to a suitable solid support (e.g., a nitrocellulose, nylon, or derivatized nylon membrane), and incubating the sample with an antibody that specifically binds to the T2R protein. The anti-T2R polypeptide antibody then specifically binds to the T2R polypeptide on the solid support. These antibodies may be labeled directly, or subsequently detected by a labeled antibody that specifically binds to the anti-T2R antibody (e.g., a labeled sheep anti-mouse antibody).

In addition, assay formats include Liposome Immunoassays (LIA) which utilize liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated agents or markers. The released reagents can then be detected using standard techniques (see Monroe et al, am. Clin. prod. Rev., 5: 34-41 (1986)).

e. Reduction of non-specific binding

It will be appreciated by those skilled in the art that it is often desirable to reduce non-specific binding in immunoassays. Particularly when the assay involves an antigen or antibody immobilized on a solid phase matrix, it is desirable to reduce the amount of non-specific binding to the solid phase matrix. Means for reducing non-specific binding are well known in the art. Generally, this technique involves coating a substrate with a proteinaceous component. In particular, components such as Bovine Serum Albumin (BSA), skim milk powder, gelatin are widely used, and the most preferable is milk powder.

f. Marker (1abels)

The particular label or detectable group used in the assay is not critical to the invention, so long as it does not significantly affect the specific binding of the antibody in the assay. The detectable group can be any substance having a detectable physical or chemical property. Such detectable labels are well developed in the field of immunoassays, and in general, most of the labels useful in these methods can be applied to the present invention. Thus, a label refers to any component that can be detected spectroscopically, photochemically, biochemically, immunochemically, electrically, optically, or chemically. Useful labels of the invention include magnetic beads (e.g., DYNABAEDSTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red (Texas red), rhodamine, etc.), radiolabels (e.g., fluorescent beads, etc.), and radioactive labels (e.g., fluorescent beads, fluorescent³H、¹²⁵I、³⁵S、¹⁴C or³²P), enzymes (such as horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and chromogenic labels such as colloidal gold or colored glass or plastic beads (e.g., polyphenylenes, polypropylenes, latex, etc.).

The label may be coupled directly or indirectly to the component of interest in the assay according to methods known in the art. As noted above, a wide variety of labels can be used depending on the sensitivity desired, ease of conjugation to the compound, stability desired, existing instrumentation, and waste disposal provisions.

Non-radioactive labels are typically labeled using an indirect method. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to another molecule (e.g., streptavidin) which itself can be detected or covalently linked to a signaling system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. The ligands and their targets may be used in any suitable combination with an antibody recognizing T2R protein, or a secondary antibody recognizing anti-T2R.

The molecule may also be conjugated directly to a signal generating compound, for example to an enzyme or fluorophore. The enzymes of interest as labels are primarily hydrolases, in particular phosphatases, esterases and glycosidases, or oxidases (oxidases), in particular peroxidases. The fluorescent compound comprises fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl compounds, umbelliferone and the like. Chemiluminescent compounds include luciferin (luciferin), and 2, 3-dihydrophenazine diones (2, 3-dihydrophthalazinediones), such as luminol. A variety of markers or signal generating systems that may be used are reviewed in U.S. Patent No.4,391,904.

Methods for detecting labels are well known in the art. Thus, for example, where the label is a radiolabel, the detection means may comprise a scintillation counter or autoradiography of the photosensitive film. When the label is a fluorescent label, the fluorophore can be excited by light of an appropriate wavelength and the resulting fluorescence detected. Fluorescence can be detected visually, through a photosensitive film, or by electron detectors such as Charge Coupled Devices (CCDs) or photomultipliers, etc. Similarly, an enzyme label may be detected by detecting the reaction product after providing a suitable substrate for the enzyme. Finally, simple chromogenic labels can be achieved by simply observing the color associated with the label. Thus, in a series of dipstick (dipstick) tests, conjugated gold typically shows a pink colour, whereas the various conjugated beads show the colour of the respective bead itself.

Some assay formats do not require the use of labeled components. For example, agglutination assays can be used to detect the presence or absence of target antibodies. In this assay, antigen-coated particles agglutinate with a sample containing the target antibody. In this format, none of the participating components need to be labeled, and the presence or absence of the target antibody can be determined by simple visual inspection.

E. Detection of taste modulators

Methods and compositions for determining whether a test compound is capable of specifically binding to a T2R polypeptide of the invention in vivo and in vitro are described below. Many indicators of cell physiology can be monitored to assess the effect of ligand binding to naturally occurring or chimeric T2R. These assays can be performed on intact cells expressing the T2R polypeptide, on permeabilized cells, or on membrane components produced by standard methods.

Taste receptors bind to tastants and trigger a chemical stimulus that is converted into an electrical signal. The activated or inhibited G protein in turn alters the properties of the target enzyme, channel, or other effector protein. Some examples are the activation of cGMP phosphodiesterase by transducins of the visual system, adenylate cyclase by stimulatory G proteins, activation of phospholipase C by Gq or other similar G proteins, and the modulation of different channels by Gi and other G proteins, among others. Downstream results can also be examined, such as the production of diacylglycerol and IP3 by phospholipase C, which in turn can also be examined for calcium migration by IP 3.

The T2R protein or polypeptide in the assay is generally encoded by a nucleotide sequence having SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, and 24, or a fragment thereof or conservatively modified variant sequence.

Alternatively, the T2R protein or polypeptide in the assay may be derived from a eukaryotic host cell comprising a nucleic acid sequence having a sequence identical to SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, and 24, or conservatively modified variants thereof. Generally, amino acid sequences are at least 30%, preferably 30-40%, more specifically 50-60, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. Alternatively, the T2R protein or polypeptide in the assay may comprise a region of the T2R polypeptide, such as an extracellular domain, a transmembrane region, a cytoplasmic domain, a ligand binding domain, and the like. Alternatively, a T2R polypeptide, or a portion thereof, may be covalently linked to a heterologous protein to produce a chimeric protein useful in the assays described herein.

Modulators of T2R activity may be tested using T2R proteins or polypeptides, whether recombinant or naturally occurring, as described above. Recombinant or naturally occurring T2R proteins and polypeptides may be isolated, expressed in cells, expressed on membranes derived from cells, expressed in animals or tissues. For example, sections of the tongue, cells isolated from the tongue, transformed cells, or membranes can be used. Modulation may be tested using one of the in vivo or in vitro assays described herein.

1. In vitro binding assays

Using a T2R polypeptide or chimeric molecule, e.g., an extracellular domain of T2R, a transmembrane region, or a combination thereof, covalently linked to a heterologous signaling domain; or a heterologous extracellular domain and/or transmembrane region covalently bound to the transmembrane and/or cytoplasmic domain of a T2R protein or polypeptide, and taste transduction can be assayed in vitro using soluble or solid phase reactions. In addition, the ligand binding region of the T2R polypeptide can be used to test its ligand binding in vitro soluble or solid phase reactions. In most embodiments, the chimeric receptor can be made to include all or a portion of the T2R polypeptide, as well as sequences that facilitate localization of T2R to the membrane, such as rhodopsin, e.g., an N-terminal fragment of rhodopsin protein.

Binding of the T2R protein, ligand binding domain, or chimeric protein to the ligand can be detected in solution, bilayer membranes (attached to a solid phase), lipid monolayers, or vesicles. Binding of the modulator can be tested using, for example, changes in spectral characteristics (e.g., fluorescence, absorption, refractive index), hydrodynamics (e.g., shape), chromatography, or solubility.

T2R-G protein interactions can also be tested. For example, binding of the G protein to the T2R polypeptide, or its release from the polypeptide, can be assayed. In the absence of GTP, the activator (activator) will lead to the formation of a tight complex of the G protein (all three subunits) and T2R. The complex can be detected by a variety of methods as described above. This assay can be modified to look for inhibitors (inhibitors), for example by adding activators to the tight complex of G protein and T2R in the absence of GTP, and then screening for inhibitors by observing the dissociation of the T2R-G protein complex. In the presence of GTP, the release of the G protein a subunit from the other two G protein subunits can serve as an activation criterion.

In another embodiment of the invention, a GTP γ S assay may be used. As described above, upon activation of GPCRs, the G.alpha.subunit of the G protein complex is stimulated to exchange bound GDP for GTP. Stimulation of ligand-mediated G protein exchange activity can be measured by biochemical assays in which the radiolabelled GTP γ added in the presence of the putative ligand is determined³⁵Binding of S to G protein. Typically, a membrane containing the chemosensory receptor of interest is mixed with the G protein complex. Potential inhibitors and/or activators and GTP γ S are added to the assay and binding of GTP γ S to the G protein is measured. Binding can be measured by liquid scintillation counting or other means known in the art, including Scintillation Proximity Assay (SPA). In another assay format, fluorescently labeled GTP γ S may be used.

In a particularly preferred embodiment, T2R-gustducin interaction is detected as a function of T2R receptor activation. For example, mouse T2R5 shows a strong actinone-dependent coupling with gustducin. This ligand-dependent coupling of the T2R receptor to gustducin can be used as a marker to identify modulators (modulators) of any member of the T2R family.

2. Fluorescence polarization assay

In another embodiment, fluorescence polarization ("FP") based assays can be used to detect and monitor ligand binding. Fluorescence polarization assays are a common laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzyme activity. The fluorescence polarization assay is homogeneous in that it does not require separation steps such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These tests are carried out directly in real time in solution, without the need for an immobilization stage. The polarization value can be measured repeatedly, and the measurement speed is very fast after adding the reagent, so that the sample can not be damaged. In general, the present techniques can be used to measure polarization values down to picomolar (picolor) to micromolar (micromolar) level fluorophores (fluorophores). This section describes how to apply fluorescence polarization, simply and quantitatively, to measure binding of the T2R polypeptide ligands of the invention.

When excited by plane polarized light, the fluorescent-labeled molecules emit light having a degree of polarization that is inversely proportional to their molecular rotation. Large fluorescent marker molecules retain a degree of stability in the excited state (4 nanoseconds for fluorescein) and the polarization of light remains somewhat constant between excitation and emission. Small fluorescent marker molecules rotate rapidly in the excited state, with a significant change in the polarization of light between excitation and emission. Thus, the polarization values of small molecules are lower and the polarization values of large molecules are higher. For example, a single-stranded fluorescein-labeled oligonucleotide has a relatively low polarization value, but has a higher polarization value when it is hybridized to the complementary strand. When using FP to detect and monitor tastant binding interactions that may activate or inhibit chemosensory receptors of the invention, fluorescently labeled tastants or autofluorescent tastants may be used.

Fluorescence polarization (P) is defined as:

where n is the emitted light intensity parallel to the excitation light plane and # is the emitted light intensity perpendicular to the excitation light plane. P, which is a ratio of light intensities, is a number without units. For example,and Beacon2000^TMThe system may be used in conjunction with these assays. Such systems typically express polarization values in milli-polarization units (1 polarization unit-1000 mP units).

The relationship between molecular rotation and size is described by the Perrin equation. In summary, the Perrin equation considers polarization in direct proportion to rotational relaxation (rotational relaxation) time, which is the time required for a molecule to rotate through an angle of about 68.5 °. The spin relaxation time is related to the viscosity (η), absolute temperature (T), molecular volume (V), and gas constant (R) by the following equation:

the spin relaxation time is very small (. apprxeq.1 ns) for small molecules such as fluorescein and very large (. apprxeq.100 ns) for large molecules such as immunoglobulin. The rotational relaxation time, i.e. the polarization, is directly related to the molecular volume if the viscosity and temperature are kept constant. The change in molecular volume may be due to interaction with other molecules, dissociation, polymerization, degradation, hybridization, or conformational change of a fluorescently labeled molecule, among others. For example, fluorescence polarization has been used to measure enzymatic cleavage of macromolecular fluorescein-labeled polymers, such as cleavage by proteases, dnases, and rnases. It is also used to measure equilibrium binding of protein/protein interactions, antibody/antigen binding, and DNA/protein binding.

3. Solid phase and soluble high throughput assay

In another embodiment, the invention provides solubility assays using a T2R polypeptide, or a cell or tissue expressing a T2R polypeptide. In another embodiment, the invention provides solid phase-based in vitro assays in a high throughput format in which a T2R polypeptide, or cells or tissues expressing a T2R polypeptide, is attached to a solid phase substrate.

In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands within one day. In particular, each well of the microplate may be used to perform a separate assay for a selected potential modulator, or if the concentration or incubation time effect is observed, one modulator may be tested every 5-10 wells. Thus, a standard microplate can test approximately 100 (e.g., 96) modulators. If 1536 well plates are used, one plate can easily analyze 1000 to about 1500 different compounds. Multiple compounds can also be analyzed in each plate well. In addition, several different microplates can be analyzed each day, making it possible to analyze approximately 6,000-20,000 compounds each day; recently, microfluidic methods of reagent manipulation have been developed.

The molecule of interest may be bound to the solid state component directly or indirectly by covalent or non-covalent bonds, such as via a label (tag). The label can be any of a wide variety of compositions. Generally, the label-binding molecules (label conjugates) are immobilized on a solid support, and the labeled molecule of interest (e.g., taste transduction molecule of interest) is immobilized on the solid support through interaction of the label and the label conjugate.

Many labels and label conjugates can be used based on known molecular interactions described in the literature. For example, where a tag has a native binder, such as biotin, protein A, or protein G, it can be used in conjunction with an appropriate tag binder (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.). Antibodies against molecules with native binders such as biotin are also widely available and are suitable marker binders (see, SigmaImmunochemicals 1998 Catalogue, Sigma, st. louis MO).

Similarly, any hapten or antigenic compound can be conjugated to an appropriate antibody to form a label/label conjugate pair. Thousands of specific antibodies have been commercialized and many others have been described in the literature. For example, in one common method, the label is a primary antibody and the label conjugate is a secondary antibody that specifically recognizes the primary antibody. In addition to antibody-antigen interactions, receptor-ligand interactions may also serve as suitable label and label conjugate pairs. For example, cell membrane receptor agonists and antagonists (e.g., cell receptor-ligand interactions such as transferrin, c-box, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, cadherin family, integrin family, selectin family, etc., see, e.g., Pogott & Power, The Adhesion Molecule facts Book I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opioids, steroids, etc.), intracellular receptors (e.g., substances that mediate the effects of various small ligands, including steroids, thyroid hormones, retinoids and vitamin D, and peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids, and antibodies, etc., can interact with different cellular receptors.

Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates may likewise form suitable markers or marker combinations. Many other marker/marker combination pairs are equally beneficial to the assay systems described herein, as will be apparent to those skilled in the art upon review of this disclosure.

General linkers such as peptides, polyethers and the like can likewise serve as labels and include polypeptide sequences, for example, poly-glycine sequences of about 5 to 200 amino acids. Such elastic joints are known to those skilled in the art. For example, poly (ethylene glycol) linkers are available from shearwater polymers, inc. These linkers optionally have amide, sulfhydryl, or heterofunctional linkages, and the like.

The label conjugate can be immobilized on the solid phase substrate using various methods known in the art. Typically, all or part of the solid phase substrate is exposed to a chemical reagent (which immobilizes a chemical group to the surface that is capable of reacting with a portion of the label conjugate) to accomplish its derivatization or functionalization. For example, groups suitable for attachment to a long chain moiety may include amine, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes (aminoalkylsilanes) and hydroxyalkylsilanes (hydroxyalkylsilanes) may be used for the functionalization of a range of surfaces, such as glass surfaces. Methods for constructing such solid-phase biopolymer membrane arrays are described in detail in the literature. See, for example, Merrifield, j. Am. chem. soc., 85: 2149-2154(1963) (synthesis of solid phases such as peptides is described); geysen et al, J. Immun. meth, 102: 259-274(1987) (synthesis of solid phase components on needles is described); frank & Doring, Tetrahedron, 44: 6031-6040 (1988) (describes the synthesis of various peptide sequences on cellulose disks); fodor et al, Science, 251: 767 777 (1991); sheldon et al, Clinical Chemistry, 39 (4): 718 719 (1993); and Kozal et al, Nature medicine, 2 (7): 753-759 (1996) (both describe biopolymer arrays immobilized on a solid support). Non-chemical methods of fixing the label conjugate to the substrate include other common methods such as heating, UV radiation crosslinking, and the like.

4. Computer-based testing

Another assay for compounds that modulate the activity of T2R polypeptides involves computer-aided compound design in which a computer system is used to generate the three-dimensional structure of a T2R polypeptide based on structural information encoded by the amino acid sequence. The input amino acid sequence interacts directly and positively with pre-established algorithms in a computer system to generate secondary, tertiary and quaternary structural models of the protein. The protein structural model is then examined to determine the structural regions that have the ability to bind, for example, a ligand. These regions are then used to identify ligands that can bind to the protein.

A three-dimensional model of the structure of a protein is generated by inputting into a computer system the amino acid sequence of the protein having at least 10 amino acid residues or the corresponding nucleic acid sequence encoding a T2R polypeptide. The nucleic acid sequence encoding the T2R polypeptide, or the amino acid sequence thereof, may be any of the sequences disclosed herein and conservatively modified variants thereof.

The amino acid sequence represents the primary sequence or subsequence of a protein, which encodes structural information of the protein. Information distributed via the internet and RAM is entered into a computer system via a keyboard, computer readable substrates including, but not limited to, electronic storage media (e.g., disks, tapes, cartridges, and chips), optical media (e.g., CD ROM), and the like, an amino acid sequence of at least 10 residues (or a nucleotide sequence encoding 10 amino acids). A three-dimensional structural model of the protein is then generated by interaction of the amino acid sequence with a computer system, using software known to those skilled in the art.

The amino acid sequence represents the primary structure, which encodes the information necessary to form the secondary, tertiary and quaternary structure of the protein of interest. The software generates a structural model based on certain parameters of the primary sequence encoding. These parameters, referred to as "energy parameters," include primarily electrostatic potential, hydrophobic potential, solvent-accessible surface, and hydrogen bond formation. The secondary energy parameter includes van der waals potential. Biomolecules form structures that minimize the value of this energy parameter in a cumulative fashion. The computer program then uses these parameter values encoded by the primary structure or amino acid sequence to generate a secondary structure model.

On the basis of the secondary structure energy parameter, the tertiary structure of the protein encoded by the secondary structure is formed. The user may then enter additional variables such as whether the protein is membrane bound or soluble, its location within the body, its intracellular location such as the cytoplasm, surface or nucleus, etc. These variables, as well as the secondary structure energy parameters, are used to generate a tertiary structure model. In the tertiary structure modeling, a computer program matches a secondary structure hydrophobic surface with the like, and matches a secondary structure hydrophilic surface with the like.

Once the structure is generated, the computer identifies potential ligand binding regions. By inputting amino acid or nucleotide sequences or chemical structural formulas of compounds as described above, three-dimensional structures of potential ligands can be generated. The three-dimensional structure of the potential ligand is then compared to the three-dimensional structure of the T2R polypeptide, thereby identifying the ligand that binds to the protein. The binding affinity between the protein and the ligand is determined by the energy parameter, from which it is possible to determine which ligands have an increased probability of binding to the protein.

Computer systems are also useful for screening for mutations, polymorphic variants, alleles, and interspecies homologs of the T2R gene. Such mutations may be associated with a disease state or a genetic trait. As described above, Gene chip (GeneChip)^TM) The same can be used to screen for mutations, polymorphic variants, alleles, and interspecies homologs as is the case with related techniques. Once the variants are identified, diagnostic tests can be used to identify patients with mutated genes. Identification of a mutant T2R gene involves receiving an input of a first nucleic acid or amino acid sequence of the T2R gene or conservatively modified variants thereof. The sequence is entered into the computer system using the method described above. The first nucleic acid or amino acid sequence is then compared to a second nucleic acid or amino acid sequence that is substantially identical to the first sequence. The second sequence is entered into the computer system using the method described above. Once the first and second sequences are compared, nucleotide or amino acid differences between the two sequences can be determined. These sequences can represent allelic differences in the different T2R genes, mutations associated with disease states and genetic traits.

5. Cell-based binding assays

In a preferred embodiment, the T2R protein or polypeptide is expressed in eukaryotic cells as a chimeric receptor with a heterologous chaperone sequence capable of promoting its maturation and targeting through the secretory pathway. In a preferred embodiment, the heterologous sequence is a rhodopsin sequence, such as an N-terminal fragment of rhodopsin. Such chimeric T2R proteins can be expressed in any eukaryotic cell, such as HEK-293 cells. Preferably, these cells comprise a functional G protein, such as G α 15, which may be coupled to a chimeric receptor to an intracellular signaling pathway, or to a signaling protein such as phospholipase C. Activation of such chimeric receptors in cells can be detected by standard methods, such as by detecting changes in intracellular calcium by measuring intracellular FURA-2 dependent fluorescence.

The activated GPCR receptor becomes a substrate for kinases that are capable of phosphorylating the C-terminal tail of the receptor (and possibly other positions as well). Thus, activators can promote³²The transfer of P from gamma-labeled GTP to the receptor can be analyzed with a scintillation counter. C-terminal tail phosphorylation promotes the binding of a capture (arrestin) -like protein, thereby interfering with G protein binding. The kinase/captin pathway plays an important role in desensitization of many GPCRs. For example, compounds that modulate the duration of taste receptor maintenance activity can be used to prolong a desired taste or to remove an unpleasant taste. For a general review of GPCR signaling and Methods of assaying signaling, see Methods in Enzymology, vols.237 and 238 (1994) and volume 96 (1983); bourne et al, Nature, 10: 349: 117-27 (1991); bourne et al, Nature, 348: 125-32 (1990); pitcher et al, annu. rev. biochem., 67: 653-92 (1998).

T2R modulation was analyzed by comparing the response of T2R polypeptide treated with putative modulator with the response of untreated control samples. Such putative T2R modulators may include tastants that inhibit or activate the activity of T2R polypeptide. In one embodiment, the relative activity value of T2R for the control sample (not treated with either an activator or inhibitor) is assigned to 100. Inhibition of the T2R polypeptide is obtained when the T2R activity value is about 90%, alternatively 50% or 25-0% relative to the control. Activation of the T2R polypeptide is obtained when the T2R activity value is 110%, alternatively 150%, 200-500% or 1000-2000% relative to the control.

Changes in ion flux can be assessed by determining changes in ion polarization (i.e., electrical potential) of cells or membranes expressing the T2R polypeptide. One means of determining changes in cell polarization is to measure changes in current (and thus detect changes in polarization) using voltage-clamp (voltage-clamp) and patch-clamp (patch-clamp) techniques (see, for example, "cell attachment" mode, "endo-exo" mode, and "whole cell" mode, e.g., Ackerman et al, New Engl. JMed., 336: 1575-. The whole-cell current can be conveniently measured by known standard methods. Other known tests include: radiolabelled ion flux assays and fluorescence assays using voltage sensitive dyes (see, for example, Vestergarrd-Bogind et al, J. Membrane biol., 88: 67-75 (1988); Gonzales & Tsien, chem. biol., 4: 269-277 (1997); Daniel et al, J. Pharmacol. meth., 25: 185-193 (1991); Holevinsky et al, J. Membrane biol., 137: 59-70 (1994)). Generally, the compound to be tested is present in a concentration of from 1pM to 100 mM.

The effect of a compound to be tested on the function of the T2R polypeptide can be measured by examining any of the parameters described above. Any appropriate physiological change that affects GPCR activity can be used to assess the effect of a test compound on a T2R polypeptide of the invention. When the functional result is determined using intact cells or animals, a range of effects such as transmitter release, hormone release, transcriptional changes of known and unidentified genetic markers (e.g., Northern blots), changes in cellular metabolism such as changes in cell growth or pH, and intracellular second messengers such as Ca can also be measured²⁺IP3, changes in cGMP or cAMP, etc.

Preferred assays for GPCRs include cells loaded with ion or voltage sensitive dyes capable of reporting receptor activity. Assays for determining the activity of such receptors may also use known agonists and antagonists of other G protein-coupled receptors as negative or positive controls to evaluate the activity of the test compound. In assays to identify compounds that modulate activity (e.g., agonists, antagonists), changes in the level of cytoplasmic ions or membrane potential can be monitored using ion sensitive or membrane potential fluorescent indicators, respectively. Ion sensitive indicators and voltage Probes that may be used are described in Molecular Probes1997 Catalot. For GPCRs, promiscuous G proteins such as G.alpha.15 and G.alpha.16 can be used in selection experiments (Wilkie et al, PNAS, 88: 10049-10053 (1991)). Such promiscuous G proteins allow the coupling of a wide range of receptor molecules.

Receptor activation typically initiates subsequent intracellular events, such as the increase of second messengers, such as IP3, which are capable of releasing intracellular calcium ion stores. Activation of some GPCRs stimulates the formation of inositol triphosphate (IP3) by phospholipase C mediated hydrolysis of phosphatidylinositol (Berridge & Irvine, Nature, 312: 315-21 (1984)). IP3 in turn stimulates the release of intracellular calcium ion stores. Thus, changes in the level of cytosolic calcium ions, or changes in the level of second messengers such as IP3, can be used to assess GPCR function. Cells expressing such GPCRs may exhibit increased levels of cytosolic calcium ions due to intracellular inventory release and activation by ion channels, in which case it is desirable, although not essential, to conduct the assay in a buffer free of calcium ions, optionally with the addition of a chelating agent such as EGTA in solution to distinguish fluorescence reactions caused by internal inventory calcium release.

Other assays may include determining receptor activity, which upon activation results in a change in intracellular cyclic nucleotide levels, such as cAMP or cGMP, by activating or inhibiting enzymes, such as adenylate cyclase. Cyclic nucleotide-gated ion channels, such as the pyramidal photoreceptor cell channel and the olfactory neuron channel, are permeable to cations when activated by binding cAMP or cGMP (see, e.g., Altenhofen et al, PNAS, 88: 9868-. In the case where activation of the receptor results in a decrease in cyclic nucleotide levels, it may be preferred to expose the cells to an agent capable of increasing intracellular cyclic nucleotide levels, such as forskolin, prior to adding the receptor activating compound to the test cells. Cells for use in this type of assay can be prepared by co-transfecting host cells with DNA encoding a cyclic nucleotide-gated ion channel, GPCR phosphatase, and DNA encoding receptors (e.g., certain glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, etc.), which upon activation will cause a change in the level of intracytoplasmic cyclic nucleotides.

In a preferred embodiment, the activity of the T2R polypeptide is measured by expressing the T2R gene in a heterologous cell having a promiscuous G protein linking the receptor to the phospholipase C signaling pathway (see offermann)&Simon, j. biol. chem., 270: 15175-15180 (1995)). Alternatively the cell line is HEK-293 (which does not express the T2R gene in its native state) and the promiscuous G protein is G.alpha.15 (O)ffermanns &Simon, supra). By measuring intracellular Ca²⁺Modulation of taste transduction is analyzed by changes in levels (which change in response to modulation of the T2R signaling pathway by uptake of molecules that bind to the T2R polypeptide). Ca²⁺The change in level is optionally caused by fluorescence Ca²⁺Indicator dyes and fluorescence imaging techniques.

In one embodiment, changes in cAMP or cGMP within the cell can be measured using an immunoassay. Offermanns & Simon, J. Bio1 chem., 270: 15175-15180 (1995) can be used to determine cAMP levels. Similarly, Felley-Bosco et al, Am. J. Resp. Celland mol. biol., 1 l: 159-164 (1994) can be used to determine cGMP levels. In addition, test kits for measuring cAMP and/or cGMP are described in U.S. Pat. No.4,115,538, which is incorporated herein by reference.

In another embodiment, Phosphatidylinositol (PI) hydrolysis can be analyzed according to U.S. Patent5,436,128, which is incorporated herein by reference. Briefly, the test involves³The cells were labeled with H-inositol for 48 hours or more. The labeled cells are treated with the test compound for 1 hour. The treated cells were lysed and extracted with chloroform-methanol-water, after which inositol phosphate was isolated by ion exchange chromatography and quantified by scintillation counting. Fold stimulation was determined by calculating the ratio of cpm in the presence of agonist to cpm in the presence of buffer control. Similarly, fold inhibition can be determined by calculating the ratio of cpm in the presence of antagonist to cpm in the presence of a buffer control (which may or may not contain agonist).

In another embodiment, the transcript level can be measured for assessing the effect of the test compound on signaling. The host cell containing the T2R polypeptide can be contacted with the test compound for a sufficient period of time to effect any interaction, and the level of gene expression of the protein of interest can then be measured. The time to achieve these interactions can be determined empirically, for example by performing a time course and measuring the transcript level as a function of time. The amount of transcription can be measured using suitable methods known to those skilled in the art. For example, expression of the mRNA of the protein of interest can be detected by Northern blot detection or by identifying its polypeptide product using an immunoassay. Alternatively, transcription-based assays using reporter genes can be used, as described in U.S. Patent5,436,128, which is incorporated herein by reference. The reporter gene may be, for example, chloramphenicol acetyltransferase, luciferase, 3' -galactosidase and alkaline phosphatase. In addition, the protein of interest can be indirectly used as a reporter after attachment to a second reporter, such as green fluorescent protein (see, e.g., Mistii & Spector, Nature Biotechnology, 15: 961-.

The amount of transcription is then compared to the amount of transcription in the same cell in the absence of the test compound, or to the amount of transcription in substantially the same cell in the absence of the T2R polypeptide. Substantially identical cells may be derived from the same cells used to prepare the recombinant cells, but without the introduction of heterologous DNA modifications. Any difference in the amount of transcription means that the test compound changes the activity of the target T2R polypeptide to some extent.

6. Transgenic non-human animals expressing taste receptors

Non-human animals expressing one or more of the T2R polypeptides of the invention are also useful in assays. Such expression can be used to determine whether a test compound can specifically bind to a mammalian T2R polypeptide in vivo by contacting a non-human animal stably or transiently transfected with a nucleic acid encoding a T2R polypeptide or a ligand binding region thereof with a test compound and determining whether the animal reacts to the test compound by specifically binding the polypeptide.

Animals transfected or infected with the vectors of the invention are particularly useful for assays to identify and identify tastants/ligands that bind to a specific or a panel of receptors. Such animals infected with the vector, which express human chemosensory receptor sequences, can be used to screen for tastants in vivo and to analyze the effect of tastants on cell physiology (e.g., on taste neurons), on the CNS or on behavior.

Means for infecting/expressing nucleic acids and vectors, either alone or as a library format, are well known in the art. Parameters of a wide variety of individual cells, organs, or whole animals can be measured by a variety of means. For example, the T2R sequences of the invention can be expressed in animal taste tissues by delivery using infectious agents such as adenoviral expression vectors.

The endogenous chemosensory receptor gene may remain functional and wild-type (native) activity is still present. In other cases, where it is desired that all chemosensory receptor activity be elicited by the introduced heterologous hybrid receptor, it is preferred to use a knock-out line. Methods for the selection and preparation of recombinant constructs for the construction of non-human transgenic animals, particularly transgenic mice, and for the generation of transformed cells are well known in the art.

Construction of "knock-out" cells and animals is based on the premise that the expression level of a particular gene in a mammalian cell can be reduced or completely eliminated by introducing into the genome a new DNA sequence that interferes with a portion of the DNA sequence of the gene to be inhibited. Likewise, "gene trap insertion" (gene transfer) can be used to disrupt host genes, and mouse embryonic stem cells (ES) can be used to generate knock-out Transgenic animals (see, e.g., Holzschu, Transgenic Res. 6: 97-106 (1997)). Insertion of heterologous sequences is typically accomplished by homologous recombination between complementary nucleic acid sequences. Heterologous sequences are part of the target gene to be modified, such as exons, introns, or transcriptional regulatory sequences, or any genomic sequence that can affect the level of expression of the target gene; or a combination thereof. Gene targeting via homologous recombination in pluripotent embryonic stem cells can allow for precise modification of genomic sequences of interest. Any technique can be used to generate, screen, breed knockout animals, see, for example, Bijvoet, hum. 53-62 (1998); moreadith, j. mol. med. 75: 208-216 (1997); tojo, Cytotechnology 19: 161-165 (1995); mudgett, methodsmol, biol, 48: 167-; longo, Transgenic Res.6: 321-328 (1997); U.S. Patents nos. 5,616,491; 5,464,764; 5,631,153, respectively; 5,487,992; 5,627,059, respectively; 5,272,071; WO 91/09955; WO 93/09222; WO 96/29411; WO 95/31560; WO 91/12650.

The nucleic acids of the invention are also useful as agents for generating "knock-out" human cells and their progeny. Similarly, the nucleic acids of the invention can also be used as reagents for generating mouse "knock-ins". The human or rat T2R gene sequence may replace orthologous T2R in the mouse genome. By this route, mice expressing human or rat T2R can be generated. The mouse can then be used to analyze the function of human or rat T2R, and to identify ligands for such T2R.

F. Regulator

The compounds tested as modulators of T2R family members may be any small chemical compound, or biological entity such as a protein, sugar, nucleic acid, or lipid. Alternatively, the modulator may be a genetic variant of a member of the T2R family. In general, test compounds can be small chemical molecules and peptides. Essentially any chemical compound can serve as a potential modulator or ligand in the assays of the invention, although in most cases the compound is dissolved in water or an organic solvent (especially based on DMSO). By automating the assay steps, and providing the compounds from any convenient source for assays that can be used to screen large chemical libraries, the assays are typically performed in parallel (e.g., in microtiter format on microtiter plates in machine 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-biochemicala Analytika (Buchs, Switzerland), and others.

In one embodiment, the high throughput screening method involves providing a combinatorial chemistry or peptide library comprising a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more of the above assays to identify those library members (particularly chemical classes or subclasses) that exhibit the desired characteristic activity. The compounds thus identified may serve as conventional "lead compounds" or may themselves serve as potential or actual consumables.

Combinatorial chemical libraries are collections of different chemical compounds (produced by chemical synthesis or biosynthesis) that are combined by a large number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide library, is formed from a series of chemical building blocks (amino acids) combined in various possible ways with a specified compound length (i.e., the number of amino acids in the polypeptide compound). Millions of chemical compounds can be synthesized by such combinatorial mixing of chemically structured components.

The preparation and screening of combinatorial chemical libraries is well known to those skilled in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No.5,010,175, Furka, int. J. Pept. prot. Res., 37: 487-493 (1991) and Houghton et al, Nature, 354: 84-88 (1991)). Other chemicals that generate chemically diverse libraries may also be used. Such chemicals include, but are not limited to: peptoids (e.g., WO91/19735), encoded peptides (e.g., WO 93/20242), random bioligomers (PCT e.g., WO 92/00091), benzodiazepines(e.g., U.S. Pat. No.5,288,514), hydantoins and benzodiazepinesAnd heteropolymers of dipeptides (diversomers) (Hubbs et al, PNAS 90: 6906-6913 (1993)), divinyl (vinylogous) polypeptides (Hagihara et al, J.Amer.chem. Soc., 114: 6568 (1992)), non-peptide mimetics with glucose scaffolds (nonippepidated peptidomimetics) (Hirschmann et al, J.Amer.chem. Soc., 114: 9217-9218(1992)), libraries of similar organic synthetic small molecule compounds (Chen et al, J.Amer.chem. Soc., 116: 2661 (1994)), oligocarbamates (oligocarbamates) (Cho et al, Science, 261: 1303 (1993)), peptidylphosphonates (Campbeber et al, J.org. chem., 59: 658, nucleic acid libraries (Saccharoboran., Becko et al., and prepro peptides, 1994), peptidyl peptides (peptide, and peptide)Nucleic acid libraries (U.S. Patent5,539,083), antibody libraries (Vaughn et al, Nature Biotechnology, 14 (3): 309-，Baum，C&EN, Jan 18, page 33 (1993); thiazolidinones (thiazolididinones) and methidazinones, U.S. Patent5,549,974; pyranosides, u.s.patents 5,525,735 and 5,519,134; morpholino compounds, U.S. patent5,506,337; benzodiazepine5,288,514, etc.).

Devices for making combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS (Advanced Chem Tech, Louisville KY), Symphony (Rainin, Woburn, Mass.), 433A (Applied Biosystems, Foster City, Calif.), 9050 Plus (Millipore, Bedford, Mass)). In addition, many combinatorial libraries are also commercially available per se (see, e.g., ComGenex, Princeton, NJ; Tripos, Inc., St. Louis, MO; 3DPharmaceuticals, Exton, PA; Martek Biosciences; Columbia, MD, etc.).

One aspect of the present invention is that the T2R modulator may be used in any edible product, confectionery, pharmaceutical composition, or ingredient thereof to modulate the taste of the product, composition, or ingredient in a desired manner. For example, T2R modulators that enhance the perception of bitter taste may be added to increase the bitter taste of the product or composition, while T2R modulators that block the perception of bitter taste may be added to improve the taste of the product or composition.

G. Methods for describing and predicting taste perception

The present invention also preferably provides methods of describing taste perception and/or predicting taste perception in a mammal, particularly a human. Preferably, the methods can be accomplished by using the receptors disclosed herein and using the genes encoding the T2R polypeptides disclosed herein.

The invention also includes a method of screening one or more compounds to detect the presence of a taste detectable by a mammal comprising: contacting one or more of the compounds referred to above with the disclosed receptor, preferably wherein the mammal is a human. The present invention may also include a method of demonstrating taste perception of a particular taste in a mammal comprising the steps of: providing X1 to Xn values representing quantitative stimulation of each of said vertebrate n taste receptors, wherein n is greater than or equal to 4; from these values, a quantitative description of taste perception is generated. The taste receptor may be a taste receptor as disclosed herein, and the description may comprise a volume of a point or n-dimensional space, may comprise a graph or a spectrum, and may comprise a quantitative description matrix. Also, the step of providing may comprise contacting a plurality of recombinantly produced taste receptors with a test composition and quantitatively measuring the effect of the composition on the receptors.

The invention also encompasses a method of predicting taste perception produced in a mammal by one or more molecules or combinations of molecules that produce an unknown taste perception in a mammal, comprising the steps of: providing X1 to Xn values representing the amount of stimulation of each of n taste receptors of the vertebrate for one or more molecules or combinations of molecules that produce a known taste perception in the mammal, wherein n is greater than or equal to 4; producing a quantitative description of taste perception from said value for one or more molecules or combinations of molecules that produce known taste perception in a mammal, providing X1 to Xn values representative of the stimulation of each of n taste receptors in said vertebrate for one or more molecules or combinations of molecules that produce unknown taste perception in a mammal, wherein n is greater than or equal to 4; generating an unknown taste perception in the mammal for one or more molecules or combinations of molecules that produce the unknown taste perception in the mammal, generating a quantitative description of taste perception from the value, predicting the unknown taste perception generated in the mammal by comparing the quantitative description in the mammal that is generated in the mammal by the one or more molecules or combinations of molecules that produce the unknown taste perception in the mammal to the quantitative description in the mammal that is generated in the mammal by the one or more molecules or combinations of molecules that produce the known taste perception in the mammal. Taste receptors useful in the present methods can include taste receptors disclosed herein.

In another embodiment, by determining the value of taste perception in a mammal of a known molecule or combination of molecules as described above; determining a taste perception value in the mammal of one or more unknown molecules or combinations of molecules as described above; comparing the taste perception value of the one or more unknown compositions in the mammal to the taste perception value of the one or more known compositions in the mammal; selecting a molecule or combination of molecules capable of eliciting a particular taste perception in a mammal; and combining two or more unknown molecules or combinations of molecules to form a molecule or combination of molecules capable of eliciting a predetermined taste perception in a mammal, thereby obtaining novel molecules or combinations of molecules that elicit a predetermined taste perception in a mammal. The combining step results in a single molecule or combination of molecules capable of eliciting a particular taste perception in the mammal.

Another embodiment of the present invention is a method of stimulating taste perception comprising the steps of: for each of the cloned taste receptor populations, preferably human receptor populations, determining the extent of receptor interaction with the tastant; combining a population of compounds each having a predetermined interaction with one or more of said receptors in amounts that together provide a receptor-stimulation profile that mimics the profile of taste cells. Interaction of tastants with taste receptors can be determined by any of the binding or reporter assays described herein. The plurality of compounds may then be combined to form a mixture. If desired, one or more of the plurality of compounds may be covalently bound. The combined compounds substantially stimulate at least 75%, 80%, or 90% of the receptors substantially stimulated by the tastant.

In another preferred embodiment of the invention, a plurality of standard compounds are tested against a plurality of taste receptors to determine the extent of interaction between each receptor and 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 residing on a data storage medium. The method may further comprise providing a desired receptor stimulation profile of taste; comparing the desired receptor stimulation profile to a relational database; determining the combination of one or more standardized compounds that most closely matches the desired receptor stimulation profile. The method may further comprise combining the standard compounds into one or more defined combinations to stimulate taste.

H. Reagent kit

The T2R gene and their homologues are advantageous tools for identifying taste receptor cells, forensic and paternity testing, and testing taste transduction. T2R family member specific reagents, such as T2R probes and primers, capable of specifically hybridizing to T2R nucleic acids, and T2R specific reagents, such as T2R antibodies, capable of specifically binding to T2R proteins, can be used to examine taste cell expression and taste transduction modulation.

Nucleic acid assays for determining the presence of T2R family member DNA and RNA in a sample include a number of techniques well known in the art, such as Southern analysis, Northern analysis, dot blotting, RNase protection, S1 analysis, amplification techniques such as PCR, and in situ hybridization. For example, in situ hybridization, the target nucleic acid is released from its intracellular environment to allow hybridization within the cell while maintaining cell morphology for subsequent interpretation and analysis. The following article provides an overview of in situ hybridization: singer et al, Biotechniques, 4: 230-250 (1986); haase et al, methods in Virology, vol.VII, 189-226 (1984); and Names et al, eds., nucleic Hybridization: a Practical Approach (1987). In addition, the T2R protein can be detected using various immunoassay techniques as described above. The test sample is typically compared to both a positive control (e.g., a sample expressing recombinant T2R protein) and a negative control.

The invention also provides kits for screening for modulators of T2R family members. These kits can be prepared from readily available materials and reagents. For example, these kits may comprise any one or more of the following materials: T2R nucleic acids or proteins, reaction tubes, guidelines for testing T2R activity, and the like. Optionally, the kit comprises a functional T2R polypeptide. A wide variety of kits and components can be prepared in accordance with the present invention, depending on the particular user of the kit and the particular needs of the user.

Examples

The following examples outline the isolated nucleic acid molecules of the invention, and the polypeptide sequences which correspond to the conceptual translation of the nucleic acid molecules. In the protein sequences provided herein, the single letter X or Xaa refers to any of the twenty common amino acid residues. In the DNA sequences provided herein, the single letter N or N represents any of the four common nucleotide bases A, T, C, G.

hT2R51 full-length cDNA (BAC AC011654) (SEQ ID NO: 1)

ATGTTGACTCTAACTCGCATCCGCACTGTGTCCTATGAAGTCAGGAGTACATTTCTGTTCA

TTTCAGTCCTGGAGTTTGCAGTGGGGTTTCTGACCAATGCCTTCGTTTTCTTGGTGAATTTT

TGGGATGTAGTGAAGAGGCAGGCACTGAGCAACAGTGATTGTGTGCTGCTGTGTCTCAGC

ATCAGCCGGCTTTTCCTGCATGGACTGCTGTTCCTGAGTGCTATCCAGCTTACCCACTTCCA

GAAGTTGAGTGAACCACTGAACCACAGCTACCAAGCCATCATCATGCTATGGATGATTGCA

AACCAAGCCAACCTCTGGCTTGCTGCCTGCCTCAGCCTGCTTTACTGCTCCAAGCTCATCC

GTTTCTCTCACACCTTCCTGATCTGCTTGGCAAGCTGGGTCTCCAGGAAGATCTCCCAGAT

GCTCCTGGGTATTATTCTTTGCTCCTGCATCTGCACTGTCCTCTGTGTTTGGTGCTTTTTTA

GCAGACCTCACTTCACAGTCACAACTGTGCTATTCATGAATAACAATACAAGGCTCAACTG

GCAGATTAAAGATCTCAATTTATTTTATTCCTTTCTCTTCTGCTATCTGTGGTCTGTGCCTC

CTTTCCTATTGTTTCTGGTTTCTTCTGGGATGCTGACTGTCTCCCTGGGAAGGCACATGAGG

ACAATGAAGGTCTATACCAGAAACTCTCGTGACCCCAGCCTGGAGGCCCACATTAAAGCCC

TCAAGTCTCTTGTCTCCTTTTTCTGCTTCTTTGTGATATCATCCTGTGTTGCCTTCATCTCTG

TGCCCCTACTGATTCTGTGGCGCGACAAAATAGGGGTGATGGTTTGTGTTGGGATAATGGC

AGCTTGTCCCTCTGGGCATGCAGCCATCCTGATCTCAGGCAATGCCAAGTTGAGGAGAGCT

GTGATGACCATTCTGCTCTGGGCTCAGAGCAGCCTGAAGGTAAGAGCCGACCACAAGGCA

GATTCCCGGACACTGTGCTGA(SEQ ID NO：1)

hT2R51 conceptual translation (BAC AC011654) (SEQ ID NO: 2)

MLTLTRIRTVSYEVRSTFLFISVLEFAVGFLTNAFVFLVNFWDVVKRQALSNSDCVLLCLSISRL

FLHGLLFLSAIQLTHFQKLSEPLNHSYQAIIMLWMIANQANLWLAACLSLLYCSKLIRFSHTFLI

CLASWVSRKISQMLLGIILCSCICTVLCVWCFFSRPHFTVTTVLFMNNNTRLNWQIKDLNLFYS

FLFCYLWSVPPFLLFLVSSGMLTVSLGRHMRTMKVYTRNSRDPSLEAHIKALKSLVSFFCFFVIS

SCVAFISVPLLILWRDKIGVMVCVGIMAACPSGHAAILISGNAKLRRAVMTILLWAQSSLKVRA

DHKADSRTLC(SEQ ID NO：2)

hT2R54 full-length cDNA (BAC AC024156) (SEQ ID NO: 3)

ATGACTAAACTCTGCGATCCTGCAGAAAGTGAATTGTCGCCATTTCTCATCACCTTAATTTT

AGCAGTTTTACTTGCTGAATACCTCATTGGTATCATTGCAAATGGTTTCATCATGGCTATAC

ATGCAGCTGAATGGGTTCAAAATAAGGCAGTTTCCACAAGTGGCAGGATCCTGGTTTTCCT

GAGTGTATCCAGAATAGCTCTCCAAAGCCTCATGATGTTAGAAATTACCATCAGCTCAACC

TCCCTAAGTTTTTATTCTGAAGACGCTGTATATTATGCATTCAAAATAAGTTTTATATTCTT

AAATTTTTGTAGCCTGTGGTTTGCTGCCTGGCTCAGTTTCTTCTACTTTGTGAAGATTGCCA

ATTTCTCCTACCCCCTTTTCCTCAAACTGAGGTGGAGAATTACTGGATTGATACCCTGGCTT

CTGTGGCTGTCCGTGTTTATTTCCTTCAGTCACAGCATGTTCTGCATCAACATCTGCACTGT

GTATTGTAACAATTCTTTCCCTATCCACTCCTCCAACTCCACTAAGAAAACATACTTGTCTG

AGATCAATGTGGTCGGTCTGGCTTTTTTCTTTAACCTGGGGATTGTGACTCCTCTGATCATG

TTCATCCTGACAGCCACCCTGCTGATCCTCTCTCTCAAGAGACACACCCTACACATGGGAA

GCAATGCCACAGGGTCCAACGACCCCAGCATGGAGGCTCACATGGGGGCCATCAAAGCTA

TCAGCTACTTTCTCATTCTCTACATTTTCAATGCAGTTGCTCTGTTTATCTACCTGTCCAAC

ATGTTTGACATCAACAGTCTGTGGAATAATTTGTGCCAGATCATCATGGCTGCCTACCCTG

CCAGCCACTCAATTCTACTGATTCAAGATAACCCTGGGCTGAGAAGAGCCTGGAAGCGGCT

TCAGCTTCGACTTCATCTTTACCCAAAAGAGTGGACTCTGTGA(SEQ ID NO：3)

hT2R54 conceptual translation (BAC AC024156) (SEQ ID NO: 4)

MTKLCDPAESELSPFLITLILAVLLAEYLIGIIANGFIMAIHAAEWVQNKAVSTSGRILVFLSVSRI

ALQSLMMLEITISSTSLSFYSEDAVYYAFKISFIFLNFCSLWFAAWLSFFYFVKIANFSYPLFLKL

RWRITGLIPWLLWLSVFISFSHSMFCINICTVYCNNSFPIHSSNSTKKTYLSEINVVGLAFFFNLGI

VTPLIMFILTATLLILSLKRHTLHMGSNATGSNDPSMEAHMGAIKAISYFLILYIFNAVALFIYLS

NMFDINSLWNNLCQIIMAAYPASHSILLIQDNPGLRRAWKRLQLRLHLYPKEWTL(SEQ IDNO：4)

hT2R55 full-length cDNA (BAC AC024156) (SEQ ID NO: 5)

ATGGCAACGGTGAACACAGATGCCACAGATAAAGACATATCCAAGTTCAAGGTCACCTTC

ACTTTGGTGGTCTCCGGAATAGAGTGCATCACTGGCATCCTTGGGAGTGGCTTCATCACGG

CCATCTATGGGGCTGAGTGGGCCAGGGGCAAAACACTCCCCACTGGTGACCGCATTATGTT

GATGCTGAGCTTTTCCAGGCTCTTGCTACAGATTTGGATGATGCTGGAGAACATTTTCAGT

CTGCTATTCCGAATTGTTTATAACCAAAACTCAGTGTATATCCTCTTCAAAGTCATCACTGT

CTTTCTGAACCATTCCAATCTCTGGTTTGCTGCCTGGCTCAAAGTCTTCTATTGTCTTAGAA

TTGCAAACTTCAATCATCCTTTGTTCTTCCTGATGAAGAGGAAAATCATAGTGCTGATGCC

TTGGCTTCTCAGGCTGTCAGTGTTGGTTTCCTTAAGCTTCAGCTTTCCTCTCTCGAGAGATG

TCTTCAATGTGTATGTGAATAGCTCCATTCCTATCCCCTCCTCCAACTCCACGGAGAAGAA

GTACTTCTCTGAGACCAATATGGTCAACCTGGTATTTTTCTATAACATGGGGATCTTCGTTC

CTCTGATCATGTTCATCCTGGCAGCCACCCTGCTGATCCTCTCTCTCAAGAGACACACCCTA

CACATGGGAAGCAATGCCACAGGGTCCAGGGACCCCAGCATGAAGGCTCACATAGGGGCC

ATCAAAGCCACCAGCTACTTTCTCATCCTCTACATTTTCAATGCAATTGCTCTATTTCTTTC

CACGTCCAACATCTTTGACACTTACAGTTCCTGGAATATTTTGTGCAAGATCATCATGGCT

GCCTACCCTGCCGGCCACTCAGTACAACTGATCTTGGGCAACCCTGGGCTGAGAAGAGCCT

GGAAGCGGTTTCAGCACCAAGTTCCTCTTTACCTAAAAGGGCAGACTCTGTGA(SEQ IDNO：5)

hT2R55 conceptual translation (BAC AC024156) (SEQ ID NO: 6)

MATVNTDATDKDISKFKVTFTLVVSGIECITGILGSGFITAIYGAEWARGKTLPTGDRIMLMLSF

SRLLLQIWMMLENIFSLLFRIVYNQNSVYILFKVITVFLNHSNLWFAAWLKVFYCLRIANFNHP

LFFLMKRKIIVLMPWLLRLSVLVSLSFSFPLSRDVFNVYVNSSIPIPSSNSTEKKYFSETNMVNLV

FFYNMGIFVPLIMFILAATLLILSLKRHTLHMGSNATGSRDPSMKAHIGAIKATSYFLILYIFNAI

ALFLSTSNIFDTYSSWNILCKIIMAAYPAGHSVQLILGNPGLRRAWKRFQHQVPLYLKGQTL(SEQ ID NO：6)

hT2R61 full-length cDNA (BAC AC018630) (SEQ ID NO: 7)

ATGATAACTTTTCTACCCATCATTTTTTCCAGTCTGGTAGTGGTTACATTTGTTATTGGAAA

TTTTGCTAATGGCTTCATAGCACTGGTAAATTCCATTGAGTGGTTCAAGAGACAAAAGATC

TCCTTTGCTGACCAAATTCTCACTGCTCTGGCGGTCTCCAGAGTTGGTTTGCTCTGGGTATT

ATTATTAAACTGGTATTCAACTGTGTTGAATCCAGCTTTTAATAGTGTAGAAGTAAGAACT

ACTGCTTATAATATCTGGGCAGTGATCAACCATTTCAGCAACTGGCTTGCTACTACCCTCA

GCATATTTTATTTGCTCAAGATTGCCAATTTCTCCAACTTTATTTTTCTTCACTTAAAGAGG

AGAGTTAAGAGTGTCATTCTGGTGATGTTGTTGGGGCCTTTGCTATTTTTGGCTTGTCATCT

TTTTGTGATAAACATGAATGAGATTGTGCGGACAAAAGAATTTGAAGGAAACATGACTTG

GAAGATCAAATTGAAGAGTGCAATGTACTTTTCAAATATGACTGTAACCATGGTAGCAAA

CTTAGTACCCTTCACTCTGACCCTACTATCTTTTATGCTGTTAATCTGTTCTTTGTGTAAAC

ATCTCAAGAAGATGCAGCTCCATGGTAAAGGATCTCAAGATCCCAGCACCAAGGTCCACA

TAAAAGCTTTGCAAACTGTGATCTCCTTCCTCTTGTTATGTGCCATTTACTTTCTGTCCATA

ATGATATCAGTTTGGAGTTTTGGAAGTCTGGAAAACAAACCTGTCTTCATGTTCTGCAAAG

CTATTAGATTCAGCTATCCTTCAATCCACCCATTCATCCTGATTTGGGGAAACAAGAAGCT

AAAGCAGACTTTTCTTTCAGTTTTTTGGCAAATGAGGTACTGGGTGAAAGGAGAGAAGACT

TCATCTCCATAG(SEQ ID NO：7)

hT2R61 conceptual translation (BAC AC018630) (SEQ ID NO: 8)

MITFLPIIFSSLVVVTFVIGNFANGFLALVNSIEWFKRQKISFADQILTALAVSRVGLLWVLLLNW

YSTVLNPAFNSVEVRTTAYNIWAVINHFSNWLATTLSIFYLLKIANFSNFIFLHLKRRVKSVILV

MLLGPLLFLACHLFVINMNEIVRTKEFEGNMTWKIKLKSAMYFSNMTVTMVANLVPFTLTLLS

FMLLICSLCKHLKKMQLHGKGSQDPSTKVHIKALQTVISFLLLCAIYFLSIMISVWSFGSLENKP

VFMFCKAIRFSYPSIHPFILIWGNKKLKQTFLSVFWQMRYWVKGEKTSSP(SEQ ID NO：8)

hT2R63 full-length cDNA (BAC AC018630) (SEQ ID NO: 9)

ATGATGAGTTTTCTACACATTGTTTTTTCCATTCTAGTAGTGGTTGCATTTATTCTTGGAAA

TTTTGCCAATGGCTTTATAGCACTGATAAATTTCATTGCCTGGGTCAAGAGACAAAAGATC

TCCTCAGCTGATCAAATTATTGCTGCTCTGGCAGTCTCCAGAGTTGGTTTGCTCTGGGTAA

TATTATTACATTGGTATTCAACTGTGTTGAATCCAACTTCATCTAATTTAAAAGTAATAATT

TTTATTTCTAATGCCTGGGCAGTAACCAATCATTTCAGCATCTGGCTTGCTACTAGCCTCAG

CATATTTTATTTGCTCAAGATCGTCAATTTCTCCAGACTTATTTTTCATCACTTAAAAAGGA

AGGCTAAGAGTGTAGTTCTGGTGATAGTGTTGGGGTCTTTGTTCTTTTTGGTTTGTCACCTT

GTGATGAAACACACGTATATAAATGTGTGGACAGAAGAATGTGAAGGAAACGTAACTTGG

AAGATCAAACTGAGGAATGCAATGCACCTTTCCAACTTGACTGTAGCCATGCTAGCAAACT

TGATACCATTCACTCTGACCCTGATATCTTTTCTGCTGTTAATCTACTCTCTGTGTAAACAT

CTGAAGAAGATGCAGCTCCATGGCAAAGGATCTCAAGATCCCAGCACCAAGATCCACATA

AAAGCTCTGCAAACTGTGACCTCCTTCCTCATATTACTTGCCATTTACTTTCTGTGTCTAAT

CATATCGTTTTGGAATTTTAAGATGCGACCAAAAGAAATTGTCTTAATGCTTTGCCAAGCT

TTTGGAATCATATATCCATCATTCCACTCATTCATTCTGATTTGGGGGAACAAGACGCTAA

AGCAGACCTTTCTTTCAGTTTTGTGGCAGGTGACTTGCTGGGCAAAAGGACAGAACCAGTC

AACTCCATAG(SEQ ID NO：9)

hT2R63 conceptual translation (BAC AC018630) (SEQ ID NO: 10)

MMSFLHIVFSILVVVAFILGNFANGFIALINFIAWVKRQKISSADQIIAALAVSRVGLLWVILLH

WYSTVLNPTSSNLKVIIFISNAWAVTNHFSIWLATSLSIFYLLKIVNFSRLIFHHLKRKAKSVVLV

IVLGSLFFLVCHLVMKHTYINVWTEECEGNVTWKIKLRNAMHLSNLTVAMLANLIPFTLTLISF

LLLIYSLCKHLKKMQLHGKGSQDPSTKIHIKALQTVTSFLILLAIYFLCLIISFWNFKMRPKEIVL

MLCQAFGIIYPSFHSFILIWGNKTLKQTFLSVLWQVTCWAKGQNQSTP(SEQ ID NO：10)

hT2R64 full-length cDNA (BAC AC018630) (SEQ ID NO: 11)

ATGACAACTTTTATACCCATCATTTTTTCCAGTGTGGTAGTGGTTCTATTTGTTATTGGAAA

TTTTGCTAATGGCTTCATAGCATTGGTAAATTCCATTGAGCGGGTCAAGAGACAAAAGATC

TCTTTTGCTGACCAGATTCTCACTGCTCTGGCGGTCTCCAGAGTTGGTTTGCTCTGGGTATT

ATTATTAAATTGGTATTCAACTGTGTTTAATCCAGCTTTTTATAGTGTAGAAGTAAGAACT

ACTGCTTATAATGTCTGGGCAGTAACCGGCCATTTCAGCAACTGGCTTGCTACTAGCCTCA

GCATATTTTATTTGCTCAAGATTGCCAATTTCTCCAACCTTATTTTTCTTCACTTAAAGAGG

AGAGTTAAGAGTGTCATTCTGGTGATGCTGTTGGGGCCTTTACTATTTTTGGCTTGTCAAC

TTTTTGTGATAAACATGAAAGAGATTGTACGGACAAAAGAATATGAAGGAAACTTGACTT

GGAAGATCAAATTGAGGAGTGCAGTGTACCTTTCAGATGCGACTGTAACCACGCTAGGAA

ACTTAGTGCCCTTCACTCTGACCCTGCTATGTTTTTTGCTGTTAATCTGTTCTCTGTGTAAA

CATCTCAAGAAGATGCAGCTCCATGGTAAAGGATCTCAAGATCCCAGCACCAAGGTCCAC

ATAAAAGCTTTGCAAACTGTGATCTTTTTCCTCTTGTTATGTGCCGTTTACTTTCTGTCCAT

AATGATATCAGTTTGGAGTTTTGGGAGTCTGGAAAACAAACCTGTCTTCATGTTCTGCAAA

GCTATTAGATTCAGCTATCCTTCAATCCACCCATTCATCCTGATTTGGGGAAACAAGAAGC

TAAAGCAGACTTTTCTTTCAGTTTTGCGGCAAGTGAGGTACTGGGTGAAAGGAGAGAAGC

CTTCATCTCCATAG(SEQ ID NO：11)

hT2R64 conceptual translation (BAC AC018630) (SEQ ID NO: 12)

MTTFIPIIFSSVVVVLFVIGNFANGFLALVNSIERVKRQKISFADQILTALAVSRVGLLWVLLLNW

YSTVFNPAFYSVEVRTTAYNVWAVTGHFSNWLATSLSIFYLLKIANFSNLIFLHLKRRVKSVIL

VMLLGPLLFLACQLFVINMKEIVRTKEYEGNLTWKIKLRSAVYLSDATVTTLGNLVPFTLTLLC

FLLLICSLCKHLKKMQLHGKGSQDPSTKVHIKALQTVIFFLLLCAVYFLSIMISVWSFGSLENKP

VFMFCKAIRFSYPSIHPFILIWGNKKLKQTFLSVLRQVRYWVKGEKPSSP(SEQ ID NO：12)

hT2R65 full-length cDNA (BAC AC018630) (SEQ ID NO: 13)

ATGATGTGTTTTTCTGCTCATCATTTCATCAATTCTGGTAGTGTTTGCATTTGTTCTTGGAAA

TGTTGCCAATGGCTTCATAGCCCTAGTAAATGTCATTGACTGGGTTAACACACGAAAGATC

TCCTCAGCTGAGCAAATTCTCACTGCTCTGGTGGTCTCCAGAATTGGTTTACTCTGGGTCAT

GTTATTCCTTTGGTATGCAACTGTGTTTAATTCTGCTTTATATGGTTTAGAAGTAAGAATTG

TTGCTTCTAATGCCTGGGCTGTAACGAACCATTTCAGCATGTGGCTTGCTGCTAGCCTCAG

CATATTTTGTTTGCTCAAGATTGCCAATTTCTCCAACCTTATTTCTCTCCACCTAAAGAAGA

GAATTAAGAGTGTTGTTCTGGTGATACTGTTGGGGCCCTTGGTATTTCTGATTTGTAATCTT

GCTGTGATAACCATGGATGAGAGAGTGTGGACAAAAGAATATGAAGGAAATGTGACTTGG

AAGATCAAATTGAGGAATGCAATACACCTTTCAAGCTTGACTGTAACTACTCTAGCAAACC

TCATACCCTTTACTCTGAGCCTAATATGTTTTCTGCTGTTAATCTGTTCTCTTTGTAAACAT

CTCAAGAAGATGCGGCTCCATAGCAAAGGATCTCAAGATCCCAGCACCAAGGTCCATATA

AAAGCTTTGCAAACTGTGACCTCCTTCCTCATGTTATTTGCCATTTACTTTCTGTGTATAAT

CACATCAACTTGGAATCTTAGGACACAGCAGAGCAAACTTGTACTCCTGCTTTGCCAAACT

GTTGCAATCATGTATCCTTCATTCCACTCATTCATCCTGATTATGGGAAGTAGGAAGCTAA

AACAGACCTTTCTTTCAGTTTTGTGGCAGATGACACGCTGA(SEQ ID NO：13)

hT2R65 conceptual translation (BAC AC018630) (SEQ ID NO: 14)

MMCFLLIISSILVVFAFVLGNVANGFIALVNVIDWVNTRKISSAEQILTALVVSRIGLLWVMLFL

WYATVFNSALYGLEVRIVASNAWAVTNHFSMWLAASLSIFCLLKIANFSNLISLHLKKRIKSVV

LVILLGPLVFLICNLAVITMDERVWTKEYEGNVTWKIKLRNAIHLSSLTVTTLANLIPFTLSLICF

LLLICSLCKHLKKMRLHSKGSQDPSTKVHIKALQTVTSFLMLFAIYFLCIITSTWNLRTQQSKLV

LLLCQTVAIMYPSFHSFILIMGSRKLKQTFLSVLWQMTR(SEQ ID NO：14)

hT2R67 full-length cDNA (BAC AC018630) (SEQ ID NO: 15)

ATGATAACTTTTCTATACATTTTTTTTTCAATTCTAATAATGGTTTTATTTGTTCTCGGAAA

CTTTGCCAATGGCTTCATAGCACTGGTAAATTTCATTGACTGGGTGAAGAGAAAAAAGATC

TCCTCAGCTGACCAAATTCTCACTGCTCTGGCGGTCTCCAGAATTGGTTTGCTCTGGGCATT

ATTATTAAATTGGTATTTAACTGTGTTGAATCCAGCTTTTTATAGTGTAGAATTAAGAATT

ACTTCTTATAATGCCTGGGTTGTAACCAACCATTTCAGCATGTGGCTTGCTGCTAACCTCA

GCATATTTTATTTGCTCAAGATTGCCAATTTCTCCAACCTTCTTTTTCTTCATTTAAAGAGG

AGAGTTAGGAGTGTCATTCTGGTGATACTGTTGGGGACTTTGATATTTTTGGTTTGTCATC

TTCTTGTGGCAAACATGGATGAGAGTATGTGGGCAGAAGAATATGAAGGAAACATGACTG

GGAAGATGAAATTGAGGAATACAGTACATCTTTCATATTTGACTGTAACTACCCTATGGAG

CTTCATACCCTTTACTCTGTCCCTGATATCTTTTCTGATGCTAATCTGTTCTCTGTGTAAAC

ATCTCAAGAAGATGCAGCTCCATGGAGAAGGATCGCAAGATCTCAGCACCAAGGTCCACA

TAAAAGCTTTGCAAACTCTGATCTCCTTCCTCTTGTTATGTGCCATTTTCTTTCTATTCCTA

ATCGTTTCGGTTTGGAGTCCTAGGAGGCTGCGGAATGACCCGGTTGTCATGGTTAGCAAGG

CTGTTGGAAACATATATCTTGCATTCGACTCATTCATCCTAATTTGGAGAACCAAGAAGCT

AAAACACACCTTTCTTTTGATTTTGTGTCAGATTAGGTGCTGA(SEQ ID NO：15)

hT2R67 conceptual translation (BAC AC018630) (SEQ ID NO: 16)

MITFLYIFFSILIMVLFVLGNFANGFIALVNFIDWVKRKKISSADQILTALAVSRIGLLWALLLNW

YLTVLNPAFYSVELRITSYNAWVVTNHFSMWLAANLSIFYLLKIANFSNLLFLHLKRRVRSVIL

VILLGTLIFLVCHLLVANMDESMWAEEYEGNMTGKMKLRNTVHLSYLTVTTLWSFIPFTLSLIS

FLMLICSLCKHLKKMQLHGEGSQDLSTKVHIKALQTLISFLLLCAIFFLFLIVSVWSPRRLRNDP

VVMVSKAVGNIYLAFDSFILIWRTKKLKHTFLLILCQIRC(SEQ ID NO：16)

hT2R71 full-length cDNA (BAC AC073264) (SEQ ID NO: 17)

ATGCAAGCAGCACTGACGGCCTTCTTCGTGTTGCTCTTTAGCCTGCTGAGTCTTCTGGGGA

TTGCAGCGAATGGCTTCATTGTGCTGGTGCTGGGCAGGGAGTGGCTGCGATATGGCAGGT

TGCTGCCCTTGGATATGATCCTCATTAGCTTGGGTGCCTCCCGCTTCTGCCTGCAGTTGGTT

GGGACGGTGCACAACTTCTACTACTCTGCCCAGAAGGTCGAGTACTCTGGGGGTCTCGGCC

GACAGTTCTTCCATCTACACTGGCACTTCCTGAACTCAGCCACCTTCTGGTTTTGCAGCTGG

CTCAGTGTCCTGTTCTGTGTGAAGATTGCTAACATCACACACTCCACCTTCCTGTGGCTGA

AGTGGAGGTTCCCAGGGTGGGTGCCCTGGCTCCTGTTGGGCTCTGTCCTGATCTCCTTCAT

CATAACCCTGCTGTTTTTTTGGGTGAACTACCCTGTATATCAAGAATTTTTAATTAGAAAAT

TTTCTGGGAACATGACCTACAAGTGGAATACAAGGATAGAAACATACTATTTCCCATCCCT

GAAACTGGTCATCTGGTCAATTCCTTTTTCTGTTTTTCTGGTCTCAATTATGCTGTTAATTA

ATTCTCTGAGGAGGCATACTCAGAGAATGCAGCACAACGGGCACAGCCTGCAGGACCCCA

GCACCCAGGCTCACACCAGAGCTCTGAAGTCCCTCATCTCCTTCCTCATTCTTTATGCTCTG

TCCTTTCTGTCCCTGATCATTGATGCCGCAAAATTTATCTCCATGCAGAACGACTTTTACTG

GCCATGGCAAATTGCAGTCTACCTGTGCATATCTGTCCATCCCTTCATCCTCATCTTCAGCA

ACCTCAAGCTTCGAAGCGTGTTCTCGCAGCTCCTGTTGTTGGCAAGGGGCTTCTGGGTGGC

CTAG(SEQ ID NO：17)

hT2R71 conceptual translation (BAC AC073264) (SEQ ID NO: 18)

MQAALTAFFVLLFSLLSLLGIAANGFIVLVLGREWLRYGRLLPLDMILISLGASRFCLQLVGTVH

NFYYSAQKVEYSGGLGRQFFHLHWHFLNSATFWFCSWLSVLFCVKIANITHSTFLWLKWRFPG

WVPWLLLGSVLISFIITLLFFWVNYPVYQEFLIRKFSGNMTYKWNTRIETYYFPSLKLVIWSIPFS

VFLVSIMLLINSLRRHTQRMQHNGHSLQDPSTQAHTRALKSLISFLILYALSFLSLIIDAAKFISM

QNDFYWPWQIAVYLCISVHPFILIFSNLKLRSVFSQLLLLARGFWVA(SEQ ID NO：18)

hT2R75 full-length cDNA (SEQ ID NO: 19)

ATGATAACTTTTCTGCCCATCATTTTTTCCATTCTAATAGTGGTTACATTTGTGATTGGAAA

TTTTGCTAATGGCTTCATAGCATTGGTAAATTCCATTGAGTGGTTCAAGAGACAAAAGATC

TCTTTTGCTGACCAAATTCTCACTGCTCTGGCAGTCTCCAGAGTTGGTTTACTCTGGGTATT

AGTATTAAATTGGTATGCAACTGAGTTGAATCCAGCTTTTAACAGTATAGAAGTAAGAATT

ACTGCTTACAATGTCTGGGCAGTAATCAACCATTTCAGCAACTGGCTTGCTACTAGCCTCA

GCATATTTTATTTGCTCAAGATTGCCAATTTCTCCAACCTTATTTTTCTTCACTTAAAGAGG

AGAGTTAAGAGTGTTGTTCTGGTGATACTATTGGGGCCTTTGCTATTTTTGGTTTGTCATCT

TTTTGTGATAAACATGAATCAGATTATATGGACAAAAGAATATGAAGGAAACATGACTTG

GAAGATCAAACTGAGGAGTGCAATGTACCTTTCAAATACAACGGTAACCATCCTAGCAAA

CTTAGTTCCCTTCACTCTGACCCTGATATCTTTTCTGCTGTTAATCTGTTCTCTGTGTAAAC

ATCTCAAAAAGATGCAGCTCCATGGCAAAGGATCTCAAGATCCCAGCATGAAGGTCCACA

TAAAAGCTTTGCAAACTGTGACCTCCTTCCTCTTGTTATGTGCCATTTACTTTCTGTCCATA

ATCATGTCAGTTTGGAGTTTTGAGAGTCTGGAAAACAAACCTGTCTTCATGTTCTGCGAAG

CTATTGCATTCAGCTATCCTTCAACCCACCCATTCATCCTGATTTGGGGAAACAAGAAGCT

AAAGCAGACTTTTCTTTCAGTTTTGTGGCATGTGAGGTACTGGGTGAAAGGAGAGAAGCCT

TCATCTTCATAG(SEQ ID NO：19)

hT2R75 conceptual translation (SEQ ID NO: 20)

MITFLPIIFSILIVVTFVIGNFANGFIALVNSIEWFKRQKISFADQILTALAVSRVGLLWVLVLNW

YATELNPAFNSIEVRITAYNVWAVINHFSNWLATSLSIFYLLKIANFSNLIFLHLKRRVKSVVLVI

LLGPLLFLVCHLFVINMNQIIWTKEYEGNMTWKIKLRSAMYLSNTTVTILANLVPFTLTLISFLL

LICSLCKHLKKMQLHGKGSQDPSMKVHIKALQTVTSFLLLCAIYFLSIIMSVWSFESLENKPVF

MFCEAIAFSYPSTHPFILIWGNKKLKQTFLSVLWHVRYWVKGEKPSSS(SEQ ID NO：20)

hT2R59 pseudogroup (BAC AC018630) (SEQ ID NO: 21)

ATGGTATATTTTCTGCTCATCATTTTATCAATTCTGGTAGTGTTTGCATTTGTTCTTGGAAA

TTTTTCCAATGGCTTCATAGCTCTAGTAAATGTCATTGACTGGGTTAAGACACGAAAGATC

TCCTCAGCTGACCAAATCCTCACTGCTCTGGTGGTCTCCAGAATTGGTTTACTCTGGGTCAT

ATTATTACATTGGTATGCAAATGTGTTTAATTCAGCTTTATATAGTTCAGAAGTAGGAGCT

GTTGCTTCTAATATCTCAGCAATAATCAACCATTTCAGCATCTGGCTTGCTGCTAGCCTCAG

CATATTTTATTTGCTCAAGATTGCCAATTTCTCCAACCTTATTTTTCTCCACCTAAAGAAGA

GAATTAGGAGTGTTGTTCTGGTGATACTGTTGGGTCCCTTGGTATTTTTGATTTGTAATCTT

GCTGTGATAACCATGGATGACAGTGTGTGGACAAAAGAATATGAAGGAAATGTGACTTGG

AAGATCAAATTGAGGAATGCAATACACCTTTCAAACTTGACTGTAAGCACACTAGCAAACC

TCATACCCTTCATTCTGACCCTAATATGTTTTCTGCTGTTAATCTGTTCTCTGCATAAACAT

CTCAAGAAGATGCAGCTCCATGGCAAAGGATCTCAAGATCTCAGCACCAAGGTCCACATA

AAAGCTTTGCAAACTGTGATCTCCTTCCTCATGTTATATGCCATTTACTTTCTGTATCTAAT

CACATTAACCTGGAATCTTTGAACACAGCAGAACAAACTTGTATTCCTGCTTTGCCAAACT

CTTGGAATCATGTATCCTTCATTCCACTCATTCTTCCTGATTATGGGAAGCAGGAAACTAA

AACAGACGTTTCTTTCAGTTTTATGTCAGGTCACATGCTTAGTGAAAGGACAGCAACCCTC

AACTCCATAG(SEQ ID NO：21)

hT2R69 pseudogene (BAC AC018630) (SEQ ID NO: 22)

ATGATATGTTTTCTGCTCATCATTTTATCAATTCTGGTAGTGTTTGCATTTGTTCTTGGAAA

TGTTGCCAATGGCTTCATAGCTCTAGTAGGTGTCCTTGAGTGGGTTAAGACACAAAAGATC

TCATCAGCTGACCAAATTTCTCACTGCTCTGGTGGTGTCCAGAGTTGGTTTACTCTGGGTC

ATATTATTACATTGGTATGCAACTGTGTTTAATTTGGCTTCACATAGATTAGAAGTAAGAA

TTTTTGGTTCTAATGTCTCAGCAATAACCAAGCATTTCAGCATCTGGGTGTTACTAGCCTCA

GCATATTTCATTTGCTCAAGACTGCCAATTTCTCCAACCTTATTTTTCTCCACCTAAAGAAA

AGGATTAAGAATGTTGGTTTGGTGATGCTGTTGGGGCCCTTGGTATTTTTCATTTGTAATC

TTGCTCTGATAACCACGGGTGAGAGTGTGTGGACAAAAGAATATGAAGGAAATTTGTCTT

GGATGATCAAATTGAGGAATGCAATACAGCTTTCAAACTTGACTGTAACCATGCCAGCAA

ACGTCACACCCTGCACTCTGACACTAATATCTTTTCTGCTGTTAATCTATTCTCCATGTAAA

CATGTCAAGAAGATGCAGCTCCATGGCAAAGGATCTCAACATCTCAGCACCAAGGTGCAC

ATAAAAGCTTTGCAAACTGTGATCTCCTTCCTTATGTTATTTGCCATTTACTTTCTGTGTCT

AATCACATCAACTTGGAATCCTAGGACTCAGCAGAGCAAACTTGTATTCCTGCTTTACCAA

ACTCTTGGATTCATGTATCTTTTGTTCCACTCATTCATCCTGACTATGGGAAGTAGGAAGCC

AAAACAGACCTTTCTTTCAGCTTTGTGA(SEQ ID NO：22)

mT2R33 full-length cDNA (BAC AC020619) (SEQ ID NO: 23)

ATGACCTCCCCTTTCCCAGCTATTTATCACATGGTCATCATGACAGCAGAGTTTCTCATCGG

GACTACAGTGAATGGATTCCTTATCATTGTGAACTGCTATGACTTGTTCAAGAGCCGAACG

TTCCTGATCCTGCAGACCCTCTTGATGTGCACAGGGCTGTCCAGACTCGGTCTGCAGATAA

TGCTCATGACCCAAAGCTTCTTCTCTGTGTTCTTTCCATACTCTTATGAGGAAAATATTTAT

AGTTCAGATATAATGTTCGTCTGGATGTTCTTCAGCTCGATTGGCCTCTGGTTTGCCACATG

TCTCTCTGTCTTTTACTGCCTCAAGATTTCAGGCTTCACTCCACCCTGGTTTCTTTGGCTGA

AATTCAGAATTTCAAAGCTCATATTTTGGCTGCTTCTGGGCAGCTTGCTGGCCTCTCTGGG

CACTGCAACTGTGTGCATCGAGGTAGGTTTCCCTTTAATTGAGGATGGCTATGTCCTGAGA

AACGCAGGACTAAATGATAGTAATGCCAAGCTAGTGAGAAATAATGACTTGCTCCTCATC

AACCTGATCCTCCTGCTTCCCCTGTCTGTGTTTGTGATGTGCACCTCTATGTTATTTGTTTC

TCTTTACAAGCACATGCACTGGATGCAAAGCGAATCTCACAAGCTGTCAAGTGCCAGAACC

GAAGCTCATATAAATGCATTAAAGACAGTGACAACATTCTTTTGTTTCTTTGTTTCTTACTT

TGCTGCCTTCATGGCAAATATGACATTTAGAATTCCATACAGAAGTCATCAGTTCTTCGTG

GTGAAGGAAATCATGGCAGCATATCCCGCCGGCCACTCTGTCATAATCGTCTTGAGTAACT

CTAAGTTCAAAGACTTATTCAGGAGAATGATCTGTCTACAGAAGGAAGAGTGA(SEQ IDNO：23)

mT2R33 conceptual translation (BAC AC020619) (SEQ ID NO: 24)

MTSPFPAIYHMVIMTAEFLIGTTVNGFLIIVNCYDLFKSRTFLILQTLLMCTGLSRLGLQIMLMT

QSFFSVFFPYSYEENIYSSDIMFVWMFFSSIGLWFATCLSVFYCLKISGFTPPWFLWLKFRISKLIF

WLLLGSLLASLGTATVCIEVGFPLIEDGYVLRNAGLNDSNAKLVRNNDLLLINLILLLPLSVFVM

CTSMLFVSLYKHMHWMQSESHKLSSARTEAHINALKTVTTFFCFFVSYFAAFMANMTFRIPYR

SHQFFVVKEIMAAYPAGHSVIIVLSNSKFKDLFRRMICLQKEE(SEQ ID NO：24)

While the foregoing detailed description has described several embodiments of the present invention, it should be understood that the above description is illustrative of the invention and is not to be taken in a limiting sense. The invention is limited only by the following claims.

Sequence listing

<110>SENOMYX，INC.

<120> T2R taste receptor and genes encoding same

<130>078003-0128589

<140>PCT/US01/10739

<141>2001-04-04

<150>60/195,532

<151>2000-04-07

<150>60/247,014

<151>2000-11-13

<160>31

<170>PatentIn Ver.2.1

<210>1

<211>1002

<212>DNA

<213> human (Homo sapiens)

<400>1

<210>2

<211>333

<212>PRT

<213> human

<400>2

<210>3

<211>966

<212>DNA

<213> human

<400>3

<210>4

<211>321

<212>PRT

<213> human

<400>4

<210>5

<211>972

<212>DNA

<213> human

<400>5

<210>6

<211>323

<212>PRT

<213> human

<400>6

<210>7

<211>930

<212>DNA

<213> human

<400>7

<210>8

<211>309

<212>PRT

<213> human

<400>8

<210>9

<211>930

<212>DNA

<213> human

<400>9

<210>10

<211>309

<212>PRT

<213> human

<400>10

<210>11

<211>930

<212>DNA

<213> human

<400>11

<210>12

<211>309

<212>PRT

<213> human

<400>12

<210>13

<211>900

<212>DNA

<213> human

<400>13

<210>14

<211>299

<212>PRT

<213> human

<400>14

<210>15

<211>900

<212>DNA

<213> human

<400>15

<210>16

<211>299

<212>PRT

<213> human

<400>16

<210>17

<211>924

<212>DNA

<213> human

<400>17

<210>18

<211>307

<212>PRT

<213> human

<400>18

<210>19

<211>930

<212>DNA

<213> human

<400>19

<210>20

<211>309

<212>PRT

<213> human

<400>20

<210>21

<211>930

<212>DNA

<213> human

<400>21

<210>22

<211>885

<212>DNA

<213> human

<400>22

<210>23

<211>912

<212>DNA

<213>Mus sp.

<400>23

<210>24

<211>303

<212>PRT

<213>Mus sp.

<400>24

<210>25

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: T2R family consensus sequence 1

<220>

<221>MOD_RES

<222>(2)

<223> Phe or Ala

<220>

<221>MOD_RES

<222>(3)

<223> Ile, Val or Leu

<220>

<221>MOD_RES

<222>(4)

<223> Val or Leu

<220>

<221>MOD_RES

<222>(6)

<223> Ile or Val

<220>

<221>MOD_RES

<222>(7)

<223> Leu or Val

<220>

<221>MOD_RES

<222>(10)

<223> Gly or Thr

<220>

<221>MOD_RES

<222>(13)

<223> Val or Ala

<220>

<221>MOD_RES

<222>(18)

<223> Ile or Met

<400>25

<210>26

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: T2R family consensus sequence 2

<220>

<221>MOD_RES

<222>(1)

<223> Asp or Gly

<220>

<221>MOD_RES

<222>(2)

<223> Phe or Leu

<220>

<221>MOD_RES

<222>(3)

<223> Ile or Leu

<220>

<221>MOD_RES

<222>(5)

<223> Thr or Ile

<220>

<221>MOD_RES

<222>(6)

<223> Gly, Ala or Ser

<220>

<221>MOD_RES

<222>(13)

<223> Cys, Gly or Phe

<400>26

<210>27

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: T2R family consensus sequence 3

<220>

<221>MOD_RES

<222>(3)

<223> Leu or Phe

<220>

<221>MOD_RES

<222>(4)

<223> Ser, Thr or Asn

<220>

<221>MOD_RES

<222>(5)

<223> Leu, Ile or Val

<220>

<221>MOD_RES

<222>(7)

<223> Phe or Leu

<220>

<221>MOD_RES

<222>(8)

<223> Ala or Thr

<220>

<221>MOD_RES

<222>(10)

<223> Cys, Ser or Asn

<220>

<221>MOD_RES

<222>(12)

<223> Ser, Asn or Gly

<220>

<221>MOD_RES

<222>(13)

<223> Ile or Val

<400>27

<210>28

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: T2R family consensus sequence 4

<220>

<221>MOD_RES

<222>(3)

<223> Phe or Cys

<220>

<221>MOD_RES

<222>(8)

<223> Asn or Ser

<220>

<221>MOD_RES

<222>(11)

<223> His or Asn

<220>

<221>MOD_RES

<222>(12)

<223> Pro or Ser

<220>

<221>MOD_RES

<222>(13)

<223> Leu, Ile or Val

<220>

<221>MOD_RES

<222>(16)

<223> Trp or Tyr

<400>28

<210>29

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: T2R family consensus sequence 5

<220>

<221>MOD_RES

<222>(4)

<223> Ile, Phe or Val

<220>

<221>MOD_RES

<222>(8)

<223> Lys or Arg

<220>

<221>MOD_RES

<222>(10)

<223> Ser or Thr

<220>

<221>MOD_RES

<222>(11)

<223> Lys or Arg

<220>

<221>MOD_RES

<222>(12)

<223> Gln or Lys

<220>

<221>MOD_RES

<222>(13)

<223> Met or Ile

<220>

<221>MOD_RES

<222>(14)

<223> Gln or Lys

<400>29

<210>30

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: T2R family consensus sequence 6

<220>

<221>MOD_RES

<222>(3)

<223> Phe or Leu

<220>

<221>MOD_RES

<222>(4)

<223> Ile or Val

<220>

<221>MOD_RES

<222>(7)

<223> Leu or Met

<220>

<221>MOD_RES

<222>(8)

<223> Gly, Ser or Thr

<220>

<221>MOD_RES

<222>(10)

<223> Pro, Ser or Asn

<220>

<221>MOD_RES

<222>(13)

<223> Lys or Arg

<220>

<221>MOD_RES

<222>(14)

<223> Gln or Arg

<400>30

<210>31

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: exemplary translocation Domain

<400>31

Claims (46)

1. An isolated nucleic acid sequence encoding a bitter taste receptor polypeptide, wherein said polypeptide hybridizes to a sequence selected from the group consisting of SEQ ID NOS: 2, 4,6, 18, and 24 have at least 80% sequence identity.

2. An isolated nucleic acid sequence encoding a bitter taste receptor polypeptide, wherein said polypeptide hybridizes to a sequence selected from the group consisting of SEQ ID NOS: 2, 4,6, 18, and 24 have at least 85% sequence identity.

3. An isolated nucleic acid sequence encoding a bitter taste receptor polypeptide, wherein said polypeptide hybridizes to a sequence selected from the group consisting of SEQ ID NOS: 2, 4,6, 18, and 24 have at least 90% sequence identity.

4. An isolated nucleic acid sequence encoding a bitter taste receptor polypeptide, wherein said polypeptide hybridizes to a sequence selected from the group consisting of SEQ ID NOS: 2, 4,6, 18, and 24 have at least 95% sequence identity.

5. An isolated nucleic acid sequence encoding a bitter taste receptor polypeptide, wherein said polypeptide hybridizes to a sequence selected from the group consisting of SEQ ID NOS: 2, 4,6, 18 and 24 have at least 97-99% sequence identity.

6. An isolated nucleic acid sequence encoding a bitter taste receptor polypeptide, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 2, 4,6, 18 and 24.

7. An isolated nucleic acid sequence encoding a bitter taste receptor polypeptide capable of hybridizing under stringent hybridization conditions to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 17 and 23, respectively.

8. An isolated nucleic acid sequence encoding a bitter taste receptor polypeptide selected from the group consisting of SEQ ID NOS: 1, 3, 5, 17 and 23.

9. The isolated nucleic acid sequence of claim 7, wherein the polypeptide is at least 250 amino acids in length.

10. An isolated nucleic acid sequence encoding a bitter taste receptor polypeptide, wherein said polypeptide is a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4,6, 18 and 24.

11. An isolated taste receptor polypeptide having an amino acid sequence encoded by an isolated nucleic acid sequence according to any one of claims 1-10.

12. The isolated taste receptor polypeptide of claim 11 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4,6, 18 and 24.

13. A cell transfected or transformed with a nucleic acid sequence comprising at least one isolated nucleic acid sequence of any of claims 1-10.

14. The cell of claim 13, wherein the isolated nucleic acid sequence encodes a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 2, 4,6, 18 and 24.

15. The cell of claim 13, wherein the isolated nucleic acid sequence is selected from the group consisting of SEQ id nos: 1, 3, 5, 17 and 23.

16. The cell of any one of claims 12-15, which is selected from the group consisting of a bacterial cell, a yeast cell, an insect cell, a mammalian cell and an amphibian cell.

17. The cell of claim 16, which is a human cell.

18. The cell of claim 16, which is selected from the group consisting of taste cells, chinese hamster ovary cells, baby hamster kidney cells, myeloma cells, and human embryonic kidney cells.

19. An expression vector comprising at least one isolated nucleic acid sequence of any one of claims 1-10.

20. The expression vector of claim 19 selected from the group consisting of mammalian, yeast, bacterial and insect expression vectors.

21. The expression vector of any one of claims 19-20, wherein the isolated nucleic acid sequence is operably linked to a constitutive or regulated promoter.

22. A solid phase comprising at least one isolated nucleic acid sequence of any one of claims 1-10.

23. A solid phase comprising an array of isolated nucleic acid sequences encoding a taste receptor, said array comprising at least one isolated nucleic acid sequence according to any one of claims 1-10.

24. A fusion polypeptide comprising a polypeptide sequence according to claim 11 or 12.

25. A solid phase comprising a cell or membrane extract obtained from a cell of any one of claims 13-18.

26. A solid phase having immobilized thereon at least one polypeptide of claim 11, 12 or 24.

27. A method of identifying a taste receptor modulating compound comprising contacting the bitter taste receptor polypeptide of claim 11, 12 or 24 with a putative taste modulating compound and detecting whether the compound specifically binds to the taste receptor polypeptide or inhibits or activates specific binding of another compound to the taste receptor polypeptide.

28. The method of claim 27 wherein said taste receptor polypeptide is expressed by a cell.

29. The method of claim 27 wherein said taste receptor polypeptide is contained in a membrane extract obtained from a cell expressing said taste receptor polypeptide.

30. A method according to claim 27, wherein the further compound is a bitter tasting compound

31. The method of claim 30, wherein the bitter tasting compound is selected from the group consisting of 6-n-propylthiouracil, sucrose octaacetate, melitriose undecanoate, cycloheximide, denatonium, copper glycinate, and quinine.

32. The method of claim 27 wherein said putative taste receptor compound is comprised in a library of compounds.

33. The method of claim 32, wherein the library is a peptide library, a coded peptide library, a combinatorial chemical library, a library of small molecule organic compounds, a benzodiazepineLibraries, non-peptide peptidomimetic libraries.

34. The method of claims 27-33, comprising high throughput screening.

35. A cell-based method for identifying a taste modulating compound comprising:

(i) contacting a cell expressing at least one taste receptor polypeptide encoded by an isolated nucleic acid sequence of any of claims 1-10 with at least one putative taste modulating compound;

(ii) detecting whether said putative regulatory compound modulates the activity of said taste receptor polypeptide; and

(iii) if said compound modulates the activity of said taste receptor polypeptide, it is identified as a taste modulating compound.

36. The method of claim 35, which detects the effect of said compound on intracellular ion concentration.

37. The method of claim 36, wherein the ion is calcium.

38. The method of claim 35, which detects the effect of the composition on intracellular cyclic nucleotides.

39. The method of claim 35, which detects the effect of said compound on GTP to GDP conversion.

40. The method of claim 35 which detects the effect of said compound on the transfer of radioactive P from gamma-labeled GTP to taste receptor polypeptides.

41. The method of claim 38, which detects the level of cAMP or cGMP.

42. The method of claim 35, wherein the effect of said compound on the level of inositol triphosphate is examined.

43. The method of claim 35 wherein said cell expresses a G protein coupled to said taste receptor polypeptide.

44. The method of claim 43, wherein said G protein is G α 15 or G α 16.

45. The method of claim 43 which detects the effect of said compound on the coupling of said G protein to said taste receptor polypeptide.

46. The method of claim 35 which detects the effect of said compound on the transcription of a nucleic acid sequence encoding said taste receptor polypeptide.