Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/142—Amino acids; Derivatives thereof
  • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K40/00—Shaping or working-up of animal feeding-stuffs
  • A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/86—Addition of bitterness inhibitors
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/18—Peptides; Protein hydrolysates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
  • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

• the taste system provides sensory information about the chemical composition of the external world. Mammals are believed to have at least five basic taste modalities: sweet, bitter, sour, salty, and umami. Each taste modality is thought to be mediated by a distinct protein receptor or receptors that are expressed in taste receptor cells found on the surface of the tongue.
• the taste receptors that recognize bitter, sweet, and umami taste stimuli belong to the G-protein-coupled receptor (GPCR) superfamily. Subtle differences in a receptor may alter which ligands bind and what signal is generated once the receptor is stimulated.
• GPCR G-protein-coupled receptor
• GPCR superfamily mediate many other physiological functions, such as endocrine function, exocrine function, heart rate, lipolysis, and
• bitter taste modality is usually described as disagreeable.
• Many natural and synthetic toxins have been characterized as bitter tastants.
• bitter taste perception has evolved as a means to discourage the consumption of toxic compounds often found in plants.
• Estimates for the number of bitter tasting compounds are in the tens of thousands.
• Compounds that block bitter taste perception have also been identified, for example p-(dipropylsulfamoyl)benzoic acid (probenecid) which acts on a subset of Taste Receptor, Type 2 (“TAS2R”) proteins, a family of monomeric G protein- coupled receptors, embedded in the surface of taste cells.
• TAS2R Type 2
• hTAS2Rs human TAS2Rs
• hTAS2Rs human TAS2Rs
• hTAS2Rs are currently believed to be promiscuous, i.e., activated by multiple ligands belonging to several chemical classes, while other hTAS2Rs bind ligands of only particular chemical classes.
• hTAS2Rs are orphan receptors, with no compounds identified as yet that stimulate them.
• hTAS2R38 is the most extensively studied bitter taste receptor.
• PTC phenylthiocarbamide
• SNPs polymorphisms
• TAS2R repertoire Much different from that of humans.
• the mouse has 34 full-length TAS2Rs encoded in its genome, while the chicken has only 3 (Go et ah, Genetics. 2005 May; 170(l):313-26).
• TAS2Rs Although some compounds can be detected by multiple TAS2Rs, it is almost certain that differences in TAS2R repertoire across species result in differences in bitter taste perception.
• GPCRs G protein-coupled receptors
• TAS2R G protein-coupled receptors
• the TAS2R genes encode a family of related seven transmembrane G-protein coupled receptors involved in taste transduction, which interact with a G-protein to mediate taste signal transduction.
• TAS2Rs interact in a ligand- specific manner with the G protein Gustducin.
• hTAS2Rs human TAS2Rs
• the human genome encodes about 26 functional TAS2Rs that are glycoproteins. All hTAS2Rs share a conserved site for Asn-linked glycosylation within the center of the second extracellular loop.
• the hTAS2Rs also have the ability to form homo- and hetero-oligomers with other GPCR when expressed in vitro, however at present no evidence exists that TAS2R receptor oligomerization has functional implications.
• bitter taste receptor cells represent a distinct subpopulation of chemosensory cells characterized by the expression of TAS2R genes and completely segregated from those receptor cells devoted to the detection of other taste stimuli. Each bitter taste receptor cell expresses multiple bitter taste receptors, although the extent of co-expression is still a matter of debate.
• TAS2Rs are found in non- gustatory tissues. Among these extra-oral sites are the respiratory epithelia, gastrointestinal tissues, reproductive organs, and brain. Bitter taste receptors are implicated in differentiation or maturation of sperm in mice. The non-gustatory expression of TAS2Rs is known to be used to regulate digestion and respiration.
• Activation of TAS2R receptors in an enteroendocrine cell line results in release of the peptide hormone cholecystokinin (CCK), which can reduce gut motility. Consequently, intake of a potential toxin that activates the TAS2R pathway may decrease the rate at which food passes through the stomach and lower the drive for continued eating.
• CCK cholecystokinin
• the release of CCK also excites sensory nerve processes of the vagus nerve to carry the signal to the brain, suggesting that regulation of food intake involves both peripheral and central controls.
• Activation of the TAS2R signaling network may also or alternatively indirectly increase elimination of absorbed toxins from gut epithelium before the toxins can enter circulation since some data suggest that the CCK-secreting enteroendocrine cells are involved in a paracrine signaling system that reduces transfer of toxic substances from the gut into the circulation. Lower in the gut, activation of TAS2R receptors has a different effect. When some bitter-tasting ligands are applied to the colonic epithelium, they induce the secretion of anions, which leads to fluid secretion by the epithelium which may flush out any noxious irritant from the colon.
• SCCs Solitary chemosensory cells
• TAS2R receptors PLCp2, gustducin
• TrpM5 the transduction channel TrpM5.
• the SCCs synapse onto polymodal pain fibers of the trigeminal nerve. Inhalation of a toxin that activates TAS2R receptors of the SCCs will be irritating and evoke trigeminally-mediated reflex changes in respiration. Additionally, the activated trigeminal nerve fibers release peptide modulators that result in local neurogenic inflammation of the respiratory epithelium, activating the immune system in response to the presence of the toxins.
• the human bitter taste receptors, hTAS2R2, hTAS2R41, hTAS2R42, hTAS2R45, hTAS2R48, and hTAS2R60 are still considered orphan GPCRs since ligands have not yet been identified for these receptors.
• hTAS2R2 was annotated as a pseudogene due to a two base deletion at codon 160 found in sequences collected from 10 human populations (Karitiana, Surui, Waorani Indians from South America, Russians from Eastern Europe, Druze from the Middle East, Atayal, Chinese, Japanese from Eastern Asia, and Khmers and Melanesians from Southeast Asia) and from GenBank resources.
• hTAS2R2 has been found to be polymorphic with respect to that deletion, with the intact gene found in the Adygei (Eastern European), Mbuti (African Pygmies), and Biaka (African Pygmies) (Go Y et ah, Genetics May 1, 2005, 170 (1): 313-326).
• feline genome has been sequenced with minimal coverage (Mullikin et al. BMC Genomics 2010 11: 406; Pontius et al, Genome Research 2007 17: 1675-1689).
• major gaps exist in the feline genome sequence and only slightly over 2000 feline genes have been annotated to date.
• the human genome has about 25,000 genes annotated.
• the sequences prior to a gap in the genomic assembly are of poor quality, so in addition to information that is missing, a large portion of the data present is of poor quality. Consequently, there is much to be discovered within feline genomics and in determining the molecular basis of feline taste perception.
• fTAS2R feline TAS2R
• an isolated feline TAS2R (fTAS2R) receptor polypeptide comprises an extracellular domain of a feline TAS2R receptor; a transmembrane region of a feline TAS2R receptor, or an intracellular domain of a feline TAS2R receptor, wherein the fTAS2R receptor comprises a sequence selected from SEQ ID NO: 18, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26, wherein the isolated fTAS2R receptor polypeptide does not consist of the amino acid sequence of SEQ ID NOs: 2, 4, 6, or 10.
• the isolated fTAS2R receptor polypeptide comprises an extracellular domain of a feline TAS2R receptor polypeptide comprising amino acids 1, 68- 84; 146-179; or 249-257 of SEQ ID NO:2; amino acids 1-10, 73-88; 151-186; or 256-264 of SEQ ID NO:4; amino acids 1-8; 72-88; 150-186; or 256-265 of SEQ ID NO:6; amino acids 1, 68- 84; 146-179; or 249-257 of SEQ ID NO:2; amino acids 1-10, 73-88; 151-186; or 256-264 of SEQ ID NO:4; amino acids 1-8; 72-88; 150-186; or 256-265 of SEQ ID NO:6; amino acids
• a polynucleotide encoding the novel feline TAS2R receptor, fragment thereof, is also disclosed.
• the polynucleotide comprises a nucleotide sequence selected from: the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26; a nucleotide sequence that hybridizes to the complement of the polynucleotide having SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
• Expression vectors and host cells comprising the polynucleotides, as well as oligonucleotides are also disclosed.
• Antibodies and kits for detecting the fTAS2R receptor are also disclosed.
• the method comprises contacting a TAS2R receptor polypeptide herein with a test compound, and detecting interaction between the receptor polypeptide and the test compound.
• the method comprises contacting a TAS2R receptor polypeptide disclosed herein with a receptor ligand in the presence or absence of a test compound, and determining whether the test compound modulates binding of the ligand to the receptor or activation of the receptor by the ligand.
• a method of preparing edible compositions comprises contacting an edible composition or a component thereof with a feline TAS2R receptor polypeptide for a time sufficient to reduce the amount of a bitter compound from the edible composition or component thereof.
• a method of preparing edible compositions for controlling palatability to an animal comprises adding a compound to an edible composition to decrease the palatability of the edible composition to an animal, wherein the compound is an agonist of or a positive modulator of a feline TAS2R receptor polypeptide.
• a method of formulating an edible composition with enhanced palatability comprises determining the presence of a compound which is an agonist, antagonist, or modulator of a feline TAS2R receptor polypeptide in an edible composition; and enhancing palatability of the edible composition by: if the compound is an agonist or a positive modulator, increasing the amount of an antagonist for the receptor in the edible composition or reducing the amount of the compound in the edible composition, or if the compound is an antagonist or a negative modulator, increasing the amount of the compound in the edible composition.
• a method of administering a bitter compound to an animal in need thereof comprises administering an edible composition to an animal, wherein the edible composition comprises a bitter compound and a compound that is an antagonist, or modulator of a feline TAS2R receptor polypeptide that alters acceptance of the edible composition by the animal compared to acceptance of the edible composition without the compound.
• the bitter compound can comprise a pharmaceutical, an oral care material, a nutritional supplement, or a repellant.
• flavor compositions for coating or incorporating into an edible composition to be administered to an animal and methods of manufacture thereof.
• the flavor composition comprises an agonist or an antagonist of a feline TAS2R receptor polypeptide, wherein the agonist is denatonium, aloin, or PTC and the antagonist is probenecid; optionally, a palatability enhancer; optionally, a compound to help adhere the flavor composition to the edible composition; and optionally, a compound for providing color or aroma; wherein the flavor composition is a liquid, solid, powder, paste, gel, spreadable formulation, granule, or sprayable formulation.
• the method of making the flavor composition comprises mixing an agonist or an antagonist of a feline TAS2R receptor polypeptide, wherein the agonist is denatonium, aloin, or PTC and the antagonist is probenecid; optionally, a palatability enhancer; optionally, a compound to help adhere the flavor composition to the edible composition; and optionally, a compound for providing color or aroma with an ingredient selected from the group consisting of meat products, meat by-products, fish products, fish by-products, dairy products, dairy by-products, sources of microbial proteins, vegetable proteins, carbohydrates and amino acids to obtain a flavor composition, wherein the flavor composition is a liquid, solid, powder, paste, gel, spreadable formulation, granule, or sprayable formulation.
• Fig. 1 is a sequence alignment displaying the 3rd through the 7th transmembrane (TM) region (transmembrane regions in grey) of several human and feline bitter receptors: human TAS2R16 (SEQ ID NO:30), TAS2R4 (SEQ ID NO:27), TAS2R9 (SEQ ID NO:28), TAS2R10 (SEQ ID NO:29) AND TAS2R38 (SEQ ID NO:31); and feline bitter receptors, TAS2R4 (SEQ ID NO:8), 9 (SEQ ID NO: 12), 10 (SEQ ID NO: 14), 12 (SEQ ID NO: 16), and 38 (SEQ ID NO: 18).
• TM transmembrane
• Fig. 2 shows a sequence alignment for human TAS2R38 polypeptide (SEQ ID NO:31) and feline TAS2R38 polypeptide (SEQ ID NO:18) determined from sequencing of genomic DNA of five individual cats.
• a family of novel feline bitter taste receptors feline TAS2R (fTAS2R), are disclosed herein.
• GPCRs G-protein coupled receptors
• These G-protein coupled receptors are components of the feline taste transduction pathway, specifically, part of the bitter taste transduction pathway, and are involved in feline taste detection of bitter substances such as 6-n-propylthiouracil, sucrose octaacetate, raffinose undecaacetate, cycloheximide, denatonium, copper glycinate, and quinine.
• Polynucleotides encoding the novel feline bitter taste receptors are also disclosed, as are expression vectors and host cells for expression of the novel feline bitter taste receptors. Methods of expressing and isolating the nucleic acids and encoded polypeptides are also disclosed.
• the nucleic acids provide probes for identification of cells in which the nucleic acids are expressed, e.g., taste cells.
• probes for expression of TAS2R polypeptides can be used to identity taste cells present in foliate, circumvallate, and fungiform papillae.
• the TAS2R probes are useful to identify bitter sensing cells and can serve as tools for the generation of anatomical maps that elucidate the relationship between the bitter sensing cells and their projections into the central nervous system.
• fTAS2R members of the fTAS2R family act as direct or indirect reporter molecules to identify modulators of taste receptor expressing cellular activity. Such compounds are useful for modulation of feline bitter taste receptor activity. Modulating the activity of feline bitter receptors receptor can be achieved by agonists, antagonists, inhibitors, and/or enhancers. These modulatory compounds can be used in the food and pharmaceutical industries to customize taste of foods or drugs, for example, to decrease the bitter taste of foods or drugs. Thus, the methods disclosed herein are useful for designing or formulating food, food palatants, treats, and medications in which aversive compounds are avoided or blocked, particularly for felines.
• An "agonist”, or “receptor agonist” as used herein, refers to a molecule that has an affinity for and stimulates functional activity of a cell receptor.
• the level of stimulation of the functional activity at the receptor can be, e.g., at least 5%, at least 10%, at least 30%, at least 50%, at least 80%, at least 100%, at least 200%, at least 300%, at least 500%, at least 1,000%, at least 10,000% over baseline.
• amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
• Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
• Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
• Amino acid mimetics means chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three- letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
• a receptor "antagonist” as used herein refers to a type of receptor ligand that binds to the receptor at the same site as an agonist, but does not activate the functional response initiated by the active form of the receptor. Once bound, an antagonist will block agonist binding thereby inhibiting the functional response produced by agonist binding. Since agonists and antagonists "compete" for the same binding site on the receptor, the level of activity of the receptor will be determined by the relative affinity of each molecule for the site and their relative concentrations.
• the inhibition of the functional response elicited by an agonist by an antagonist applied prior, concomitantly or after the application of the agonist can be e.g., at least by 10%, at least 15%; at least 20%; at least 30%; at least 40%; at least 50%; at least 60%; at least 70%; at least 80%; at least 90%; at least 95%; at least 98%; at least 99%; at least 99.5%; or at least 100%.
• the antagonist and agonist are applied at the same molar concentration.
• Antibody refers to a polypeptide that specifically binds and recognizes an antigen.
• the term “monoclonal antibody” means an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In some embodiments, the term “monoclonal antibody” refers to an antibody derived from a single cell clone.
• An “antibody” comprises at least one heavy (H) chain and one light (L) chain.
• these heavy and light chains are inter-connected by disulfide bonds and there are two paired heavy and light chains; these two are also interconnected by disulfide bonds.
• Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
• the heavy chain constant region is comprised of three domains, CHI, CH2, and CH3.
• Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
• the light chain constant region is comprised of one domain, CL.
• the VH and VL regions can be further subdivided into regions of hypervariability, termed
• CDR complementarity determining regions
• FR framework regions
• J Joining regions
• Each VH and VL is composed of three CDRs three FRs and a J domain, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, J.
• the variable regions of the heavy and light chains bind with an antigen.
• the constant regions of the antibodies may mediate the binding of the
• immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) or humoral factors such as the first component (Clq) of the classical complement system.
• various cells of the immune system e.g., effector cells
• humoral factors such as the first component (Clq) of the classical complement system.
• antigen-binding portion or "antigen-binding fragment” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of an antibody.
• binding fragments denoted as an antigen-binding portion or fragment of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al.
• the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions are paired to form monovalent molecules (such a single chain cognate of an immunoglobulin fragment is known as a single chain Fv (scFv).
• scFv single chain Fv
• single chain antibodies are also encompassed within the term "antibody fragment.”
• Antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same general manner as are intact antibodies.
• Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins .
• an "anti-TAS2R” or a “TAS2R” antibody is an antibody or antibody fragment that specifically binds a polypeptide encoded by a TAS2R gene, cDNA, or a subsequence thereof.
• the term "chimeric polypeptide” refers to a molecule, which does not occur in nature, in which all or a portion of an fTAS2R polypeptide sequence is part of the linear chimeric polypeptide sequence.
• the portion of an fTAS2R polypeptide sequence can be the amino acid sequence of one or more domains of the complete fTAS2R polypeptide.
• the portion can be an extracellular domain of a fTAS2R polypeptide.
• the chimeric polypeptide can be made by any method known in the art.
• the chimeric polypeptide can be made by a recombinant expression system or can be synthesized.
• Codon optimization describes a method applied to nucleotide sequences encoding a polypeptide to modify the nucleotide sequence for enhanced expression of the polypeptide in the cells of a non-feline organism of interest, e.g. Drosophila melanogaster or Saccharomyces cerevisae, by replacing at least one, more than one, or all, codons of the native feline sequence with codons that are more frequently or most frequently used in the genes of the expression organism without changing the amino acids of the expressed polypeptide.
• Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
• mRNA messenger RNA
• tRNA transfer RNA
• Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the
• nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
• each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
• TGG which is ordinarily the only codon for tryptophan
• C terminal domain refers to the region that spans the end of the last
• transmembrane domain and the C-terminus of the protein, and which is normally located within the cytoplasm.
• Cytoplasmic domains or “intracellular domains” refers to the domains of TAS2R proteins that face the inside 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, the intracellular loops between transmembrane regions 3 and 4, and the intracellular loops between transmembrane regions 5 and 6.
• extracellular domains refers to the domains of TAS2R polypeptides that protrude from the cellular membrane and are exposed to the extracellular face of the cell. Such domains include the "N terminal domain” that is exposed to the extracellular face of the cell, as well as the extracellular loops of the transmembrane domain that are exposed to the extracellular face of the cell, i.e., the loops between transmembrane regions 2 and 3, and between transmembrane regions 4 and 5.
• the "N terminal domain” region starts at the N- terminus and extends to a region close to the start of the transmembrane domain.
• feline refers herein to any member of the Felidae family, including domestic cats and nondomestic cats.
• felines can include both wild or captive cats, including wild and exotic cats, such as cougars, cheetah, lynxes, ocelots, lions, tigers, jaguars, panthers, and leopards.
• heterologous means that the sequence or cell originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention, or that the sequence is designed de novo without reference to any natural sequence.
• a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same or an analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.
• Heterologous sequences are those that are not operatively linked or are not contiguous to each other in nature.
• a “heterologous polypeptide” as used herein refers to a polypeptide which is not naturally included in the polypeptide sequence of the fTAS2R receptor polypeptide.
• a “heterologous cell” for expression of a polypeptide or nucleic acid refers to a cell that does not normally express that polypeptide or nucleic acid.
• Homology refers to the percent identity between polynucleotide or polypeptide molecules.
• Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50% , specifically at least about 75%, more specifically at least about 80%-85%, at least about 90%, and most specifically at least about 95%-98% sequence identity over a defined length of the molecules.
• substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
• identity refers to an exact nucleotide-to-nucleotide or amino acid- to- amino acid correspondence of two polynucleotides or polypeptide sequences
• immunoassay is an assay that uses an antibody to specifically bind an antigen.
• the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
• inhibiting means that the functional response of a TAS2R receptor, or fragment, to an agonist is reduced or prevented when in the presence of the inhibitor, for example the TAS2R receptor interacts with an intracellular signaling pathway to produce a smaller functional response, e.g. the TAS2R receptor interacts with a G-protein to promote signal transduction that produces a smaller increase in intracellular Ca2+ than is elicited by the agonist in the absence of inhibition.
• Interaction of a compound with a TAS2R receptor can mean binding of the compound to the receptor or modulation of a functional response of the receptor by the compound.
• isolated refers to a nucleic acid, a polypeptide, or other biological moiety that is removed from components with which it is naturally associated.
• isolated can refer to a polypeptide that is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro-molecules of the same type.
• isolated with respect to a polynucleotide can refer to a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome. Purity and homogeneity are typically determined using analytical chemistry techniques, for example polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated TAS2R nucleic acid is separated from open reading frames that flank the TAS2R gene and encode proteins other than a TAS2R. In some embodiments, the term “purified” means that the nucleic acid or protein is at least 85% pure, specifically at least 90% pure, more specifically at least 95% pure, or yet more specifically at least 99% pure.
• a "ligand” as used herein refers to a molecule that binds to a macromolecule, such as a TAS2R receptor.
• the ligand can be a small molecule, or a biological moiety, such as a protein, a sugar, nucleic acid or lipid.
• the ligand can be a molecule that modulates TAS2R receptor activity.
• a molecule that modulates activity of a receptor can be an agonist, an antagonist, or a modulator as defined herein.
• Ligands for various TAS2R receptors are known in the art.
• ligands of a mammalian TAS2R1 can include adhumulone, adlupulone, amarogentin, arborescin, cascarillin, chloramphenicol, cis-isocohumulone, cis-isoloadhumulone, cohumulone, colupulone, dextromethorphan, diphenidol (diphenylthiourea, sulfocarbanilide, sym- diphenylthiourea, or thiocarbanilide), humulon (humulone), isoxanthohumol, lupulon, lupulone, parthenolide, picrotoxinin, sodium cyclamate, sodium thiocyanate, thiamine, trans-isoadhumulone, trans-isocohumulone, trans-isohumulone, xanth
• Ligands of a mammalian TAS2R3 can include chloroquine.
• the mammalian TAS2R3 can be from a human, a rodent, a canine, or a feline.
• the mammalian TAS2R3 is a feline TAS2R3.
• Ligands of a mammalian TAS2R4 can include amarogentin, arborescin, artemorin, azathioprine, brucine, campher, chlorpheniramine, colchicine, dapsone, denatonium benzoate, diphenidol, parthenolide, quassin, quinine, and yohimbine.
• the mammalian TAS2R4 can be from a human, a rodent, a canine, or a feline. In an embodiment, the mammalian TAS2R4 is a feline TAS2R4.
• Ligands of a mammalian TAS2R7 can include caffeine, chlorpheniramine, cromolyn, diphenidol, papaverine, and quinine.
• the mammalian TAS2R7 can be from a human, a rodent, a canine, or a feline. In an embodiment, the mammalian TAS2R7 is a feline TAS2R7.
• Ligands of a mammalian TAS2R9 can include ofloxacin, pirenzapin, and procainamid.
• the mammalian TAS2R9 can be from a human, a rodent, a canine, or a feline.
• the mammalian TAS2R9 is a feline TAS2R9.
• Ligands of a mammalian TAS2R10 can include (-)-alpha thujone, absinthin, arborescin, arglabin, artemorin, azathioprine, benzoin , caffeine, campher, cascarillin, chloramphenicol, chloroquine, chlorpheniramine, coumarin, cucurbitacin b, cucurbitacin e, cucurbitacins, cycloheximid, cycloheximide, dapsone, denatonium benzoate,
• the mammalian TAS2R10 can be from a human, a rodent, a canine, or a feline. In an embodiment, the mammalian TAS2R10 is a feline TAS2R10.
• Ligands of a mammalian TAS2R38 can include 6-methyl-2-thiouracil, acetylthiourea, allyl isothiocyanate, caprolactam, chlorpheniramine, dimethylthioformamide, diphenidol, (diphenylthiourea, sulfocarbanilide, sym-diphenylthiourea, thiocarbanilide), ethylene thiourea, ⁇ , ⁇ -ethylene thiourea, ethylpyrazine, limonin, methimazole, n- ethylthiourea, n-methylthiourea, phenethyl isothiocyanate, phenylthiocarbamide (ptc), probenecid, propylthiouracil, sinigrin, sodium cyclamate, sodium_thiocyanate, and yohimbine.
• the mammalian TAS2R38 can be from a human, a rodent, a canine, or a feline. In an embodiment, the mammalian TAS2R38 is a feline TAS2R38.
• Ligands of a mammalian TAS2R43 can include acesulfame K, aloin, amarogentin, arborescin, arglabin, aristolochic acid, caffeine, chloramphenicol, cromolyn, denatonium benzoate, diphenidol, falcarindiol, grosheimin (grossheimin), helicin, probenecid, quinine, and saccharin.
• the mammalian TAS2R43 can be from a human, a rodent, a canine, or a feline. In an embodiment, the mammalian TAS2R43 is a feline TAS2R43.
• Ligands of a mammalian TAS2R44 can include acesulfame K, aloin, aristolochic acid, diphenidol, famotidine, parthenolide, quinine, and saccharin.
• TAS2R44 can be from a human, a rodent, a canine, or a feline.
• the mammalian TAS2R44 is a feline TAS2R44.
• ligand-binding fragment of a TAS2R receptor refers to one or more fragments of the TAS2R receptor retaining the ability to specifically bind to a ligand of the TAS2R receptor.
• a “modulator” is a molecule that modulates the functional response of a receptor by binding to a binding site that is distinct from the agonist binding site.
• a positive modulator or “enhancer” enhances the functional response of a receptor, while a negative modulator or “inhibitor” inhibits the functional response of a receptor.
• An “allosteric modulator” induces a conformational change in the receptor, which alters the affinity of the receptor for ligands, particularly at the agonist binding site. Positive allosteric modulators increase the affinity for ligands at the agonist binding site and/or enhance functional activity of a receptor, while negative allosteric modulators decrease the affinity for ligands at the agonist binding site and/or inhibit functional activity of a receptor.
• Modulators can include non-peptide molecules such as non-peptide mimetics, non-peptide allosteric effectors, and peptides.
• the "modulating" or “altering” activity of a TAS2R receptor herein can refer to any change in TAS2R receptor activity occurring in response to binding of an agonist, antagonist, or modulator to the TAS2R receptor or a ligand binding fragment thereof, that is the alteration can be stimulating, antagonizing, or modulating the functional response of the receptor.
• Non-naturally occurring in reference to a polynucleotide means that the polynucleotide sequence does not occur in nature in genomic DNA of an organism.
• nucleic acid includes DNA molecules and RNA molecules.
• a polynucleotide may be single-stranded or double- stranded.
• Polynucleotides can contain known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
• a polynucleotide can be obtained by a suitable method known in the art, including isolation from natural sources, chemical synthesis, or enzymatic synthesis. Nucleotides may be referred to by their commonly accepted single-letter codes.
• operably linked refers to a nucleic acid sequence placed into a functional relationship with another nucleic acid sequence.
• DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
• a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
• a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
• “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
• enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
• a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
• a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
• operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
• a "palatability enhancer” or “palatant” for animal edible composition is an additive that provides an aroma, taste, aftertaste, mouth feel, texture, and/or organoleptic sensation that is appealing to the target animal.
• polypeptide peptide
• protein protein
• link between one amino acid residue and the next is an amide bond and is sometimes referred to as a peptide bond.
• a polypeptide can be obtained by a suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis, or enzymatic synthesis.
• the terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
• Macromolecular structures of polypeptides can be described in terms of various levels of organization.
• Primary structure refers to the amino acid sequence of a particular peptide.
• Secondary structure refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 50 to 350 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices.
• “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer.
• Quaternary structure refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units.
• the term "primer” refers to an isolated single-stranded oligonucleotide of between about 10 to 50 nucleotides in length, preferably between about 15 to 50, more preferably 15 to 30 nucleotides in length and most preferably between about 18 and 28 nucleotides in length, that forms a duplex with a single stranded nucleic acid sequence of interest, and which is capable of acting as a point of initiation of nucleic acid synthesis to allow for polymerization of a complementary strand using a polymerase under appropriate conditions (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
• the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent.
• the exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
• the primer is an oligodeoxyribonucleotide.
• primers may have restriction enzyme site sequences appended to their 5' ends. Such enzymes and sites are well known in the art.
• the primers themselves can be synthesized using techniques which are well known in the art. Generally, the primers can be made using oligonucleotide synthesizing machines which are commercially available.
• a “primer pair” is a pair of primer sequences chosen to amplify a particular DNA target sequence by PCR.
• One primer of the pair is complementary to the 3' end of the "sense" strand of the DNA target, e.g. a cDNA, and the other is complementary to the 3' end of the "anti-sense” strand of the DNA target.
• probe refers to an oligonucleotide which is capable of hybridizing to another nucleic acid of interest.
• a probe may be single-stranded or double- stranded.
• a probe herein is an oligonucleotide of between about 10 to 100 nucleotides in length, preferably between about 15 to 80, more preferably 20 to 50 nucleotides in length. Probes are useful in the detection, identification and isolation of particular nucleic acid sequences, for example via Southern hybridization or other methods known in the art.
• any probe used in the present invention will be labeled with any "reporter molecule,” so that it is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
• the term "recombinant” can be used to describe a nucleic acid molecule and refers to a polynucleotide of genomic, RNA, DNA, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature.
• the term "recombinant” as used with respect to a protein or polypeptide can refer to a polypeptide produced by expression of a recombinant polynucleotide.
• the gene of interest is cloned and then expressed in transformed organisms, by a method known in the art. The host organism expresses the foreign gene to produce the protein under expression conditions.
• solid support refers to a material or group of materials having a rigid or semi-rigid surface or surfaces. Examples of materials include plastics (e.g.,
• the solid support can be substantially flat, although in some aspects it may be desirable to physically separate regions for different molecules with, for example, wells, raised regions, pins, etched trenches, or the like.
• the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations.
• the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
• Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
• polyclonal antibodies raised to a fTAS2R can be selected to obtain only those polyclonal antibodies that are specifically
• solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
• a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
• the terms “specific binding,” “specifically binds,” “selective binding,” and “selectively binds” mean that a receptor, such as a TAS2R receptor, exhibits appreciable affinity for a particular ligand.
• “Appreciable” binding affinity includes binding with an affinity of at least 10 4 M “1 , at least 10 5 M “1 , specifically at least 10 6 M “1 , more specifically at least 10 7 M- " 1 , yet more specifically at least 108 M- " 1 , or even yet more specifically at least 10 9 M "1 .
• a binding affinity can also be indicated as a range of affinities, for example, 10 4 M “1 to 10 10 M “1 , specifically 10 5 M “1 to 10 10 M “1 , more specifically 10 6 M “1 to 10 10 M “1 .
• Specific binding can be determined according to any art-recognized means for determining such binding. In some embodiments, specific binding is determined according to Scatchard analysis and/or competitive binding assays.
• stimulation means that the TAS2R receptor, or fragment, is placed in a state in which it produces a functional response, for example the TAS2R receptor interacts with an intracellular signaling pathway to produce the functional response, e.g. the TAS2R receptor interacts with a G-protein to promote signal transduction that produces increased intracellular Ca2+.
• substantially the same biological activity refers to a polypeptide fragment, derivative, homolog, analog, or variant retaining at least about 50%, 55%, 60%, 65%, 70%, preferably at least about 75%, 80%, 85%, 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, and most preferably at least about 96%, 97%, 98%, 99% or greater biological activity of the parent polypeptide.
• the extent to which a polypeptide fragment, derivative, homolog, analog, or variant retains the biological activity of the parent polypeptide may be assessed by any means available in the art, including, but not limited to, the assays listed or described herein.
• A"TAS2R binding partner is a compound that directly or indirectly binds a TAS2R polypeptide disclosed herein.
• a "TAS2R receptor polypeptide" for use in assays described herein to measure ligand binding or receptor activity can comprise a TAS2R receptor; a domain of a TAS2R receptor, such as an extracellular domain, transmembrane region, transmembrane domain, cytoplasmic domain, a ligand-binding fragment, subunit association domain, active site, and the like; or a chimeric protein in which either a TAS2R receptor or a domain thereof is covalently linked to a heterologous protein.
• a "tastant” means a ligand that can bind to a specific TAS2R receptor or set of TAS2R receptors.
• taste perception refers to a response (e.g., biochemical, behavioral) or sensitivity of a TAS2R receptor to a taste stimulus. Modification of taste perception includes an alteration of (enhancement of, reduction of, or change to) a
• Taste perception does not require, though it can include, transmission of a neural signal resulting in the in vivo sensation of taste by a mammal.
• transmembrane domain which comprises the seven transmembrane regions, refers to the domain of TAS2R polypeptides that lies within the plasma membrane, and may also include the corresponding cytoplasmic (intracellular) and extracellular loops, also referred to as transmembrane domain "regions.” Transmembrane regions can also bind ligand either in combination with the extracellular domain or alone, and are therefore also useful for in vitro ligand binding assays.
• transmembrane region denotes a three-dimensional protein structure which is thermodynamically stable in a membrane, e.g., a single
• transmembrane alpha helix or a transmembrane beta barrel transmembrane alpha helix or a transmembrane beta barrel.
• the term "vector” means a nucleic acid sequence to express a target gene in a host cell.
• examples include a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector.
• viral vectors include a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector.
• the vector may be an expression vector including a membrane targeting or secretion signaling sequence or a leader sequence, in addition to an expression control element such as promoter, operator, initiation codon, termination codon, polyadenylation signal, and enhancer.
• the vector may be manufactured in various ways known in the art depending on the purpose.
• An expression vector may include a selection marker for selecting a host cell containing the vector. Further, a replicable expression vector may include an origin of replication.
• the term "recombinant vector” or "expression vector” means a vector operably linked to a heterologous nucleotide sequence for the purpose of expression, production, and isolation of the heterologous nucleotide sequence.
• the heterologous nucleotide sequence can be a nucleotide sequence encoding all or part of a fTAS2R receptor or a chimeric polypeptide disclosed herein.
• hTAS2R Human TAS2R
• fTAS2R feline TAS2R
• isolated feline genomic DNA was used to clone the fTAS2R genes.
• the nucleotide sequence of the cloned fTAS2R genes of several felines was then determined by sequencing, e.g., Sanger sequencing, and used to establish a consensus nucleotide sequence for the gene, and to identify any variant sites in the sequence.
• polynucleotides encoding a fTAS2R receptor are disclosed.
• the polynucleotides are isolated.
• the polynucleotide can comprise a nucleotide sequence selected from the nucleotide sequence of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26; a nucleot
• the percent homology is at least 90%. In an embodiment, the percent homology is at least 95%, preferably at least 98%, more preferably at least 99%.
• the polynucleotide comprises a nucleotide sequence selected from: the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26; a nucleotide sequence that hybridizes to the complement of the polynucleotide having SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11,
• the polynucleotide comprises a nucleotide sequence selected from: the nucleotide sequence of SEQ ID NO: 17; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18; a nucleotide sequence that hybridizes to the complement of the polynucleotide having SEQ ID NO: 17 under high stringency conditions; and the complement of the foregoing nucleotide sequences.
• the nucleotide sequence is codon-optimized for expression in a non-feline cell.
• the non-feline cell is Escherichia coli, a Saccharomyces cerevisae cell, a Drosophila melanogaster cell, a Caenorhabditis elegans cell, or a mammalian cell.
• the mammalian cell is a human or murine cell. Examples of codon-optimized sequences for expression of the novel fTAS2R receptor polypeptides in Escherichia coli, Saccharomyces cerevisae cell, Drosophila melanogaster, Caenorhabditis elegans, human, or murine cells are disclosed in SEQ ID NOs: 58-135.
• polynucleotides comprising a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% homology with SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or the complement of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25.
• compositions comprising at least two polynucleotides disclosed herein.
• each polynucleotide encodes a portion of a different fTAS2R receptor.
• the composition comprises at least 3, 4, or 5 of the polynucleotides disclosed herein.
• the composition comprises at least 6, 7, 8, 9, 10, 11, 12, or 13 of the polynucleotides disclosed herein.
• each polynucleotide of the composition encodes a different fTAS2R receptor, or fragment thereof.
• the composition comprises a polynucleotide comprising SEQ ID NO: 17 and/or SEQ ID NO: 21.
• the composition comprises a primer pair for amplifying a portion of a nucleic acid encoding a feline TAS2R polypeptide.
• the primer pairs are selected from the primer pairs of Table 5.
• the primer pairs disclosed herein are useful for determination of the nucleotide sequence of a particular TAS2R polynucleotide, or fragment thereof, using PCR.
• the pairs of single- stranded DNA primers can be annealed to sequences within or surrounding the fTAS2R gene in order to prime amplifying DNA synthesis of the fTAS2R gene itself. Allele- specific primers can also be used. Such primers anneal only to particular fTAS2R mutant alleles, and thus will only amplify a product in the presence of the mutant allele as a template.
• a single nucleotide polymorphism in the nucleic acid sequence encoding fTAS2R38 was identified at nucleotide 220 of the cDNA sequence (SEQ ID NO: 17) from sequencing amplified feline genomic DNA from multiple subjects.
• the two alleles observed at the nucleotide 220 were G and A.
• the G220A nucleic acid variation corresponds to an amino acid variation D74N in the fTAS2R38 protein sequence (SEQ ID NO: 18).
• a disclosed polynucleotide comprises a nucleotide sequence of at least 15 contiguous nucleotides of SEQ ID NO: 17 containing nucleotide 220, wherein an A is present at nucleotide 220; or the complement of the nucleotide sequence.
• the polynucleotide comprises at least 20 contiguous nucleotides of SEQ ID NO: 17 containing nucleotide 220, wherein an A is present at nucleotide 220; or the complement of the nucleotide sequence.
• a disclosed fTAS2R38 polypeptide comprises SEQ ID NO: 18 with N present at residue 74 of the sequence, or a fragment thereof comprising the N74 residue.
• isolated fTAS2R receptor polypeptides are disclosed.
• the isolated fTAS2R polypeptide is encoded by a
• the isolated fTAS2R polypeptide can comprise the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26; or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% homology with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26.
• the isolated fTAS2R polypeptide comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID N0:16, SEQ ID N0:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26; or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% homology with one of the foregoing amino acid sequences.
• the isolated fTAS2R polypeptide comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:22.
• Sensory GPCRs such as the TAS2R bitter taste receptors, have a domain structure including an N-terminal domain; extracellular domains; a transmembrane domain comprising seven transmembrane regions, cytoplasmic, and extracellular loops; cytoplasmic domains; and a C-terminal domain.
• These domains can be structurally identified using methods known in the art, such as sequence analysis programs that identify hydrophobic and hydrophilic domains.
• Such domains are useful for making chimeric proteins and for in vitro assays disclosed herein, e.g., ligand binding assays.
• transmembrane regions and extracellular and cytoplasmic loops can be identified using standard methods known in the art.
• transmembrane regions of the fTAS2R proteins can be identified using software, TOPCONS, available on the internet from the Swiss Bioinformatics Center, Swiss University (Andreas Bernsel, et al. (2009) Nucleic Acids Research 37(Webserver issue), W465-8).
• TOPCONS available on the internet from the Swiss Bioinformatics Center, Sweden University (Andreas Bernsel, et al. (2009) Nucleic Acids Research 37(Webserver issue), W465-8).
• transmembrane regions and extracellular and cytoplasmic loops of the fTAS2R identified by TOPCONS are shown in the following table:
• Transmembrane domains 1 : 2-22, 2: 47-67, 3: 85-105, 4: 125-145, 5: 180-200, 6: 228-248, 7. 258- 278
• Extracellular domain 1, 68-84; 146-179; 249-257.
• Intracellular domain 23-46; 106-124; 201-227; 279-298 fTAS2R2 (SEQ ID NO:4)
• Transmembrane domains 1 : 11-31, 2: 52-72, 3: 89-109, 4: 130-150, 5: 187-207, 6: 235-255, 7: 265-
• Extracellular domain 1-10, 73-88; 151-186; 256-264
• Intracellular domain 32-51 ; 110-129; 208-234; 286-304 fTAS2R3 (SEQ ID NO:6)
• Transmembrane domains 1 : 9-29, 2: 51-71, 3: 89-109, 4: 129-149, 5: 187-207, 6: 235-255, 7: 266- Extracellular domain: 1-8; 72-88; 150-186; 256-265
• Intracellular domain 30-50; 110-128; 208-234; 287-316 fTAS2R4 (SEQ ID NO:8)
• Transmembrane domains 1 : 3-23, 2: 48-68, 3: 88-108, 4: 130-150, 5: 184-204, 6: 232-252, 7: 262- 282
• Intracellular domain 24-47; 109-129; 205-231 ; 283-306 fTAS2R7 (SEQ ID NO: 10)
• Transmembrane domains 1 : 9-29, 2: 51-71, 3: 89-109, 4: 129-149, 5: 188-208, 6: 236-256, 7: 266- 286
• Intracellular domain 30-50; 110-128; 209-235; 287-311 fTAS2R9(SEQ ID NO: 12)
• Transmembrane domains 1 : 7-27, 2: 51-71, 3: 89-109, 4: 129-149, 5: 184-204, 6: 232-252, 7: 263-
• Intracellular domain 28-50; 110-128; 205-231 ; 284-337 fTAS2R10 (SEQ ID NO: 14)
• Transmembrane domains 1 : 2-22, 2. 48-68, 3: 88-108, 4: 129-149, 5: 182-202, 6: 230-250, 7: 261- 281
• Extracellular domain 1 ; 69-87; 150-181 ; 251-260
• Intracellular domain 23-48; 109-128; 203-229; 282-300 fTAS2R12 (SEQ ID NO: 16)
• Transmembrane domains 1 : 9-29, 2: 48-68, 3: 89-109, 4: 129-149, 5: 186-206, 6: 231-251, 7: 262- 282
• Intracellular domain 30-47; 110-128; 207-230; 283-309 fTAS2R38 (SEQ ID NO: 18)
• Transmembrane domains 1 : 18-38, 2: 62-82, 3: 99-119, 4: 140-160, 5: 199-219, 6: 247-267, 7: 278-
• Intracellular domain 39-61 ; 120-139; 220-246; 299-334 fTAS2R42 (SEQ ID NO:20)
• Transmembrane domains 1: 2-22, 2: 48-68, 3: 89-109, 4: 129-149, 5: 186-206, 6: 234-254, 7: 265-
• Extracellular domain 1 ; 69-88; 150-185; 255-264
• Intracellular domain 23-47; 110-128; 207-233; 286-322 fTAS2R43 (SEQ ID NO:22)
• Transmembrane domains 1 : 3-23, 2: 48-68, 3: 88-108, 4: 128-148, 5: 182-202, 6: 230-250, 7: 261- 281
• Extracellular domain 1-2; 69-87; 149-181 ; 251-260
• Intracellular domain 24-47; 109-127; 203-229; 282-299 fTAS2R44 (SEQ ID NO:24)
• Transmembrane domains 1: 3-23, 2: 48-68, 3: 88-108, 4: 128-148, 5: 182-202, 6: 230-250, 7: 260-
• Intracellular domain 24-47; 109-127; 203-229; 281-308 fTAS2R67 (SEQ ID NO:26)
• Transmembrane domains 1 : 9-29, 2: 51-71, 3: 89-109, 4: 129-149, 5: 186-206, 6: 233-253, 7: 264- 284
• Intracellular domain 30-50; 110-128; 207-232; 285-312
• Additional methods known in the art to predict the structural regions include hydropathy prediction methods of Goldman-Engleman-Steitz, or Kyte-Doolittle (J. Mol. Biol. 157: 105-132 (1982), or Hopp-Woods. Secondary structure prediction methods include Garnier-Robson, or Deleage & Roux or Chou-Fasman. As known in the art, the various available algorithms may predict slightly different boundaries for transmembrane regions based on the amino acid sequence.
• the isolated TAS2R receptor polypeptide can comprise at least one extracellular domain of a feline TAS2R receptor; at least one transmembrane domain of a feline TAS2R receptor; or at least one intracellular domain of a feline TAS2R receptor, wherein the feline TAS2R receptor comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26; or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% homology, specifically at least 97% homology, more specifically at least 99% homology with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10,
• the extracellular domain of the fTAS2R polypeptide can comprise amino acids 1, 68-84; 146-179; or 249-257 of SEQ ID NO:2; amino acids 1-10, 73- 88; 151-186; or 256-264 of SEQ ID NO:4; amino acids 1-8; 72-88; 150-186; or 256-265 of SEQ ID NO:6; amino acids 1-2; 69-87; 151-183; or 253-261 of SEQ ID NO:8; amino acids 1-8; 72-88; 150-187; or 257-265 of SEQ ID NO: 10; amino acids 1-6; 72-88; 150-183; or 253- 262 of SEQ ID NO:12; amino acids 1; 69-87; 150-181; or 251-260 of SEQ ID NO:14; amino acids 1-8; 69-88; 150-185; or 252-261 of SEQ ID NO:16; amino acids 1-17: 83-98; 161-198; or 268-2
• the transmembrane domain of the fTAS2R polypeptide can comprise amino acids 2-22, 47-67, 85-105, 125-145, 180-200, 228-248, or 258-278 of SEQ ID NO:2; amino acids 11-31, 52-72, 89-109, 130-150, 187-207, 235-255, or 265-285 of SEQ ID NO:4; amino acids 9-29, 51-71, 89-109, 129-149, 187-207, 235-255, or 266-286 of SEQ ID NO:6; amino acids 3-23, 48-68, 88-108, 130-150, 184-204, 232-252, or 262-282 of SEQ ID NO:8; amino acids 9-29, 51-71, 89-109, 129-149, 188-208, 236-256, or 266-286 of SEQ ID NO:10; amino acids 7-27, 51-71, 89-109, 129-149, 184-204,
• the intracellular domain of the fTAS2R polypeptide can comprise amino acids 23-46; 106-124; 201-227; or 279-298 of SEQ ID NO:2; amino acids 32-51; 110-129; 208-234; or 286-304 of SEQ ID NO:4; amino acids 30-50; 110-128; 208- 234; or 287-316 of SEQ ID NO:6; amino acids 24-47; 109-129; 205-231; or 283-306 of SEQ ID NO:8; amino acids 30-50; 110-128; 209-235; or 287-311 of SEQ ID NO:10; amino acids 28-50; 110-128; 205-231 ; or 284-337 of SEQ ID NO:12; amino acids 23-48; 109-128; 203- 229; or 282-300 of SEQ ID NO:14; amino acids 30-47; 110-128; 207-230; or 283-309 of SEQ ID NO:16; amino acids 39-61; 120-139;
• the fTAS2R receptor polypeptide comprises a transmembrane region 2, a transmembrane region 3, a transmembrane region 4, a transmembrane region 5, a transmembrane region 6, and a transmembrane region 7, wherein each transmembrane region comprises at least 20 consecutive amino acids of the corresponding transmembrane region sequence independently selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26; or a transmembrane region 3, a transmembrane region 6, and a transmembrane region 7, wherein each transmembrane region comprises at least 20 consecutive amino acids of the corresponding transmembrane region sequence independently selected from SEQ ID NO:2, SEQ ID NO
• transmembrane region comprises at least 20 consecutive amino acids of the corresponding transmembrane region sequence independently selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26; an extracellular domain 3 comprising at least 15 consecutive amino acids selected from amino acids 146-179 of SEQ ID NO:2; amino acids 151-186 of SEQ ID NO:4; amino acids 150-186 of SEQ ID NO:6; amino acids 151-183 of SEQ ID NO:8; amino acids 150-187 of SEQ ID NO:10; amino acids 150-183 of SEQ ID NO:12; amino acids 150-181 of SEQ ID NO:14; amino acids 150-185 of SEQ ID NO:16; amino acids 161-198 of SEQ ID NO:18; amino acids 150-185 of
• polypeptide comprising at least one extracellular domain of a feline TAS2R receptor; at least one transmembrane domain of a feline TAS2R receptor; or at least one intracellular domain of a feline TAS2R receptor.
• chimeric polypeptides comprising an extracellular domain, an intracellular domain, or a transmembrane region of a feline TAS2R receptor polypeptide, and further comprising a heterologous polypeptide are disclosed.
• the intracellular domain, extracellular domain, or the transmembrane region of the feline TAS2R receptor polypeptide can be any of those disclosed herein.
• the heterologous polypeptide can be any suitable polypeptide known in the art, or a portion of such polypeptide as may be useful herein.
• the heterologous polypeptide can be, for example, a sequence to determine cellular localization and expression, to permit proper folding of the chimeric polypeptide in an expression system, and/or to facilitate isolation of the chimeric polypeptide.
• the heterologous polypeptide can be linked to any portion of the chimeric polypeptide, for example to the amino terminal end or the carboxy terminal end of the fTAS2R sequence.
• the heterologous polypeptide can be the first 45 amino acids of rat somatostatin, the FLAG® tag, a 6x histidine (his) tag, MYC, a fluorescent protein tag, V5, and/or glutathione S-transferase (GST).
• the heterologous polypeptide is the first 45 amino acids of rat somatostatin, it is typically placed at the amino terminal end of the chimeric polypeptide to permit membrane targetting.
• the heterologous polypeptide is a tag to permit easier isolation of the chimeric polypeptide, e.g., a 6x histidine tag, it can be placed at the amino terminus of the chimeric polypeptide.
• Determination of a suitable location for the heterologous polypeptide in the chimeric polypeptide relative to the amino end or the carboxy end of the fTAS2R sequence to obtain a particular functional aspect of the heterologous polypeptide on the chimeric polypeptide can be made by one of skill in the art.
• polynucleotides encoding the chimeric polypeptides are also disclosed.
• composition comprising at least two fTAS2R polypeptides disclosed herein.
• the composition comprises at least 3, 4, or 5
• the composition comprises at least 6, 7, 8, 9, 10, 11, 12, or 13 polypeptides disclosed herein.
• each polypeptide in the composition is a different fTAS2R receptor.
• the composition comprises a polypeptide comprising SEQ ID NO: 18 and a polypeptide comprising SEQ ID NO:22.
• a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
• a conservative amino acid substitution in a polypeptide sequence includes the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix.
• 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.
• degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with 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)).
• Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100.
• Readily available computer programs can be used to aid in the analysis, such as ALIGN (Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC), which adapts the local homology algorithm of Smith and Waterman 1981 Advances in Appl Math 2:482-489, for peptide analysis.
• Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the
• nucleotide homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
• DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989) or Current Protocols in
• Moderate hybridization conditions are defined as equivalent to hybridization in 2X sodium chloride/sodium citrate (SSC) at 30° C, followed by a wash in IX SSC, 0.1% SDS at 50° C.
• Highly stringent conditions include conditions equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45° C, followed by a wash in 0.2X SSC, 0.1% SDS at 65° C.
• SSC sodium chloride/sodium citrate
• the recombinant vector comprises a polynucleotide consisting of SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25; a polynucleotide consisting of the complement of SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25; or a polynucleotide consisting of a sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% homology with SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or the complement of SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25.
• the recombinant vector comprises a polynucleotide comprising a nucleotide sequence selected from: the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26; a nucleotide sequence that hybridizes to the complement of the polynucleotide having SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO
• the vector comprises a polynucleotide sequence of SEQ ID NO: 17 or SEQ ID NO:21. Also disclosed is an expression vector comprising a polynucleotide encoding a chimeric polypeptide disclosed herein.
• the recombinant vector may be constructed for use in prokaryotic or eukaryotic host cells.
• the expression vector used generally includes a strong promoter capable of initiating transcription (for example, ⁇ promoter, trp promoter, lac promoter, tac promoter, T7 promoter), a ribosome binding site for initiating translation, and a transcription/translation termination sequence.
• the vector used generally includes the origin of replication acting in the eukaryotic cell, for example fl origin of replication, SV40 origin of replication, pMBl origin of replication, adeno origin of replication, AAV origin of replication, or BBV origin of replication, but is not limited thereto.
• a promoter in an expression vector for a eukaryotic host cell may be a promoter derived from the genomes of mammalian cells (for example, a metallothionein promoter or an EF-1 alpha promoter) or a promoter derived from mammalian viruses (for example, an adenovirus late promoter, a Vaccinia virus 7.5K promoter, a Sindbis promoter, a SV40 promoter, a cytomegalovirus promoter, and a tk promoter of HSV).
• a transcription termination sequence in an expression vector for a eukaryotic host cell may be, in general, a polyadenylation sequence.
• a host cell comprising an expression vector or a
• a suitable host cell can be transformed with at least one of the recombinant vectors or at least one polynucleotide disclosed herein, for example a polynucleotide consisting of SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25.
• the host cell of the vector may be any cell that can be practically utilized by the expression vector.
• the host cell may be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell.
• the host cell may be a prokaryotic cell, such as a bacterial cell.
• a prokaryotic host cell may be a Bacillus genus bacterium, such as E. coli JM109, E. coli BL21, E. coli RRl, E. coli LE392, E. coli B, E. coli X 1776, E.
• a eukaryotic host cell may be a yeast (e.g., Saccharomyces cerevisiae), an insect cell, a plant cell, or an animal cell, for example, mouse Sp2/0, CHO (Chinese hamster ovary) Kl, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, HeLa, HEK- 293, or a MDCK cell line.
• yeast e.g., Saccharomyces cerevisiae
• insect cell e.g., a plant cell
• an animal cell for example, mouse Sp2/0, CHO (Chinese hamster ovary) Kl, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, HeLa, HEK- 293, or a MDCK cell line.
• fish cells are useful herein.
• the polynucleotide or recombinant vector including the polynucleotide may be transferred into the host cell using a method known in the art.
• a method known in the art For example, when a prokaryotic cell is used as the host cell, the transfer may be performed using a CaCl 2 method or an electroporation method, and when a eukaryotic cell is used as the host cell, the transfer may be performed by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, LIPOFECTAMINE® (Life Technologies Corporation) transfection, or gene bombardment, but is not limited thereto.
• the transfected cells can be cultured under conditions favoring expression of the fTAS2R.
• the fTAS2R can be recovered from the culture using standard techniques known in the art.
• the expression vectors disclosed herein are particularly useful for assays to identify and characterize tastants.
• Means to introduce/express the nucleic acids and vectors, either individually or as libraries, are well known in the art.
• a variety of individual cell, organ, or whole animal parameters can be measured by a variety of means.
• the disclosed fTAS2R sequences can be expressed, for example, in animal taste tissues by delivery with a transmissible agent, e.g., adenovirus expression vector.
• Nucleic acid assays for the presence of DNA and RNA for a TAS2R family member in a sample include numerous techniques known to those skilled in the art, such as Southern analysis, Northern analysis, dot blots, RNase protection, SI analysis, amplification techniques such as polymerase chain reaction (PCR) and ligase chain reaction (LCR), and in situ hybridization.
• a TAS2R protein can be detected with the various immunoassay techniques known in the art.
• the test sample is typically compared to both a positive control (e.g., a sample expressing a recombinant TAS2R protein) and a negative control.
• nucleic acid and amino acid sequence information disclosed herein also makes possible identification of binding partner compounds with which a TAS2R
• Methods to identify binding partner compounds include solution assays, in vitro assays wherein TAS2R polypeptides are immobilized, and cell-based assays.
• binding molecules including natural ligands and synthetic compounds, can be identified or developed using isolated or recombinant TAS2R products, TAS2R variants, or cells expressing such products. Binding partners are useful for purifying TAS2R products and detection or quantification of TAS2R products in fluid and tissue samples using known immunological procedures. Binding molecules are also useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of TAS2R, especially those activities involved in signal transduction. Binding molecules also are useful in methods for predicting the taste perception of an organism such as a mammal by detecting a TAS2R polypeptide in a biological sample of a feline.
• the method comprises contacting TAS2R receptor with a test compound suspected of binding TAS2R receptor; and detecting binding between the compound and the TAS2R receptor.
• Binding can be determined by any binding assay known to the skilled artisan, including gel-shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross-linking, interaction trap/two-hybrid analysis, soiled analysis, and ELISA.
• the methods may also use ligands that are attached to a label, such as a radiolabel (e. g., 125 I, 35 S,
• a fluorescence label a chemiluminescent label, an enzymic label, and an immunogenic label.
• a composition comprising a cell expressing TAS2R receptor on its surface is used in the method.
• isolated TAS2R receptor or cell membranes comprising TAS2R receptor are employed. The binding may be measured directly, e. g., by using a labeled compound, or may be measured indirectly. Compounds identified as binding a TAS2R receptor may be further tested in other assays including TAS2R activity assays and/or in vivo models, in order to confirm or quantitate their activity.
• Ligand binding to a TAS2R protein, a domain, or chimeric protein can be tested in solution, in a bilayer membrane, attached to a solid phase, in a lipid monolayer, or in vesicles.
• Ligand binding to a TAS2R receptor can be tested using, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), or in hydrodynamic (e.g., shape), chromatographic, or solubility properties.
• the TAS2R polypeptide or polynucleotide employed in such a test may either be free in solution, attached to a solid support, borne on a cell surface, located intracellularly, or associated with a portion of a cell.
• One skilled in the art can, for example, measure the formation of complexes between a TAS2R receptor or polynucleotide and the compound being tested.
• one skilled in the art can examine the diminution in complex formation between a TAS2R receptor or polynucleotide and its substrate caused by the compound being tested.
• the recognition sites of the TAS2R receptor or polynucleotide are coupled with a monitoring system, either electrical or optical.
• an appropriate chemical stimulus can bind to the receptor's ligand binding domain, changing the receptor conformation to a degree that the coupled electronics or optical changes can be observed on a read-out.
• the solid support is formulated into a feline- specific electronic tongue or biosensor.
• the methods can comprise the steps of contacting a TAS2R receptor with one or more test compound and identifying the compounds that bind to the TAS2R receptor. Identification of the compounds that bind the TAS2R receptor can be achieved by isolating the TAS2R polypeptide/binding partner complex, and separating the binding partner compound from the TAS2R polypeptide. In one aspect, the TAS2R polypeptide/binding partner complex is isolated using an antibody immuno specific for either the TAS2R receptor or the test compound.
• either the TAS2R receptor or the test compound comprises a label or tag that facilitates its isolation
• methods to identify binding partner compounds include a step of isolating the TAS2R polypeptide/binding partner complex through interaction with the label or tag.
• the method comprises the steps of contacting an immobilized TAS2R receptor with a test compound and detecting binding of the test compound to the TAS2R receptor.
• the test compound is immobilized and binding of TAS2R receptor is detected. Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead, or a chromatographic resin, as well as non-covalent, high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety.
• the support may, for example, be formulated into a feline- specific electronic tongue or biosensor.
• cell-based assays are used to identify binding partner compounds of a TAS2R receptor.
• the method comprises the steps of contacting a TAS2R receptor expressed on the surface of a cell with a test compound and detecting binding of the test compound to the TAS2R receptor.
• the detection comprises detecting a physiological event in the cell caused by the binding of the molecule.
• HTS high throughput screening
• large numbers of different test compounds are synthesized on a solid substrate.
• the test compounds are contacted with TAS2R receptor and washed. Bound TAS2R receptor is then detected by methods well known in the art.
• Purified polypeptides of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques.
• non-neutralizing antibodies can be used to capture the protein and immobilize it on the solid support.
• an expressed TAS2R receptor can be used for HTS binding assays in conjunction with a ligand, such as an amino acid or carbohydrate.
• a ligand such as an amino acid or carbohydrate.
• the identified ligand is labeled with a suitable radioisotope, including, 121 I, 3 H, 35 S or 32 P, by methods that are well known to those skilled in the art.
• the ligands may be labeled by well-known methods with a suitable fluorescent derivative (Baindur et al., Drug Dev. Res., 1994, 33, 373- 398; Rogers, Drug Discovery Today, 1997,2, 156-160).
• Radioactive ligand specifically bound to the receptor in membrane preparations made from the cell line expressing the recombinant protein can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand. Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary. Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct
• either the TAS2R receptor or the test compound comprises a label or tag that facilitates its isolation
• methods to identify test compounds include a step of isolating the TAS2R polypeptide/test compound complex through interaction with the label or tag.
• An exemplary tag of this type is a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation.
• Other labels and tags, such as the FLAG tag (Eastman Kodak, Rochester, NY), are well known and routinely used in the art.
• Detection of binding can be accomplished using a radioactive label on the compound that is not immobilized, using a fluorescent label on the non-immobilized compound, using an antibody immuno specific for the non-immobilized compound, using a label on the non- immobilized compound that excites a fluorescent support to which the immobilized compound is attached, as well as other techniques well known and routinely practiced in the art.
• assays may be used to identify specific ligands of a TAS2R receptor, including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods.
• binding interactions are evaluated indirectly using the yeast two- hybrid system, a genetic assay for detecting interactions between two proteins or polypeptides.
• the test compound in any of the methods disclosed herein, to be considered a ligand of the TAS2R receptor polypeptide, the test compound must alter the measured interaction by an amount sufficient to achieve a statistically significant difference between the responses in the presence vs. the absence of the test compound. In an embodiment, to be considered a ligand, the test compound must alter the measured interaction by an amount sufficient to achieve a statistically significant difference between the responses in the presence vs. the absence of the test compound.
• Statistical significance can be determined by any appropriate statistical test known in the art, such as a t-test. For example, to be of statistical significance, the p-value is at least 0.05, at least 0.01, or at least 0.001.
• Also disclosed are methods of identifying compounds that modulate (i.e., increase or decrease) activity of TAS2R receptor comprising contacting a TAS2R receptor with a compound, and determining whether the compound modifies activity of TAS2R receptor.
• the method comprises contacting a TAS2R receptor with a known TAS2R receptor ligand in the presence or absence of a test compound. The activity in the presence of the test compound is compared to the activity in the absence of the test compound. Where the activity of the sample containing the test compound is higher than the activity in the sample lacking the test compound, the compound is an agonist. Similarly, where the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound is an antagonist.
• TAS2R protein activity is measured by expressing a TAS2R gene in a heterologous cell with a promiscuous G-protein that links the receptor to a phospholipase C signal transduction pathway (see Offermanns & Simon, J. Biol. Chem. 270:15175-15180 (1995)).
• the cell line is a eukaryotic cell line which does not naturally express TAS2R genes (e.g., Life Technologies Cat# R700-07) and the promiscuous G-protein is Gal 5 (Offermanns & Simon, supra).
• a TAS2R polypeptide is expressed in a eukaryotic cell as a chimeric receptor with a heterologous, chaperone sequence that facilitates its maturation, targeting through the secretory pathway or membrane localization.
• a heterologous, chaperone sequence that facilitates its maturation, targeting through the secretory pathway or membrane localization.
• the heterologous sequence is a rhodopsin sequence, such as an N-terminal fragment of a rhodopsin.
• a rhodopsin sequence such as an N-terminal fragment of a rhodopsin.
• Such chimeric TAS2R receptors can be expressed in any eukaryotic cell, such as Life Technologies Cat# R700-07 cells.
• the cells comprise a functional G protein, e.g., Gal5, that is capable of coupling the receptor to an intracellular signaling pathway or to a signaling protein such as phospholipase Cp.
• Activation of such expressed receptors in such cells can be detected using any standard method, such as by detecting changes in intracellular calcium by detecting FURA-2 dependent fluorescence in the cell.
• transcription levels can be measured to assess the effects of a test compound on signal transduction.
• a host cell containing a TAS2R protein of interest is contacted with a test compound for a sufficient time to effect any interactions, and then the level of gene expression is measured.
• the amount of time to effect such interactions may be empirically determined, such as by running a time course and measuring the level of transcription as a function of time.
• the amount of transcription may be measured by using any method known to those of skill in the art to be suitable. For example, mRNA expression of the protein of interest may be detected using Northern blots or their polypeptide products may be identified using immunoassays. Alternatively, transcription based assays using a reporter gene may be used as described in U.S. Pat.
• the reporter genes can be, e.g., chloramphenicol acetyltransferase, luciferase, [beta]-galactosidase and alkaline phosphatase.
• the protein of interest can be used as an indirect reporter via attachment to a second reporter such as green fluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)).
• the amount of transcription is then compared to the amount of transcription in either the same cell in the absence of the test compound, or it may be compared with the amount of transcription in a substantially identical cell that lacks the protein of interest.
• a substantially identical cell may be derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. Any difference in the amount of transcription indicates that the test compound has in some manner altered the activity of the protein of interest.
• a method for identifying an agonist of a feline TAS2R receptor comprises contacting a feline Tas2R receptor polypeptide disclosed herein with a test compound; and detecting an increase in biological activity of the receptor in the presence of the compound relative to biological activity of the polypeptide in the absence of the compound.
• a method for identifying an antagonist of a feline Tas2R receptor comprises contacting a feline Tas2R receptor polypeptide disclosed herein with a test compound; and detecting a decrease in biological activity of the receptor in the presence of the compound relative to biological activity of the polypeptide in the absence of the compound.
• Receptor-G-protein interactions can also be examined. For example, binding of the G-protein to the receptor or its release from the receptor can be examined. For example, in the absence of GTP, an agonist will lead to the formation of a tight complex of a G protein (all three subunits) with the receptor. This complex can be detected in a variety of ways, as noted above. Such an assay can be modified to search for antagonists, e.g., by adding an agonist to the receptor and G protein in the absence of GTP, which form a tight complex, and then screen for antagonists by looking at dissociation of the receptor-G protein complex. In the presence of GTP, release of the alpha subunit of the G protein from the other two G protein subunits serves as a criterion of activation.
• TAS2R-Gustducin interactions are monitored as a function of TAS2R receptor activation.
• Ligand dependent coupling of TAS2R receptors with Gustducin can be used as a marker to identify modifiers of any member of the TAS2R family.
• G-protein An activated or inhibited G-protein will in turn alter the properties of target enzymes, channels, and other effector proteins.
• the classic examples are the activation of cGMP phosphodiesterase by transducin in the visual system, adenylate cyclase by the stimulatory G-protein, phospholipase C by Gq and other cognate G proteins, and modulation of diverse channels by Gi and other G proteins.
• Downstream consequences can also be examined such as generation of diacyl glycerol and IP3 by phospholipase C, and in turn, for calcium mobilization by IP3.
• Receptor activation typically initiates subsequent intracellular events, e.g., increases in second messengers such as IP3, which releases intracellular stores of calcium ions.
• IP3 inositol triphosphate
• IP3 phospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge & Irvine, Nature 312:315-21 (1984)).
• IP3 in turn stimulates the release of intracellular calcium ion stores.
• a change in cytoplasmic calcium ion levels, or a change in second messenger levels such as IP3 can be used to assess G-protein coupled receptor function.
• IP3 can be measured using various commercially available kits. Some exemplary kits to detect the generation of IP3 use antibodies specific for IP3 which can detect IP3 in a cell lysate in a western blot or an ELISA; alternatively the antibodies are fluorescently labeled and detected using a plate reader.
• Modulation of receptor activity can be assayed by measuring changes in intracellular Ca2+ levels, which change in response to modulation of the TAS2R signal transduction pathway via administration of a molecule that associates with a TAS2R protein. Changes in Ca2+ levels are optionally measured using fluorescent Ca2+ indicator dyes and fluorometric imaging.
• assays for G-protein coupled receptors include cells that are loaded with ion or voltage sensitive dyes to report receptor activity. Assays for determining activity of such receptors can also use known agonists and antagonists for other G-protein coupled receptors as positive or negative controls to assess activity of tested compounds. In assays for identifying modulatory compounds (e.g., agonists, antagonists, modulators), changes in the level of ions in the cytoplasm or membrane voltage will be monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively. Ion- sensitive indicators and voltage probes that may be employed are commercially available from a variety of sources. For G-protein coupled receptors, promiscuous G-proteins such as Gal5 and Gal 6 can be used in the assay of choice. Such promiscuous G-proteins allow coupling of a wide range of receptors.
• Activated GPCR proteins become substrates for kinases that phosphorylate the C-terminal tail of the receptor (and possibly other sites as well).
• agonists will promote the transfer of 32 P from gamma-labeled GTP to the receptor, which can be assayed with a scintillation counter.
• the phosphorylation of the C-terminal tail will promote the binding of arrestin-like proteins and will interfere with the binding of G-proteins.
• the kinase/arrestin pathway plays a key role in the desensitization of many GPCR proteins. For example, compounds that modulate the duration a taste receptor stays active would be useful as a means of prolonging a desired taste or cutting off an unpleasant one.
• Changes in ion flux may be assessed by determining changes in polarization (i.e., electrical potential) of the cell or membrane expressing a TAS2R protein.
• polarization i.e., electrical potential
• One means to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques, e.g., the "cell-attached" mode, the "inside-out” mode, and the "whole cell” mode. Whole cell currents are conveniently determined using standard methodology known in the art.
• Other known assays include: radiolabeled ion flux assays and fluorescence assays using voltage-sensitive dyes. Generally, the compounds to be tested are present in the range from 1 pM to 100 mM.
• Other assays can involve determining the activity of receptors which, when activated, result in a change in the level of intracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymes such as adenylate cyclase.
• cyclic nucleotide- gated ion channels e.g., rod photoreceptor cell channels and olfactory neuron channels that are permeable to cations upon activation by binding of cAMP or cGMP (see, e.g., Altenhofen et al., Proc. Natl. Acad. Sci. U.S.A.
• Cells for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-crated ion channel, GPCR phosphatase and DNA encoding a receptor (e.g., certain glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like), which, when activated, causes a change in cyclic nucleotide levels in the cytoplasm.
• a receptor e.g., certain glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like
• the changes in intracellular cAMP or cGMP can be measured using immunoassays.
• the method described in Offermanns & Simon, J. Biol. Chem. 270:15175-15180 (1995) may be used to determine the level of cAMP.
• the method described in Felley-Bosco et al., Am. J. Resp. Cell and Mol. Biol. 11:159-164 (1994) may be used to determine the level of cGMP.
• an assay kit for measuring cAMP and/or cGMP is described in U.S. Pat. No. 4,115,538, herein incorporated by reference.
• phosphatidyl inositol (PI) hydrolysis can be analyzed according to U.S. Pat. No. 5,436,128, herein incorporated by reference. Briefly, the assay involves labeling of cells with 3H-myoinositol for 48 or more hrs. The labeled cells are treated with a test compound for one hour. The treated cells are lysed and extracted in chloroform-methanol- water after which the inositol phosphates were separated by ion exchange chromatography and quantified by scintillation counting. Fold stimulation is determined by calculating the ratio of counts per minute (cpm) in the presence of agonist to cpm in the presence of buffer control. Likewise, fold inhibition is determined by calculating the ratio of cpm in the presence of antagonist to cpm in the presence of buffer control (which may or may not contain an agonist).
• cpm counts per minute
• buffer control which may or may not contain an agonist
• the effects of the test compounds upon the function of the polypeptides can be measured by examining any of the parameters described above. Any suitable physiological change that affects GPCR activity can be used to assess the influence of a test compound on the polypeptides disclosed herein.
• any suitable physiological change that affects GPCR activity can be used to assess the influence of a test compound on the polypeptides disclosed herein.
• the functional consequences are determined using intact cells, animals or animal behavior, one can also measure a variety of effects such as neurotransmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca , IP3, cGMP, or cAMP.
• Samples or assays that are treated with a test compound that is a potential TAS2R agonist are compared to control samples without the test compound, to examine the extent of modulation.
• Activation of a TAS2R protein is achieved when the TAS2R activity value relative to the control is 110%, optionally 150%, 200-500%, or 1000-2000%.
• Samples or assays that are treated with a known agonist and a test compound that is a potential TAS2R antagonist are compared to control samples treated with the known agonist without the test compound, to examine the extent of modulation.
• the control samples are assigned a relative value of 100%.
• Inhibition of a TAS2R protein is achieved when the TAS2R activity value relative to the control is about 90%, optionally 50%, optionally 25-0%.
• TAS2R receptor activity or expression also may be identified, for example, by incubating a putative modulator with a cell containing a TAS2R polypeptide or polynucleotide and determining the effect of the putative modulator on TAS2R receptor activity or expression.
• the putative modulator must alter the measured interaction by an amount sufficient to achieve a statistically significant difference between the responses in the presence vs. the absence of the putative modulator.
• Statistical significance can be determined by any appropriate statistical test known in the art, such as a t-test.
• the p- value is at least 0.05, at least 0.01, or at least 0.001.
• the selectivity of a compound that modulates the activity of TAS2R receptor can be evaluated by comparing its effects on TAS2R receptor to its effect on other TAS2R receptors.
• Selective modulators may include, for example, antibodies and other proteins, peptides, or organic molecules that specifically bind to a TAS2R polypeptide or a TAS2R receptor-encoding nucleic acid.
• Compounds identified as modulating TAS2R receptor activity may be further tested in other assays including in vivo models, in order to confirm or quantitate their activity.
• TAS2R polynucleotides and polypeptides, and their homologs are useful tools for identifying taste receptor expressing cells, for taste perception, and for examining taste transduction.
• TAS2R family member- specific reagents that specifically hybridize to TAS2R nucleic acids, such as TAS2R probes and primers, and TAS2R specific reagents that specifically bind to a TAS2R protein, e.g., TAS2R antibodies are used to examine taste cell expression and taste transduction regulation.
• a TAS2R antibody can be used to identify and/or isolate feline taste cells expressing the particular TAS2R from a mixed feline cell population.
• polynucleotide probes disclosed herein may be used in tissue distribution studies and diagnostic assays.
• kits for screening for modulators of TAS2R family members can be prepared from readily available materials and reagents.
• such kits can comprise any one or more of the following materials: TAS2R nucleic acids or proteins, reaction tubes, and instructions for testing TAS2R activity.
• the kit contains a biologically active TAS2R receptor.
• kits and components can be prepared, depending upon the intended user of the kit and the particular needs of the user.
• any technique known in the art can be used. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides disclosed herein. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens. In one embodiment isolated DNA sequences encoding a monoclonal antibody or a binding fragment thereof are obtained by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246:1275-1281 (1989).
• Monoclonal antibodies and polyclonal sera can be collected and titered against the protein immunogen in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
• an immunoassay for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
• polyclonal antisera with a titer of 10 4 or greater are selected and tested for their cross reactivity against non-TAS2R proteins, or even other TAS2R family members or other related proteins from other organisms, using a competitive binding immunoassay.
• Specific polyclonal antisera and monoclonal antibodies will usually bind with a K ⁇ j of at least about 0.1 mM, more usually at least about 1 ⁇ , specifically at least about 0.1 ⁇ or better, and more specifically 0.01 ⁇ or better.
• Immunoassays can be used to detect, qualitatively or quantitatively, a fTAS2R, e.g., to identify taste receptor cells, especially bitter taste receptor cells, and variants of TAS2R family members.
• the anti-fTAS2R antibodies can also be used to isolate feline taste cells from a mixed population of cells obtained from a feline.
• isolation of the feline taste cells bound to the anti-fTAS2R antibody can be achieved by flow cytometry. Other methods known in the art can also be used.
• taste behavior can be determined in a short term assay which directly measures taste preferences by counting licking responses of an animal, e.g., a mouse, using a multi-channel gustometer (e.g., the Davis MS160-Mouse gustometer, DiLog instruments, Tallahassee, FL).
• the mean rate that a mouse will lick a tastant relative to their sampling of an appropriate control indicates whether the stimulus is appetitive, neutral or aversive.
• the change in intake of a palatable stimulus can be evaluated in the presence of the test stimulus to assess enhancement or suppression of the palatable stimulus.
• animals can be trained to discriminate qualitatively distinct stimuli using operant testing methods known in the art. These animals can then be used to determine qualitative similarity between two stimuli, regardless of palatability or preference.
• Electrodes may be attached to these brain areas and animals tested in an awake or anesthetized state.
• positron emission tomography PET
• electroencephalography may be used to monitor neural activity associated with appetitive or aversive taste responses.
• Such methods may also be used to evaluate the impact of various factors such as age, experience or nutritional state on neural activity elicited by stimuli identified in cell-based experiments to modify receptor function.
• kits comprising at least one composition, polypeptide, or nucleic acid disclosed herein, optionally contained in a single package.
• the kits may optionally include, e.g., instructions for use of the kit components in detecting a fTAS2R receptor or a polynucleotide encoding a fTAS2R receptor, or compounds altering the activity of a TAS2R receptor.
• the kit comprises at least one anti-TAS2R antibody disclosed herein and reagents for detecting a complex between the antibody and the TAS2R antigen.
• the kit can include a buffer that enables binding reaction between the antibody and the TAS2R antigen in a biological sample, or components for producing the buffer.
• TAS2R polypeptides can be assessed using a variety of in vitro and in vivo assays to determine functional, chemical, and physical effects, e.g., measuring ligand binding (e.g., radioactive ligand binding), second messengers (e.g., cAMP, cGMP, IP3, DAG, or Ca 2+ ), ion flux, phosphorylation levels, transcription levels, neurotransmitter levels, and the like.
• ligand binding e.g., radioactive ligand binding
• second messengers e.g., cAMP, cGMP, IP3, DAG, or Ca 2+
• ion flux e.g., phosphorylation levels
• transcription levels e.g., phosphorylation levels
• neurotransmitter levels e.g., neurotransmitter levels
• assays can be used to test for inhibitors and activators of TAS2R family members.
• modulators of taste transduction activity are useful for customizing taste perception, for example
• the TAS2R protein of the assay will typically be selected from a polypeptide having a sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26; a conservatively modified variant of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26; or a sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO
• the polypeptide of the assays will comprise a domain of a TAS2R protein, such as an extracellular domain, transmembrane region, transmembrane domain, cytoplasmic domain, ligand binding domain, subunit association domain, active site, and the like.
• a TAS2R protein such as an extracellular domain, transmembrane region, transmembrane domain, cytoplasmic domain, ligand binding domain, subunit association domain, active site, and the like.
• Either the TAS2R protein or a domain thereof can be covalently linked to a heterologous protein to create a chimeric protein used in the assays described herein.
• the polypeptide has a domain from SEQ ID NO: 18 or SEQ ID NO:22.
• TAS2R polypeptides as described above, either recombinant or naturally occurring.
• the protein can be isolated, expressed in a cell, expressed in a membrane derived from a cell, expressed in tissue or in an animal, either recombinant or naturally occurring.
• sections or dissociated cells from a TAS2R-expressing tissue, transformed cells, or membranes can be used.
• Assays may also be prepared using TAS2R polypeptides in artificial / synthetic membrane systems.
• Taste transduction can also be examined in vitro with soluble or solid state reactions, using a full- length TAS2R-GPCR or a chimeric molecule such as an extracellular domain or
• ligand-binding domains of the protein of interest can be used in vitro in soluble or solid state reactions to assay for ligand binding.
• a chimeric receptor will be made that comprises all or part of a TAS2R polypeptide as well an additional sequence that facilitates the localization of the TAS2R to the membrane, such as a rhodopsin, e.g., an N-terminal fragment of a rhodopsin protein.
• a rhodopsin e.g., an N-terminal fragment of a rhodopsin protein.
• the compounds tested as modulators or ligands of a TAS2R family member can be any compound, including small molecules, or more complex molecules such as biological molecules, for example a protein, sugar, nucleic acid or lipid.
• modulators can be genetically altered versions of a TAS2R gene.
• any chemical compound can be used as a potential modulator or ligand in the assays and methods disclosed herein.
• the compounds can be dissolved in aqueous or organic solutions (for example, DMSO solutions).
• the assays are designed to screen libraries of chemicals, including large libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays).
• a flavor composition is a composition which can be added to an edible composition for an animal to improve acceptance of the edible composition for consumption by the animal.
• edible compositions include foods, treats, nutritional
• the edible composition can be in the form of a tablet, capsule, caplet, edible film, wet food, liquid food, treat or kibble.
• a flavor composition comprises a compound that is an agonist, antagonist, or modulator of a feline TAS2R receptor.
• the flavor composition further comprises a palatability enhancer; optionally, an adhesive compound to help adhere the flavor composition to the edible composition; and optionally, a compound for providing color or aroma for a human, wherein the flavor composition is a solid, liquid, powder, paste, gel, sprayable formulation or spreadable formulation.
• the flavor composition is a coating composition and further comprises the adhesive compound. Alteration or masking of perceived bitterness of an edible composition can be tested using any of the behavioral assays for palatability disclosed herein, such as a standard two bowl comparison.
• Base food composition refers to an animal food combinable with the flavor composition.
• the animal food is formulated for felines, and includes dry food, canned food, semi-dry food, edible treats, and the like, and
• basal food composition may be employed as long as the food is acceptably consumable by a recipient (such as an animal, particularly a feline) in an amount so that the animal receives a normal daily ration providing the known essential nutrients.
• a basal food composition may be uncoated, or may be coated, for example, with a coating comprising lipids. If desired, feeding may be carried out by feeding the animal one or more times per day.
• the flavor composition is combined with an edible composition, for example a basal food composition (e.g. for a feline), in an amount effective to impart increased palatability of the edible composition to the animal.
• an edible composition for example a basal food composition (e.g. for a feline)
• effective amounts of such flavor compositions are readily determined by one of ordinary skill in the art without undue experimentation, particularly in view of the general guidance provided below.
• the flavor composition may be combined with a basal food composition in a manner such that the flavor composition is incorporated into the basal food composition.
• incorporated it is meant that the flavor composition is intimately associated with the edible composition and does not become substantially dissociated, for example, during normal storage conditions.
• the flavor composition is substantially uniformly dispersed throughout the edible composition. In other embodiments, the distribution of the flavor composition may intentionally not be uniform. In such
• the flavor composition may provide bits or pieces that are intermixed with the basal food.
• the flavor composition may be deposited in the edible composition in an amount effective to provide about 0.5 wt to about to about 3 wt , specifically about 0.8 wt to about 2.5 wt , and more specifically about 1 wt to about 2 wt of the dry weight of the edible composition.
• the flavor composition is deposited on the surface of the edible composition, for example in the form of a coating.
• Coating the edible composition includes the topical deposition of the flavor composition onto the surface of the edible composition, such as by spraying, dusting, and the like.
• the coating comprising the flavor composition may comprise one or more fats to help adhere the flavor composition to the surface. It may further or alternatively comprise other components useful to facilitate adhesion of the flavor composition to the surface of the edible composition. It is possible, although not required, that the flavor composition be coated onto the edible composition uniformly or that uniform distribution of the flavor composition be achieved, for example, by repeatedly tumbling the coated food. One or more coats may be applied.
• the flavor composition may be deposited onto the surface of the edible composition in an amount effective to provide about 0.5 wt to about to about 3 wt , specifically about 0.8 wt to about 2.5 wt , and more specifically about 1 wt to about 2 wt of the dry weight of the basal animal food composition.
• the flavor composition may be both dispersed in and coated onto the edible composition, such as a dry animal food composition.
• the finished animal food product is packaged for sale and ultimately fed to the animal.
• the flavor composition may be packaged for combination with a food prior to serving.
• the animal is a feline.
• the flavor composition may further comprise an additional palatability enhancer such as a flavoring.
• Suitable flavorings include, for example, a vegetable flavoring, a meat flavoring, (e.g., liver flavoring), a cheese flavoring, yeast, sodium pyrophosphate, a fat, an acid phosphate, a phosphate salt, and/or other food or flavor ingredients utilized by the flavor industry in order to improve palatability.
• Suitable meat flavorings include, for example, meat-derived flavorings (e.g., beef, pork, bacon, lamb, ham, fish, chicken, turkey, and/or other poultry flavoring).
• Palatability or acceptance of a food refers to the overall willingness of an animal, such as a feline, to eat a certain food. Developing preferred flavorants and palatability enhancers for animals such as pets is subjective. Flavorants which work for humans do not always work with felines. Similarly, a flavorant which is effective with one animal species may not work as well with a different animal species. The skilled artisan will appreciate that palatability testing is routinely used to determine preferences for animals with respect to food and flavorants. For purposes herein, such palatability testing will be effective and straight forward to implement for testing preferences for flavorants for any animal, including felines.
• the flavor composition is a palatant for a feline food and the flavor composition exhibits improved palatability for the feline compared to the feline food without the flavor composition, as measured by improved consumption of the feline food comprising the palatant compared to the animal food in the absence of the palatant.
• the flavor composition may be used as a liquid flavor in either unconcentrated or concentrated form. If the flavor composition is to be a dry flavor composition, the flavor composition may be dried in a suitable dryer such as, for example, a spray dryer, or an oven.
• the flavor composition may comprise a variety of other useful components, for example, maltodextran, gum, or a combination which may be useful for providing the composition with one or more preferred functionalities such as the ability to bind to a food or to retain a desired texture, viscosity, flowability, color, aroma or the like. Such components and their uses will be readily understood by the skilled food scientist.
• Palatability testing can be performed by a standard two bowl comparison.
• each animal is presented with two bowls of food, each containing a measured amount of either a control ration or a test ration.
• the control and test rations contain the same basal compositions. The animal is allowed to select the food it prefers. The amount of food eaten from each bowl is measured. A direct comparison of the amount eaten from the two rations gives a reliable indication of relative palatability.
• a feline may be given two bowls with equal amounts of food, one containing the flavor composition to be tested and the other not containing the flavor composition. The amount of food in the two bowls is weighed prior to giving them to the feline. During the test, steps should be taken to ensure that the feline does not finish one bowl and continue to the other because it is still hungry. This can be accomplished, for example, by limiting the time of the feline with the two bowls, or by providing enough food in each bowl to fully satisfy the feline.
• the two bowls are weighed again to determine the amount of food eaten from each bowl. If more food is eaten from the bowl with the test flavor composition (bowl A), the ingestion ratio is recorded as a positive value to indicate that the flavor composition had a positive effect on the animal preference. If more food was eaten from the bowl with the control food (bowl B), the ratio is recorded as a negative value to indicate that the flavor composition did not perform as well as the control food.
• the flavor compositions are applied to a dry basal feline food composition and multiple felines, e.g., ten, are fed for a period of time (e.g. two days).
• the bowl position is changed daily to eliminate bias due to the animals showing a preference for right or left placement of the bowls.
• the average preference is calculated as the average value of each day for the duration of the test period.
• an IR value close to 0.5 indicates equal preference.
• IR values greater than 0.5 and typically above 0.55 indicate preference.
• the degree of preference estimation based on IR scores can be determined by number of animals used and statistical analysis of the data.
• a method for making the flavor composition for coating or incorporating into an edible composition to be administered to an animal is disclosed.
• the method comprises mixing an agonist, an antagonist, or a modulator of a feline TAS2R receptor polypeptide; optionally, a palatability enhancer;
• a compound to help adhere the flavor composition to the edible composition optionally, a compound for providing color or aroma with an ingredient selected from the group consisting of meat products, meat by-products, fish products, fish by-products, dairy products, dairy by-products, sources of microbial proteins, vegetable proteins, carbohydrates and amino acids carrier to obtain a flavor composition, wherein the flavor composition is a liquid, solid, powder, paste, gel, spreadable formulation, granule, or sprayable formulation.
• an agonist or an antagonist of the feline TAS2R receptor polypeptide is mixed into the composition.
• the agonist is denatonium, aloin, or PTC and the antagonist is probenecid.
• liquid flavor composition for example, commercially available liquid ingredients are combined in a mixer with an agonist, an antagonist, or a modulator of a feline TAS2R receptor polypeptide. Wet ingredients are ground or emulsified to a slurry and the liquid ingredients are combined therewith.
• a commercially available protease may be added to the slurry to hydrolyze proteins, and later inactivated with heat, acid or another method. Preservatives such as sorbic acid can also be added. Water is added to adjust the viscosity and the solids content of the slurry to facilitate spray application.
• a dry formulation of the flavor composition can be prepared by combining commercially available dry ingredients, including amino acids, inorganic salts and organic materials with an agonist, an antagonist, or a modulator of a feline TAS2R receptor polypeptide in the desired proportions in a batch mixer and blending to homogeneity prior to drying.
• wet and dry ingredients are combined by mixing the wet ingredients with all or some of the dry ingredients in a mixer until a homogenous mixture is formed.
• the mixture is dried by evaporation or lyophilization, for example, to form a dry, powdery product that is then blended with any remaining dry ingredients in a tumbler until a homogeneous mixture is formed.
• the method comprises contacting an edible composition or a component thereof with a fTAS2R receptor polypeptide disclosed herein for a time sufficient to reduce the amount of a bitter compound from the edible composition or component thereof.
• the time to reduce the amount of the bitter compound can be determined by one of skill in the art.
• the contacting can occur in a continuous, semi-continuous, or batch process.
• the edible composition is for a feline
• the method comprises adding a compound to an edible composition to decrease the palatability of the edible composition to an animal, wherein the compound is an agonist or a positive modulator of a feline bitter taste receptor.
• the palatability is decreased to an extent that a feline consumes 10 to 30% less of the edible compositionwith the added compound than the edible compositionwithout the added compound.
• the decrease in palatability is measured as decrease in calories of edible composition consumed, weight of edible composition consumed, or volume of edible composition consumed.
• a method of formulating an edible composition with enhanced palatability for an animal is disclosed.
• the method comprises determining the presence of a compound which is an agonist, antagonist, or modulator of a feline TAS2R receptor polypeptide in an edible composition; and enhancing palatability of the edible composition by if the compound is an agonist or a positive modulator, increasing the amount of an antagonist for the receptor in the edible composition or reducing the amount of the compound in the edible composition, or if the compound is an antagonist or a negative modulator, increasing the amount of the compound in the edible composition.
• the amount of the compound can be increased by applying a flavor composition comprising the compound to the edible composition such that the flavor composition is incorporated into or a leastleas partially coats the edible composition.
• an animal e.g. a feline
• a human or other animal may be in need of a bitter compound (e.g. a pharmaceutical, a nutrient, or the like) and that it can be challenging to administer the compound to the animal.
• the method comprises administering a feline edible composition to a feline, wherein the edible composition comprises a feline bitter compound and a compound that alters perceived bitterness of the edible composition, masks the bitter compound in the edible composition, or acts as an agonist, antagonist, or modulator of a feline TAS2R receptor in the feline to alter bitter taste perception by the feline.
• the bitter compound comprises a therapeutic, a nutritional supplement or an oral care product.
• a nutritional supplement refers to a supplement intended to provide nutrients that may otherwise not be consumed in sufficient quantities and includes vitamins, minerals, fiber, probiotics, fatty acids, and amino acids.
• a therapeutic or pharmaceutical refers to a compound, element, or mixture that when administered to a subject, alone or in combination with another compound, element, or mixture, confers, directly or indirectly, a physiological effect on the subject.
• An oral care product refers to a product used to promote healthy teeth, gums, freshen breath or prevent or treat oral disease.
• the method comprises contacting a feline food composition or a component thereof with a TAS2R receptor polypeptide herein for a time sufficient to remove a bitter compound from the food product or component.
• the TAS2R receptor is bound to a solid support that can be separated from the food composition.
• the contacting is a continuous operation.
• the food composition is contacted with a plurality of TAS2R receptor polypeptides.
• the method comprises determining the presence of one or more bitter compounds in an edible composition; determining a bitterness profile of the edible composition based on the one or more bitter compounds determined to be present; and adding a compound to or removing a compound from the edible composition to enhance the palatability of the edible composition, wherein the compound alters the bitterness profile of the edible composition, masks one or more of the bitter compounds present in the edible composition, or acts as an agonist, antagonist or modulator of a feline bitter taste receptor.
• adding the compound to the edible composition comprises applying a coating solution to the edible composition comprising the compound such that a coating at least partially surrounds the feline edible composition.
• the edible composition is a basal food, a flavor composition, a treat, a therapeutic, or a nutritional supplement.
• the presence of a bitter compound in a edible composition can be determined by a method disclosed herein, or by any other method known in the art.
• a bitterness profile of an edible composition refers to an enumeration of bitter compounds determined to be present in the edible composition, and optionally further includes the amount of a given bitter compound in the edible composition.
• the edible composition is for a feline.
• the repellent compositon can comprise a feline TAS2R receptor agonist or positive modulator in a sufficient amount to elicit rejection, for example at least 0.05% to about 30% by weight, and optionally aromatics or perfumes such as rosemary oil, mint oil, cinnamon oil, limonene, or eugenol, and one or more inert ingredients such as a liquid diluents, carriers, thickeners, surface-active agents, preservatives, aromatics, deodorizers, antibacterial agents, antifungal agents, antimicrobial agents, biocide agents, and one or more of several types of adjuvant including, but not limited to, wetting agents, spreading agents, sticking agents, foam retardants, buffers and acidifiers.
• aromatics or perfumes such as rosemary oil, mint oil, cinnamon oil, limonene, or eugenol
• inert ingredients such as a liquid diluents, carriers, thickeners, surface-active agents, preservatives, aromatics, deodor
• Suitable liquid diluents include water, petroleum distillates, or other liquid carriers with or without surface active agents.
• carriers include bentonite, fullers earth, additional clays, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth, vermiculite, highly dispersed silicic acid, alumina and silicates, calcite, marble, pumice, sepiolite and dolomite, inorganic and organic meals, sawdust, coconut shells, corn cobs and tobacco stalks.
• the repellent compositon can further comprise a propellant gas for dispensing as a spray, such as Figen 11/12 or propane/butane, e.g. in a ratio of 15:85.
• the fTAS2R agonist is denatonium, aloin, or PTC.
• TAS2R feline bitter taste receptor
• feline TAS2R genes were identified, by querying the NCBI Felis catus whole genome shotgun contigs database with human bitter receptor gene sequences. Human gene sequences used are identified by NCBI Gene IDs in Table 2. Table 2. NCBI Gene IDs for all functional and pseudogene hTAS2Rs used to identify feline bitter genes.
• Predicted functional genes were identified based on a set of rules selected to include a protein which is approximately 300 amino acids in length, the start site and stop site are in similar locations as the human protein when the blasted sequences are aligned, then the sequence was compared to the sequence of the orthologous canine bitter gene to verify that similarity was reasonable. Table 3 identifies canine bitter gene sequences used.
• Table 4 summarizes the full length feline genes identified. The % protein similarity between the feline gene and closest human homologue is presented in the table. Table 4. Full length Feline Bitter Receptor Genes Identified
• TAS2R38 The process of amplification and cloning of a representative gene, TAS2R38, is briefly described.
• the fTAS2R38 sequence was amplified via PCR using Easy A High Fidelity PCR Cloning Enzyme (Agilent, Santa Clara CA), custom primers, and feline genomic DNA as a template.
• the resulting PCR product was ligated into the pGEM-T Easy Vector (Promega, Madison WI).
• DH5-a bacterial cells (Life Technologies; Carlsbad, CA) were transformed with the vector.
• Plasmid was purified from cultures of the transformed DH5-alpha cells using the Plasmid Miniprep Kit (Omega BioTec, Norcross, GA). Sequencing of the gene using the purified plasmid DNA was performed by the Core DNA Sequencing Facility at the University of Illinois, Champaign-Urbana. The sequencing data was analyzed with SeqMan Pro (DNAStar, Madison WI) to determine the quality of the data and to edit the data.
• the feline gene is named after its homologous human counterpart, as shown in Table 7. However for a feline gene similar to many human genes, such as fTAS2R43, the feline gene is named as its homologous canine counterpart.
• TM transmembrane
• FIG. 2 A sequence alignment of human TAS2R38 polypeptide (SEQ ID NO:31) and feline TAS2R38 polypeptide (SEQ ID NO: 18) determined from sequencing genomic DNA of five individual cats is shown in Fig. 2. Amino acids in hTAS2R38 that differ from those in fTAS2R38 are boxed in Fig. 2. The positions of the human polymorphisms known to affect taste perception of 6-n-propylthiouracil (PROP), A49P, V262A, I293V (where AVI is a non- taster and PAV is a taster) are shaded grey in Fig. 2.
• PROP 6-n-propylthiouracil
• A49P A49P
• V262A V262A
• I293V where AVI is a non- taster and PAV is a taster
• This example describes generation of an expression vector for a representative feline bitter receptor, TAS2R38. An analogous process is conducted for each of the TAS2R receptors.
• the full length gene of feline TAS2R38 was amplified by polymerase chain reaction (PCR) using gene-specific primers that span the entire coding region.
• the TAS2R38 cDNA was subcloned into an expression cassette based on the plasmid/expression vector pcDNA3.
• ID- V5His (Life Technologies, Carlsbad, Calif., US), which contains within its multiple cloning sites the nucleotide sequence coding for the FLAG epitope to allow surface detection of the receptor, then the first 45 amino acids of the rat somatostatin receptor subtype 3 (RSS tag) to facilitate cell surface targeting of the transgene, and the nucleotide sequence coding for the herpes simplex virus (HSV) glycoprotein D epitope (HSV epitope) for facilitating immunocytochemical detection (HSV Tag) on the carboxy terminus.
• HSV herpes simplex virus
• HSV epitope herpes simplex virus glycoprotein D epitope
• the nucleic acid sequences encoding the FLAG tag, RSS tag, TAS2R38, and the HSV tag were fused, in that order, in frame to create a construct to allow translation into the receptor protein.
• the resulting receptor cDNA in the expression vector encodes the joined amino acid sequences of TAS2R38 preceded by the RSS tag and followed by the HSV tag.
• the expression vector including the construct is called pcDNA3.11D- FLAGV5His-TAS2R38 and allows for expression of the TAS2R38 protein (SEQ ID NO:18).
• 60,000 cells per well were plated on poly lysine coated, black 96 well plates with clear bottoms (Costar). The following day the cells were transfected with 150ng TAS2R38 expression vector, e.g., pcDNA3.1D-FLAGV5His (Invitrogen) along with 45ng of Gal6 chimera containing the last 44 amino acids of rat gustducin (Gal6gust44) with 0.5ul Lipofectamine 2000 (Invitrogen) per well. Cells were then incubated 22-44 hours at 37°C 5% C0 2 .
• TAS2R38 expression vector e.g., pcDNA3.1D-FLAGV5His (Invitrogen) along with 45ng of Gal6 chimera containing the last 44 amino acids of rat gustducin (Gal6gust44) with 0.5ul Lipofectamine 2000 (Invitrogen) per well. Cells were then incubated 22-44 hours at 37°C 5% C0 2 .
• fTAS2R38 was evaluated by testing for the presence of a functional response to a known hTAS2R38 ligand (e.g., PTC), determined via automated calcium imaging using a Fluo-4AM (Life Technologies Corporation) Calcium Assay.
• Fluo- 4AM is a fluorescent indicator of intracellular calcium dynamics (change in concentration) and allows monitoring changes in the calcium concentration, particularly an increase in response to receptor activation occurring after agonist exposure.
• the fTAS2R38 was activated 81% over baseline by 100 ⁇ PTC, but was not stimulated by 30 ⁇ PROP.
• the fTAS2R43 was activated 45% over baseline by 300 ⁇ aloin, and 17% over baseline by 1 mM denatonium, but was not stimulated by 6.7 mM saccharin. Furthermore, the responses to PTC, denatonium and aloin were inhibited by ImM probenecid.
• Testing for a functional response of each of the other fTAS2Rs disclosed herein can be performed by analogous methods using known ligands to a corresponding homolog of each fTAS2R.
• the fTAS2R38 cDNA is subcloned into an expression cassette based on the plasmid/expression vector pcDNA3.1Zeo ( Life Technologies,
• the nucleic acid sequences encoding the RSS tag, HSV tag, and fTAS2R38 are fused, in that order, in frame to create a construct to allow translation into the receptor protein.
• the resulting receptor cDNA in the expression vector encodes the joined amino acid sequences of fTAS2R38 preceded by the RSS tag and the HSV tag.
• the expression vector including the construct is called pcDNA3.1Zeo-TAS2R38 and allows for expression of the fTAS2R38 protein (SEQ ID: 18).
• Cell lines that stably express a desired fTAS2R disclosed herein are generated by transfecting the appropriate expression vector, e.g., pcDNA3.1Zeo-TAS2R38, constructed as described above in Ex. 2A into a eukaryotic host cell line (Life Technologies Cat# R700-07) transformed with the Gal 6 chimera containing the last 44 amino acids of rat gustducin (G[alpha] 16-gustducin 44 cells) as described in WO2004/055048 (US7919236).
• the appropriate expression vector e.g., pcDNA3.1Zeo-TAS2R38, constructed as described above in Ex. 2A into a eukaryotic host cell line (Life Technologies Cat# R700-07) transformed with the Gal 6 chimera containing the last 44 amino acids of rat gustducin (G[alpha] 16-gustducin 44 cells) as described in WO2004/055048 (US7919236).
• the G[alpha] 16-gustducin 44 cells are plated in a 6-well plate at a density of 900,000 cells per well and grown overnight in a selective growth media (DMEM with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin).
• DMEM selective growth media
• the cells are re-plated in selective growth medium (DMEM with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, 200 ⁇ g/ml G418 and 200 ⁇ g/ml zeocin) and are further incubated in a humidified atmosphere (37 C, 5% C02).
• DMEM selective growth medium
• fTAS2R38 is evaluated by testing for the presence of a functional response to a knownhTAS2R38 ligand (e.g., PTC and PROP), determined via automated calcium imaging using the Fluo-4AM (Life Technologies Corporation) Calcium Assay.
• Fluo-4AM is a fluorescent indicator of intracellular calcium dynamics (change in concentration) and allows monitoring changes in the calcium concentration, particularly an increase in response to receptor activation occurring after agonist exposure.
• One clone is selected resulting in the G[alpha] 16-gustducin 44/TAS2R38 cell line.
• the G[alpha] 16- gustducin 44/TAS2R38 cell line was stimulated 90% over baseline in the presence of 100 ⁇ PTC but was not stimulated with 30 ⁇ PROP.
• Identification of agonists, antagonists and modulators of feline TAS2R38 receptor is performed by a cell-based screening assay in which the effect of a test compound on cells transfected with feline TAS2R38 and Gal6gust44 is compared against the effect of the test compound on untransfected cells.
• the cells Prior to the screening assay, the cells are loaded with the calcium sensitive dye Fluo-AM (Life Technologies) for one hour at 37 °C as described in Example 2B. The dye is washed out and the cells are assayed in Hank's Balanced Salt Solution (HBSS; Life
• test compounds containing 20mM HEPES in a Flexstation ⁇ (Molecular Devices). A 10 fold dilution series O.OlmM - ImM of test compounds is used to stimulate the cells.
• PTC a known human TAS2R38 agonist, is among the test compounds
• the stimuli are injected and monitored for 100-180 seconds. Data is analyzed and graphed as a percentage over the baseline signal, which is the reading prior to stimulation. Stimulation of the fTAS2R38 expressing cell line by a particular test compound is considered to occur when the signal is greater than both the signal from the buffer alone in the receptor expressing cell line and the signal from the un-transfected cell line sample injected with the test compound.
• Exemplary dry flavor compositions for an animal comprising an agonist, an antagonist, or a modulator of a feline TAS2R receptor disclosed herein are made in general accordance with the following formulation. Table 9. Dry Flavor Composition
• the identified agonist in the dry flavor composition is denatonium, aloin, or PTC or the identified antagonist is probenecid.
• Exemplary liquid flavor compositions for an animal comprising an agonist, an antagonist, or a modulator of a feline TAS2R receptor are made in general accordance with the following formulation.
• citric acid 0%-5% specialized natural flavor spikes 0%-5%
• the identified agonist in the liquid flavor composition is denatonium, aloin, or PTC or the identified antagonist is probenecid.
• An exemplary repellent composition in the form of an aerosol for spraying onto an object to deter companion cats from chewing or eating the object is made by formulating 50% active ingredient solution, the active ingredient being a feline TAS2R agonist or positive modulator, with 50% of a propellant gas such as Frigen 11/12 (a halogenated hydrocarbon) or propane/butane (e.g., in a 15:85 ratio) in an aerosol can.
• a propellant gas such as Frigen 11/12 (a halogenated hydrocarbon) or propane/butane (e.g., in a 15:85 ratio) in an aerosol can.
• the active ingredient solution consists of from about 0.5% to about 30 % by weight of a feline TAS2R agonist or positive modulator dissolved in a liquid diluent, e.g., water, optionally 0.5 - 1.5% of an aromatic or a perfume, and up to 29.5% isopropanol.
• a liquid diluent e.g., water
• the feline TAS2R agonist is denatonium, aloin, or PTC.
• Embodiment 1 An isolated feline TAS2R (fTAS2R) receptor polypeptide comprising an extracellular domain of a feline TAS2R receptor; a transmembrane region of a feline TAS2R receptor, or an intracellular domain of a feline TAS2R receptor, wherein the fTAS2R receptor comprises a sequence selected from SEQ ID NO: 18, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26, wherein the isolated feline TAS2R (fTAS2R) receptor polypeptide does not consist of the amino acid sequence of SEQ ID NOs: 2, 4, 6, or 10.
• Embodiment 2 The polypeptide of embodiment 1 wherein: the extracellular domain of the feline TAS2R receptor polypeptide comprises: amino acids 1, 68-84; 146-179; or 249-257 of SEQ ID NO:2; amino acids 1-10, 73-88; 151-186; or 256-264 of SEQ ID NO:4; amino acids 1-8; 72-88; 150-186; or 256-265 of SEQ ID NO:6; amino acids 1-2; 69- 87; 151-183; or 253-261 of SEQ ID NO:8; amino acids 1-8; 72-88; 150-187; or 257-265 of SEQ ID NO:10; amino acids 1-6; 72-88; 150-183; or 253-262 of SEQ ID NO:12; amino acids 1; 69-87; 150-181; or 251-260 of SEQ ID NO:14; amino acids 1-8; 69-88; 150-185; or 252- 261 of SEQ ID NO:16; amino acids 1-17: 83
• transmembrane region of the feline TAS2R receptor polypeptide comprises: amino acids 2-
• Embodiment 3 The polypeptide of embodiment 1 or 2 comprising a transmembrane region 2, a transmembrane region 3, a transmembrane region 4, a
• transmembrane region 5 a transmembrane region 6, and a transmembrane region 7, wherein each transmembrane region comprises at least 20 consecutive amino acids of the
• each transmembrane region comprises at least 20 consecutive amino acids of the corresponding transmembrane region sequence independently selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26; or a transmembrane region 3, a transmembrane region 6, and a transmembrane region 7, wherein each transmembrane region comprises at least 20 consecutive amino acids of the corresponding transmembrane region sequence independently selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and
• Embodiment 4 The polypeptide of any one of embodiments 1-3, further comprising a heterologous polypeptide.
• Embodiment 5 The polypeptide of embodiment 4, wherein the heterologous polypeptide is linked to the amino terminus or the carboxy terminus of the feline TAS2R receptor polypeptide.
• Embodiment 6 The polypeptide of any one of embodiments 1-5 comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26.
• Embodiment 7 The polypeptide of any one of embodiments 1-6 consisting of the amino acid sequence of SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26.
• Embodiment 8 The polypeptide of any one of embodiments 1-7 that is non- naturally occurring.
• Embodiment 9 The polypeptide of any one of embodiments 1-8 having fTAS2R receptor activity or binding a ligand of an fTAS2R receptor.
• Embodiment 10 The polypeptide of any one of embodiments 1-9, wherein the sequence is SEQ ID NO: 18.
• Embodiment 11 The polypeptide of any one of embodiments 1-10, wherein the fTAS2R is fTAS2R38 and amino acid 74 of SEQ ID NO: 18 is N.
• Embodiment 12 The polypeptide of any one of embodiments 1-11, wherein the extracellular domain comprises a sequence of at least 15 consecutive amino acids of extracellular domain 2 or 3 or of at least 8 consecutive amino acids of extracellular domain 4 of a fTAS2R receptor sequence; the transmembrane region comprises a sequence of at least 20 consecutive amino acids of a fTAS2R receptor sequence transmembrane region, and the intracellular domain comprise a sequence of at least 17 consecutive amino acids of a fTAS2R receptor sequence intracellular domain.
• Embodiment 13 A composition comprising at least two polypeptides of any one of embodiments 1-12
• Embodiment 14 An isolated polynucleotide encoding the polypeptide of any one of embodiments 1-12.
• Embodiment 15 An isolated polynucleotide encoding a feline TAS2R
• (fTAS2R) receptor polypeptide or fragment thereof comprising a nucleotide sequence selected from: the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26; a nucleotide sequence that hybridizes to the complement of the polynucleotide having SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
• Embodiment 16 A polynucleotide comprising at least 15 contiguous nucleotides of SEQ ID NO: 17, wherein the contiguous nucleotides contain nucleotide 220 and an A is present at nucleotide 220; or the complement of the nucleotide sequence.
• Embodiment 17 The polynucleotide of embodiment 16, comprising at least 20 contiguous nucleotides.
• Embodiment 18 The polynucleotide of embodiment 16 or 17, comprising at least 25 contiguous nucleotides.
• Embodiment 19 The polynucleotide of any one of embodiments 14-18, wherein the nucleotide sequence is codon-optimized for expression in a non-feline cell.
• Embodiment 20 The polynucleotide of embodiment 19, wherein the non-feline cell is Escherichia coliE., a Saccharomyces cerevisae cellyeast, a Drosophila melanogaster cell, a Caenorhabditis elegans cell, or a mammalian cell.
• the non-feline cell is Escherichia coliE., a Saccharomyces cerevisae cellyeast, a Drosophila melanogaster cell, a Caenorhabditis elegans cell, or a mammalian cell.
• Embodiment 21 The polynucleotide of embodiment 20, wherein the mammalian cell is a murine or human cell.
• Embodiment 22 The polynucleotide of any one of embodiments 14-21 that is non-naturally occurring.
• Embodiment 23 A composition comprising at least two polynucleotides of any one of embodiments 14-22.
• Embodiment 24 A primer pair for amplifying at least a portion of a nucleic acid encoding a feline TAS2R polypeptide.
• Embodiment 25 The composition of embodiment 24 comprising a primer pair selected from the primer pairs of Table 5.
• Embodiment 26 A feline TAS2R receptor polypeptide encoded by the polynucleotide of any one of embodiments 14-22.
• Embodiment 27 An expression vector comprising the polynucleotide of any one of embodiments 14-22.
• Embodiment 28 A host cell comprising the expression vector of embodiment 27.
• Embodiment 29 The host cell of embodiment 28 wherein the cell is a mammalian cell, a fish cell, a yeast cell, a bacterial cell, or an insect cell.
• Embodiment 30 The host cell of embodiments 28 or 29 wherein the cell is a human, murine, or feline cell.
• Embodiment 31 The host cell of embodiment 28 or 29 wherein the cell is a bacterial, insect, or yeast cell.
• Embodiment 32 A cell culture comprising at least one cell of any one of embodiments 28-31.
• Embodiment 33 An oligonucleotide comprising a nucleotide sequence of at least 15 and up to 100 contiguous nucleotides of SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25; or the complement of the nucleotide sequence.
• Embodiment 34 The oligonucleotide of embodiment 33, comprising at least 18 and up to 50 contiguous nucleotides.
• Embodiment 35 The oligonucleotide of embodiment 33 or 34, comprising at least 18 and up to 30 contiguous nucleotides.
• Embodiment 36 An isolated antibody or a fragment thereof, that specifically binds an fTAS2R receptor epitope of the polypeptide of any one of embodiments 1-12 and 26.
• Embodiment 37 A method for identifying a compound that interacts with a feline TAS2R receptor polypeptide comprising: contacting a polypeptide of any one of embodiments 1-12 and 26 with a test compound, and detecting interaction between the polypeptide and the test compound.
• Embodiment 38 The method of embodiment 37, wherein detecting interaction between the polypeptide and the test compound comprises measuring an electrical property, measuring a change in an ion concentration, measuring a change in protein conformation, measuring binding of the test compound to the polypeptide, measuring a change in phosphorylation level, measuring a change in transcription level, measuring a change in second messenger level, measuring a change in neurotransmitter level, measuring a change in a spectroscopic characteristic, measuring a change in a hydrodynamic (e.g., shape) property, measuring a change in a chromatographic property, or measuring a change in solubility.
• a hydrodynamic e.g., shape
• Embodiment 39 The method of embodiment 37 or 38, further comprising identifying the test compound as a compound that interacts with the receptor.
• Embodiment 40 A method for identifying a compound which modulates a feline TAS2R receptor polypeptide which comprises: contacting the polypeptide of any one of embodiments 1 to 12 and 24 with a TAS2R receptor ligand in both the presence and absence of a test compound in separate assays, and determining whether the test compound modulates binding of the ligand to the receptor polypeptide or activation of the receptor polypeptide by the ligand.
• Embodiment 41 The method of embodiment 40, wherein determining whether the test compound modulates binding of the ligand to the receptor or activation of the receptor by the ligand comprises measuring an electrical property, measuring an ion concentration, measuring a change in protein conformation, measuring a binding of the test compound to the polypeptide, measuring a change in phosphorylation level, measuring a change in transcription level, measuring a change in second messenger level, or measuring a change in neurotransmitter level.
• Embodiment 42 The method of embodiment 40 or 41 further comprising identifying the test compound as a modulator.
• Embodiment 43 The method of any one of embodiments 37 to 42, wherein the polypeptide is bound to a solid support, expressed in a host cell, in a bilayer membrane, in a lipid monolayer, or in a vesicle.
• Embodiment 44 A method of preparing an edible composition comprising contacting an edible composition or a component thereof with a polypeptide of any one of embodiments 1 to 12 and 26 for a time sufficient to reduce the amount of a bitter compound from the edible composition or component thereof.
• Embodiment 45 The method of embodiment 44 wherein the polypeptide is bound to a solid support that can be separated from the edible composition.
• Embodiment 46 The method of embodiment 44 or 45 wherein the contacting is a continuous operation, a semi-continuous operation, or a batch operation.
• Embodiment 47 The method of any one of embodiments 44-46 wherein the edible composition is a feline food composition, and the composition or a component thereof is contacted with a plurality of different polypeptides.
• Embodiment 48 A method of formulating an edible composition with enhanced palatability comprising determining the presence of a compound which is an agonist, antagonist, or modulator of a feline TAS2R receptor polypeptide of any one of embodiments 1 to 12 and 26 in an edible composition; and enhancing palatability of the edible composition by if the compound is an agonist or a positive modulator, increasing the amount of an antagonist for the receptor in the edible composition or reducing the amount of the compound in the edible composition, or if the compound is an antagonist or a negative modulator, increasing the amount of the compound in the edible composition.
• Embodiment 49 The method of embodiment 48 wherein increasing the amount of the compound comprises applying a flavor composition comprising the compound to the edible composition such that the flavor composition is incorporated into or at least partially coats the edible composition.
• Embodiment 50 The method of embodiment 48 or 49, wherein the edible composition comprises a food, a flavor composition, a treat, a pharmaceutical, an oral care material, a nutritional supplement, a chewable product, or a drinkable product.
• Embodiment 51 A method of administering a bitter compound to an animal in need thereof comprising administering an edible composition to an animal, wherein the edible composition comprises a bitter compound and a compound that is an agonist, antagonist, or modulator of a feline TAS2R receptor polypeptide of any one of embodiments 1 to 12 and 26 that alters acceptance of the edible composition by the animal compared to acceptance of the edible composition without the compound.
• Embodiment 52 The method of embodiment 51 wherein the bitter compound comprises a pharmaceutical, oral care material, a repellant, or a nutritional supplement.
• Embodiment 53 A method of preparing an edible composition for controlling palatability of the edible composition to an animal comprising adding a compound to an edible composition to decrease the palatability of the edible composition to an animal, wherein the compound is an agonist of or a positive modulator of a feline TAS2R receptor polypeptide of any one of embodiments 1 to 12 and 26.
• Embodiment 54 The method of embodiment 53 wherein the palatability is decreased to an extent that an animal to whom the edible composition is administered consumes 10 to 30% less of the edible composition with the compound than of the edible composition without the added compound.
• Embodiment 55 The method of embodiment 53 or 54 wherein the decrease is measured in calories of edible composition consumed, weight of edible composition consumed, or volume of edible composition consumed.
• Embodiment 56 A method for making a flavor composition for coating or incorporating into an edible composition to be administered to an animal comprising: mixing an agonist or an antagonist of a feline TAS2R receptor polypeptide of any one of
• Embodiment 57 A flavor composition for coating or incorporating into an edible composition to be administered to an animal comprising: an agonist or an antagonist of a feline TAS2R receptor polypeptide of any one of embodiments 1 to 12 and 26, wherein the agonist is denatonium, aloin, or PTC and the antagonist is probenecid; optionally, a palatability enhancer; optionally, a compound to help adhere the flavor composition to the edible composition; and optionally, a compound for providing color or aroma; wherein the flavor composition is a liquid, solid, powder, paste, gel, spreadable formulation, granule, or sprayable formulation.
• Embodiment 58 The flavor composition of embodiment 57 or the method of any one of embodiments 53-56, wherein the edible composition is a food, treat, nutritional supplement, pharmaceutical, oral care material, chewable product, repellant, or drinkable product.
• Embodiment 59 The flavor composition of embodiment 57 or 58 or the method of any one of embodiments 53-56 wherein the edible composition is a dry food, a soft food, a semisoft food, a liquid, a tablet, capsule, caplet, granule, paste, colloidal mixture, dispersion, or gel.
• Embodiment 60 The method of any one of embodiments 48 to 56 or the flavor composition of any one of embodiments 57 to 58, wherein the edible composition is for administration to a feline.
• Embodiment 61 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R38.
• Embodiment 62 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R42.
• Embodiment 63 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R43.
• Embodiment 64 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R44.
• Embodiment 65 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R67.
• Embodiment 66 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R12.
• Embodiment 67 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R10.
• Embodiment 68 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R9.
• Embodiment 69 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R7.
• Embodiment 70 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R4.
• Embodiment 71 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R3.
• Embodiment 72 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2R2.
• Embodiment 73 The polypeptide of any one of embodiments 1-12 and 26, wherein the fTAS2R receptor comprises a domain of fTAS2Rl.
• the terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item.
• the term “or” means “and/or”.
• the terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”).
• the modifier "about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., includes the degree of error associated with

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• 2014
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  • 2014-03-06 AU AU2014237801A patent/AU2014237801B2/en active Active
  • 2014-03-06 BR BR112015023737A patent/BR112015023737A2/pt not_active IP Right Cessation
  • 2014-03-06 CA CA2903208A patent/CA2903208C/en active Active
  • 2014-03-06 EP EP14714028.9A patent/EP2983507B1/en active Active
• 2017
  • 2017-03-27 US US15/469,692 patent/US10174096B2/en active Active

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