US20050106571A1 - Mammalian T1R3 sweet taste receptors - Google Patents

Mammalian T1R3 sweet taste receptors Download PDF

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US20050106571A1
US20050106571A1 US10/679,102 US67910203A US2005106571A1 US 20050106571 A1 US20050106571 A1 US 20050106571A1 US 67910203 A US67910203 A US 67910203A US 2005106571 A1 US2005106571 A1 US 2005106571A1
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receptor
taste
cell
polypeptide
compound
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Isolde Erlenbach
Nicholas Ryba
Grace Zhao
Charles Zuker
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US Department of Health and Human Services
University of California
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University of California
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Priority to US10/679,102 priority Critical patent/US20050106571A1/en
Priority to JP2006534237A priority patent/JP2007507544A/ja
Priority to CA002540837A priority patent/CA2540837A1/fr
Priority to EP04794142A priority patent/EP1684789A4/fr
Priority to PCT/US2004/032678 priority patent/WO2005033125A2/fr
Priority to AU2004278425A priority patent/AU2004278425A1/en
Assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA reassignment THE REGENTS OF THE UNIVERSITY OF CALIFORNIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ZHAO, GRACE, ZUKER, CHARLES S.
Assigned to THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES reassignment THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ERLENBACH, ISOLDE, RYBA, NICHOLAS J.P.
Publication of US20050106571A1 publication Critical patent/US20050106571A1/en
Priority to US11/402,202 priority patent/US7459277B2/en
Priority to US12/257,290 priority patent/US7939279B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention provides isolated nucleic acid and amino acid sequences of sweet taste receptors, the receptors comprising consisting of a monomer or homodimer of a T1R3 G-protein coupled receptor polypeptide, antibodies to such receptors, methods of detecting such nucleic acids and receptors, and methods of screening for modulators of sweet taste receptors.
  • the sense of taste is responsible for detecting and responding to sweet, bitter, sour, salty and umami (amino acid) stimuli. It is also capable of distinguishing between these various taste modalities to generate innate behavioral responses. For instance, animals are vigorously averse to bitter-tasting compounds, but are attracted to sweet and umami stimuli.
  • sweet, umami and bitter taste receptors To examine taste signal detection and information processing, we have focused on the isolation and characterization of sweet, umami and bitter taste receptors. These receptors provide powerful molecular tools to delineate the organization of the taste system, and to help define the logic of taste coding.
  • T1Rs and T2Rs Two families of candidate mammalian taste receptors, the T1Rs and T2Rs, have been implicated in sweet, umami and bitter detection.
  • the T2Rs are a family of ⁇ 30 taste-specific GPCRs distantly related to opsins, and clustered in regions of the genome genetically linked to bitter taste in humans and mice (Adler et al., Cell 100, 693-702 (2000); Matsunami et al., Nature, 404, 601-604 (2000)).
  • T2Rs have been shown to function as bitter taste receptors in heterologous expression assays, substantiating their role as bitter sensors (Chandrashekar et al., Cell, 100, 703-711 (2000); Bufe et al., Nat Genet, 32, 397-401 (2002)). Most T2Rs are co-expressed in the same subset of taste receptor cells (Adler, E. et al., Cell 100, 693-702 (2000)), suggesting that these cells function as generalized bitter detectors.
  • the T1Rs are a small family of 3 GPCRs expressed in taste cells of the tongue and palate epithelium, distantly related to metabotropic glutamate receptors, the calcium sensing receptor and vomeronasal receptors (Hoon et al., Cell., 96, 541-551 (1999); Kitagawa et al., Biochem Biophys Res Commun, 283, 236-242 (2001); Max et al.,
  • T1Rs combine to generate at least two heteromeric receptors: T1R1 and T1R3 form an L-amino acid sensor, which in rodents recognizes most amino acids, and T1R2 and T1R3 associate to function as a broadly tuned sweet receptor (Nelson, G. et al., Cell, 106, 381-390 (2001); Nelson, G. et al., Nature, 416, 199-202 (2002); Li, X. et al., Proc Natl Acad Sci U S A, 99, 4692-4696 (2002); see also WO 00/06592, WO 00/06593, and WO 03/004992).
  • T1R1+3 may be the mammalian umami receptor (Nelson. et al., Nature, 416, 199-202 (2002); Li.
  • taste receptor cells are assembled into taste buds that are distributed in different papillae in the tongue epithelium.
  • Each taste bud contains 50-150 cells, including precursor cells, support cells, and taste receptor cells (Lindemann, Physiol Rev, 76, 718-766 (1996)).
  • the receptor cells are innervated by afferent fibers that transmit information to the taste centers of the cortex through synapses in the brain stem and thalamus.
  • each taste modality would be encoded by a unique population of cells expressing specific receptors (e.g. sweet cells, bitter cells, salt-sensing cells, etc.).
  • T1Rs and T2Rs are expressed in completely non-overlapping populations of receptor cells in the lingual epithelium (Nelson et al., Cell, 106, 381-390 (2001)), and demonstrated that bitter-receptor expressing cells mediate responses to bitter but not to sweet or amino acid tastants (Zhang et al., Cell, 112, 293-301 (2003)). Together, these results argued that taste receptor cells are not broadly tuned across all modalities, and strongly supported a labeled line model of taste coding at the periphery. A fundamental question we address now is how many types of cells and receptors are necessary to mediate sweet and umami, the two principal attractive taste modalities.
  • T1R1 sweet and umami tastes are exclusively mediated by T1Rs, and demonstrate that genetic ablation of individual T1R subunits selectively affects these two attractive taste modalities.
  • the identification of cells and receptors for sweet and umami sensing also allowed us to devise a strategy to separate the role of receptor activation from cell stimulation in encoding taste responses.
  • animals engineered to express a modified k-opioid receptor in T1R2+3-expressing cells become specifically attracted to a k-opioid agonist, and prove that activation of sweet-receptor expressing cells, rather than the T1R receptors themselves, is the key determinant of behavioral attraction to sweet tastants.
  • T1R1 alone either as a monomer or as a homodimer, acts as a receptor for naturally occurring sugars.
  • the present invention thus provides for the first time a homodimeric sweet taste receptor, the receptor comprising or consisting of two T1R3 polypeptides.
  • the present invention also provides a monomeric sweet taste receptor comprising or consisting of one T1R3 polypeptide.
  • the receptors transduce a signal in response to sweet taste ligands when T1R3 is expressed in a cell.
  • the sweet taste ligands are naturally occurring sweet tasting molecules.
  • the T1R3 polypeptides of the homodimer are non-covalently linked.
  • the present invention provides a sweet taste receptor comprising a T1R3 polypeptide, the T1R3 polypeptide comprising greater than about 80% amino acid sequence identity to an amino acid sequence of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31 or encoded by a nucleotide sequence hybridizing under moderately or highly stringent hybridization conditions to a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31.
  • the T1R3-comprising receptor specifically binds to polyclonal antibodies generated against SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31.
  • the receptor has G-protein coupled receptor activity.
  • the T1R3 polypeptide has an amino acid sequence of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31.
  • the receptor is from a human, a rat, or a mouse.
  • the sweet receptor comprises a T1R3 polypeptide and recognizes natural sugars, e.g., glucose, galactose, fructose, maltose, lactose, and sucrose.
  • the present invention provides an isolated polypeptide comprising an extracellular, a transmembrane domain, or a cytoplasmic domain of a sweet T1R3-comprising homodimeric or monomeric taste receptor, the extracellular, a transmembrane domain, or a cytoplasmic domain comprising greater than about 80% amino acid sequence identity to the extracellular, a transmembrane domain, or a cytoplasmic domain of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31.
  • the extracellular, transmembrane, or cytoplasmic domain hybridize under highly stringent conditions to an extracellular, transmembrane, or cytoplasmic domain of an amino acid sequence of SEQ ID NO:15, 20, 23, 25, or 31.
  • the polypeptide encodes the extracellular, a transmembrane domain, or a cytoplasmic domain of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31.
  • the extracellular, a transmembrane domain, or a cytoplasmic domain is covalently linked to a heterologous polypeptide, forming a chimeric polypeptide.
  • the chimeric polypeptide has G-protein coupled receptor activity.
  • the present invention provides an antibody that selectively binds to a homodimeric or monomeric sweet taste receptor, the receptor comprising one or two T1R3 polypeptides but no T1R1 or T1R2 polypeptides, the antibody raised against a receptor comprising a T1R3 polypeptide comprising greater than about 80% amino acid sequence identity to an amino acid sequence of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:31 or encoded by a nucleotide sequence hybridizing under highly stringent hybridization conditions to a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31.
  • the present invention provides a method for identifying a compound that modulates sweet taste signaling in taste cells, the method comprising the steps of: (i) contacting the compound with a homodimeric or monomeric receptor comprising a T1R3 polypeptide but not a T1R1 or a T1R2 polypeptide, the polypeptide comprising greater than about 80% amino acid sequence identity to SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31; or encoded by a nucleotide sequence hybridizing under highly stringent hybridization conditions to a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31; and (ii) determining the functional effect of the compound upon the receptor.
  • the functional effect is determined in vitro.
  • the polypeptide is expressed in a cell or cell membrane.
  • the receptor is linked to a solid phase, either covalently or non-covalently.
  • the present invention provides a method for identifying a compound that modulates sweet taste signaling in taste cells, the method comprising the steps of: (i) contacting a cell with the compound, the cell expressing a homodimeric or monomeric receptor comprising a T1R3 polypeptide but not expressing a T1R1 or a T1R2 polypeptide, the T1R3 polypeptide comprising greater than about 80% amino acid sequence identity to SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31; or encoded by a nucleotide sequence hybridizing under highly stringent hybridization conditions to a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:31; and (ii) determining the functional effect of the compound upon the receptor.
  • the functional effect is determined by measuring changes in intracellular cAMP, IP3, or Ca2+. In another embodiment, the functional effect is a chemical or phenotypic effect. In another embodiment, the functional effect is a physical effect. In another embodiment, the functional effect is determined by measuring binding of the compound to the extracellular domain of the receptor. In another embodiment, the polypeptide is recombinant. In another embodiment, the cell is a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell. In another embodiment, the cell expresses G protein G ⁇ 15.
  • FIG. 1 Targeted KO of T1R1, T1R2 and T1R3.
  • FIG. 2 T1R mutants respond normally to sour, salty and bitter stimuli
  • FIG. 3 T1R1+3 functions as the mammalian umami receptor (a-d) Taste preferences of wild-type (open circles, dashed lines), T1R1 KO (blue circles and bars), T1R2 KO (gray circles and bars) and T1R3 KO mice (brown circles and bars) were measured relative to water using a brief access taste test. T1R2 KO mice are equivalent to wild type controls. In contrast, T1R1 and T1R3 knockout animals exhibit a complete loss in preference for umami tastants (a) MSG+1 mM IMP, (b) MSG, (c) IMP, and (d) L-Asp (100 mM), and AP4 (30 mM).
  • both knockout have marked impairments in other amino acid responses.
  • L-Asn (100 mM) and L-Arg were used at 100 mM each.
  • e-f Integrated chorda tympani responses to umami tastants and amino acids.
  • T1R1 and T1R3 knockouts have a complete loss of responses to (e) umami agonists and L-amino acids if salt effects are avoided by using either amiloride or the potassium salt of MSG (MPG). In contrast, (f) if high concentrations of salt are used (e.g. 100 mM MSG), residual responses are detected.
  • FIG. 4 T1R2 and T1R3 are essential for sweet taste perception
  • T1R1 KO mice are equivalent to wild type controls.
  • T1R2 and T1R3 knockout animals exhibit a complete loss in preference for artificial sweeteners and D-amino acids, but retain residual responses to high concentration of natural sugars. These are highlighted in (b) as dose responses in expanded scale for maltose, sucrose and glucose.
  • FIG. 5 T1R2 and T1R3 encode the mammalian sweet taste receptors
  • Panel (a) shows integrated chorda tympani responses to natural sugars, artificial sweeteners and D-amino acids in wild type (WT) and T1R knockout animals (1-KO, 2-KO, 3-KO).
  • T1R2 and T1R3 knockouts have a complete loss of responses to artificial sweeteners and D-amino acid (red traces), but show small neural responses to high concentrations of natural sugars. These, however, are completely abolished in T1R2/T1R3 double KO mice (bottom red traces).
  • Panel (b) shows average neural responses to an expanded panel of tastants; wild type, white bars; T1R2 KO, green bars; T1R3 KO, brown bars; T1R2/T1R3 double KO, red bars.
  • FIG. 6 T1R3 responds to high concentrations of natural sugars
  • HEK-293 cells co-expressing the promiscuous G protein G gust-25 (see Experimental Procedures) and the mouse T1R3 GPCR, or co-transfected with both T1R2 plus T1R3, were stimulated with various sweet compounds.
  • Upper panels show increases in [Ca 2+ ]i upon stimulation of T1R3-expressing cells with 500 mM, but not 300 mM sucrose. No responses were detected with artificial sweeteners (300 mM saccharin, right panel), or in cells without receptors or G gust-25 ; scale indicates [Ca 2+ ]i (nM) determined from FURA-2 F 340 /F 380 ratios.
  • control cells expressing T1R2+3 respond robustly to lower concentrations of natural (300 mM sucrose) and artificial sweeteners (30 mM saccharin).
  • FIG. 7 Activation of T1R2-expressing cells triggers behavioral attraction
  • the human T1R2 taste receptor is (a) selectively expressed in T1R2-cells, and (b) effectively rescues sweet taste responses of T1R2 KO mice.
  • the presence of the transgene (c-d) humanizes the sweet taste preferences of the transgenic animals. See text for details.
  • FIG. 8 provides a nucleotide sequence of hT1R1 (SEQ ID NO:26).
  • FIG. 9 provides an amino acid sequence of hT1R1 (SEQ ID NO:27).
  • FIG. 10 provides a nucleotide sequence of hT1R2 (SEQ ID NO:28).
  • FIG. 11 provides a amino acid sequence of hT1R2 (SEQ ID NO:29).
  • FIG. 12 provides a nucleotide sequence of hT1R3 (SEQ ID NO:30).
  • FIG. 13 provides an amino acid sequence of hT1R3 (SEQ ID NO:31).
  • T1Rs and T2Rs are two families of G-protein-coupled receptors (GPCRs) selectively expressed in subsets of taste receptor cells (Hoon et al., Cell 96:541-551 (1999); Adler et al., Cell 100:693-702 (2000); Chandrashekar et al., Cell 100:703-711 (2000); Matsunami et al., Nature 404:601-604 (2000); Nelson et al., Cell 106:381-390 (2001); Kitagawa et al., Biochem. Biophys. Res. Cummun. 283:236-242 (2001); Montmayeur et al., Nature Neurosci.
  • T2Rs are involved in bitter taste detection (Adler et al., Cell 100:693-702 (2000); Chandrashekar et al. Cell, 100:703-711 (2000)); T1R2 and T1R3 combine to function as a sweet taste receptor (see also Nelson et al., Cell 106:381-390 (2001); and T1R1 and T1R3 combine to function as an amino acid taste receptors, as described herein (see also Nelson et al., Nature 24 Feb. 2002 and WO 03/004992))).
  • T1R3 combine to function as a sweet taste receptor.
  • the monomeric form of T1R3 also acts as a sweet receptor.
  • T1R3 combines with itself and also acts as a monomer to function as a sweet receptor, recognizing sweet-tasting molecules such as sucrose, galactose, fructose, glucose, maltose, and lactose.
  • Candidate receptors are expressed in human embryonic kidney (HEK) cells containing the G ⁇ 16 -G ⁇ z and G ⁇ 15 promiscuous G proteins (Offermanns et al., J. Biol. Chem. 270:15175-15180 (1995); Mody et al., Mol. Pharmacol. 57:13-23 (2000)), and assayed for stimulus-evoked changes in intracellular calcium.
  • PLC- ⁇ phospholipase C ⁇
  • release of calcium from internal stores which can be monitored at the single-cell level using calcium-indicator dyes (Chandrashekar et al., Cell 100:703-711 (2000); Nelson et al., Cell 106:381-390 (2001); Tsien et al., Cell Calcium 6:145-157 (1985)).
  • nucleic acids and proteins encoding the receptors provide valuable probes for the identification of taste cells, as the nucleic acids are specifically expressed in taste cells.
  • the receptors are useful for assaying for novel tastants, such as artificial sweetener molecules.
  • probes for GPCR polypeptides and proteins can be used to identity subsets of taste cells such as foliate cells, palate cells, and circumvallate cells, or specific taste receptor cells, e.g., sweet taste receptor cells. They also serve as tools for the generation of taste topographic maps that elucidate the relationship between the taste cells of the tongue and taste sensory neurons leading to taste centers in the brain.
  • the nucleic acids and the proteins they encode can be used as probes to dissect taste-induced behaviors.
  • the invention also provides methods of screening for modulators, e.g., activators, inhibitors, stimulators, enhancers, agonists, and antagonists, of these novel monomeric or homodimeric sweet taste receptors comprising T1R3.
  • modulators e.g., activators, inhibitors, stimulators, enhancers, agonists, and antagonists
  • the monomeric or homodimeric T1R3-comprising receptors of the invention can be used to screen for naturally occurring or artificial sweet tasting molecules or modulators of sweet taste transduction, e.g., small organic molecules, amino acids, peptides, carbohydrates, lipids, polysaccharides, etc.
  • homodimeric or monomeric T1R3-comprising receptors of the invention recognize naturally occurring sweet tastants, as described below in the example section.
  • Such receptors can be used to screen for artificial sweeteners, or altered naturally occurring sweeteners, that mimic the naturally occurring sugar ligands of the homodimeric or monomeric T1R3-comprising receptor.
  • modulators of sweet taste transduction are useful for pharmacological and genetic modulation of sweet taste signaling pathways, and for the discovery of novel sweet taste ligands. These methods of screening can be used to identify agonists and antagonists of sweet taste cell activity. These modulatory compounds can then be used in the food and pharmaceutical industries to customize taste.
  • the invention provides assays for taste modulation, where the T1R3-comprising receptor acts as an direct or indirect reporter molecule for the effect of modulators on sweet taste transduction.
  • GPCRs can be used in assays, e.g., to measure changes in ligand binding, G-protein binding, regulatory molecule binding, ion concentration, membrane potential, current flow, ion flux, transcription, signal transduction, receptor-ligand interactions, neurotransmitter and hormone release; and second messenger concentrations, in vitro, in vivo, and ex vivo.
  • a receptor comprising T1R3 can be used as an indirect reporter via attachment to a second reporter molecule such as green fluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)).
  • a receptor comprising T1R3 is recombinantly expressed in cells that do not express either T1R1 or T1R2, and modulation of taste transduction via GPCR activity is assayed by measuring changes in Ca2+ levels.
  • Methods of assaying for modulators of taste transduction include in vitro ligand binding assays using receptors comprising T1R3, portions thereof such as the extracellular domain, or chimeric proteins comprising one or more domains of T1R3, and in in vivo (cell-based and animal) assays such as oocyte T1R3 receptor expression; tissue culture cell T1R3 receptor expression; transcriptional activation of T1R3; phosphorylation and dephosphorylation of GPCRs; G-protein binding to GPCRs; ligand binding assays; voltage, membrane potential and conductance changes; ion flux assays; changes in intracellular second messengers such as cAMP and inositol triphosphate; changes in intracellular calcium levels; and neurotransmitter release.
  • T1R family taste receptor refers to a receptor comprising a member of the T1R family of G-protein coupled receptors, e.g., T1R1, T1R2, and T1R3, or any combination thereof as a homodimer receptor, a heterodimer receptor, or a monomer receptor.
  • the T1R family receptor comprises T1R3 (a “T1R3-comprising taste receptor” or a “T1R3-comprising sweet taste receptor”).
  • the T1R family receptor comprises a first T1R3 polypeptide and a second T1R3 polypeptide, which form a homodimeric receptor, either covalently or non-covalently linked.
  • the T1R family receptor comprises a single T1R3 polypeptide and no other T1R polypeptide, and forms a monomeric receptor.
  • the T1R family receptor comprises T1R3 and a heterologous polypeptide of the T1R family.
  • the receptor comprises T1R1 and T1R3.
  • the receptor comprises T1R2 and T1R3.
  • the T1R3-comprising receptor is active when the two members of the receptor are co-expressed in the same cell, e.g., T1R3 and T1R3, or T1R1 and T1R3 or T1R2 and T1R3.
  • the T1R polypeptides are co-expressed in the same cell and form a heterodimeric or homodimeric receptor, in which the T1R polypeptides of the receptor are non-covalently linked or covalently linked.
  • the receptor has the ability to recognize, e.g., naturally occurring and/or artificial sweet tasting molecule such as sucrose, fructose, galactose, mannose, glucose, lactose, saccharin, dulcin, acesulfame-K, as well as other molecules, sweet and non-sweet. These molecules are examples of compounds that “modulate sweet taste signal transduction” by acting as ligands for the taste-transducing G protein coupled receptor comprising T1R3.
  • GPCR-B3 or T1R1 refers to nucleic acid and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that are members of the T1R family of G protein coupled receptors and: (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to an amino acid sequence encoded by SEQ ID NO:1, 2, 3, 7, 8, 9, 15, 18, 20, 23, 25, 27, or 31; (2) bind to antibodies,
  • the T1R family polypeptide of the invention e.g., T1R1, T1R2, or T1R3
  • T1R3-comprising receptor e.g., T1R3, T1R3+T1R3, T1R1+3 or T1R2+3
  • G protein coupled receptor activity either alone or when co-expressed in the same cell, or when co-expressed as a monomer, homodimer, or heterodimer with another T1R family member.
  • Accession numbers for amino acid sequences and nucleotide sequences of human, rat, and mouse T1R1, T1R2, and T1R3 can be found in GenBank (for human T1R1 amino acid sequences, see, e.g., Accession No.
  • T1R1 nucleotide sequences see, e.g., Accession No. BK000153
  • human T1R2 amino acid sequences see, e.g., Accession No. DAA00019, AAM12239, and NP — 619642.1
  • human T1R2 nucleotide sequences see, e.g., Accession No. BK000151, NM — 138697.1, AF458149S1-6
  • human T1R3 amino acid sequences see, e.g., Accession No. DAA00013, for human T1R3 nucleotide sequences, see, e.g., Accession NO.
  • T1R proteins have “G-protein coupled receptor activity,” e.g., they bind to G-proteins in response to extracellular stimuli, such as ligand binding (e.g., sweet ligands), and promote production of second messengers such as IP3, cAMP, and Ca2+ via stimulation of enzymes such as phospholipase C and adenylate cyclase.
  • ligand binding e.g., sweet ligands
  • second messengers such as IP3, cAMP
  • Ca2+ phospholipase C and adenylate cyclase.
  • Such activity can be measured in a heterologous cell, by coupling a GPCR (or a chimeric GPCR) to either a G-protein or promiscuous G-protein such as G ⁇ 15 or G ⁇ 16 -G ⁇ z and an enzyme such as PLC, and measuring increases in intracellular calcium using (Offermans & Simon, J. Biol. Chem. 270:15175-15180 (1995
  • Such GPCRs have transmembrane, extracellular and cytoplasmic domains that can be structurally identified using methods known to those of skill in the art, such as sequence analysis programs that identify hydrophobic and hydrophilic domains (see, e.g., Kyte & Doolittle, J. Mol. Biol. 157:105-132 (1982)). Such domains are useful for making chimeric proteins and for in vitro assays of the invention (see, e.g., WO 94/05695 and U.S. Pat. No. 5,508,384).
  • phrases “functional effects” in the context of assays for testing compounds that modulate activity (e.g., signal transduction) of a sweet taste receptor or protein of the invention includes the determination of a parameter that is indirectly or directly under the influence of a GPCR or sweet taste receptor, e.g., a physical, phenotypic, or chemical effect, such as the ability to transduce a cellular signal in response to external stimuli such as ligand binding, or the ability to bind a ligand. It includes binding activity and signal transduction. “Functional effects” include in vitro, in vivo, and ex vivo activities.
  • determining the functional effect is meant assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a T1R GPCR protein or a sweet taste receptor comprising one or more T1R GPCR proteins, e.g., physical and chemical or phenotypic effect.
  • Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index); hydrodynamic (e.g., shape); chromatographic; or solubility properties for the protein; measuring inducible markers or transcriptional activation of the protein; measuring binding activity or binding assays, e.g., binding to antibodies; measuring changes in ligand binding activity or analogs thereof, either naturally occurring or synthetic; measuring cellular proliferation; measuring cell surface marker expression, measurement of changes in protein levels for T1R-associated sequences; measurement of RNA stability; G-protein binding; GPCR phosphorylation or dephosphorylation; signal transduction, e.g., receptor-ligand interactions, second messenger concentrations (e.g., cAMP, cGMP, IP3, PI, or intracellular Ca 2+ ); neurotransmitter release; hormone release; voltage, membrane potential and conductance changes; ion flux; regulatory molecule binding; identification of downstream or reporter gene
  • T1R family polynucleotide and polypeptide sequences and T1R family taste receptors are used to refer to activating, inhibitory, or modulating molecules identified using in vitro and in vivo assays of T1R polynucleotide and polypeptide sequences and T1R family taste receptors, including monomeric, homodimeric and heterodimeric receptors.
  • Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of the T1R family of taste receptors such as a receptor comprising a T1R3 polypeptide, e.g., antagonists.
  • Activators are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate a T1R family taste receptor, such as a receptor comprising a T1R3 polypeptide, e.g., agonists.
  • Inhibitors, activators, or modulators also include genetically modified versions of T1R family taste receptors, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, antisense molecules, ribozymes, small chemical molecules and the like.
  • Such assays for inhibitors and activators include, e.g., expressing T1R family taste receptors in vitro, in cells, or cell membranes, applying putative modulator compounds, and then determining the functional effects on activity, as described above.
  • taste receptor comprising a T1R3 polypeptide has the ability to recognize a sweet tasting molecule such as sucrose, glucose, fructose, lactose, mannose, galactose, saccharin, dulcin, acesulfame-K.
  • a taste receptor comprising a T1R3 polypeptide has the ability to recognize other molecules, such as potential artificial sweeteners. These molecules are examples of compounds that modulate taste signal transduction by acting as extracellular ligands for the G protein coupled receptor and activating the receptor.
  • compounds that modulate taste signal transduction are molecules that act as intracellular ligands of the receptor, or inhibit or activate binding of an extracellular ligand, or inhibit or activate binding of intracellular ligands of the receptor.
  • Samples or assays comprising the T1R family of taste receptors are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition.
  • Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%.
  • Inhibition of a T1R family receptor is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%.
  • Activation of a T1R family receptor is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200-500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.
  • test compound or “drug candidate” or “modulator” or grammatical equivalents as used herein describes any molecule, such as an artificial sweetener or naturally occurring sugar, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleotide, oligonucleotide, etc., to be tested for the capacity to directly or indirectly modulation taste.
  • an artificial sweetener or naturally occurring sugar either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleot
  • the test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity.
  • Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • a fusion partner e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • new chemical entities with useful properties are generated by identifying a test compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
  • HTS high throughput screening
  • a “small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons.
  • Biological sample include sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood, sputum, tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc.
  • a biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
  • a “heterodimer” is a dimer receptor comprising two different polypeptide subunits, e.g., two different polypeptides, where the molecules are associated via either covalent, e.g., through a linker or a chemical bond, or non-covalent, e.g., ionic, van der Waals, electrostatic, or hydrogen bonds linkages.
  • the T1R3-comprising receptors of the invention function when co-expressed in the same cell, preferably when co-expressed so that they form a heterodimer, either covalently or non-covalently linked.
  • T1R1 and T1R3 form a heteromeric receptor
  • T1R2 and T1R3 form a heteromeric receptor.
  • a “homodimer” is a dimer receptor comprising two of the same polypeptide subunits, e.g., two T1R3 polypeptides, where the molecules are associated via either covalent, e.g., through a linker or a chemical bond, or non-covalent, e.g., ionic, van der Waals, electrostatic, or hydrogen bonds linkages.
  • the T1R3-comprising receptors of the invention function when co-expressed in the same cell, preferably when co-expressed so that they form a homodimer, either covalently or non-covalently linked.
  • a “monomer” is a receptor comprising one polypeptide subunit, e.g., one T1R3 polypeptide.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequences SEQ ID NO:1-25), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like).
  • a specified region e.g., nucleotide sequences SEQ ID NO:1-25
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • polypeptide “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms 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.
  • amino acid refers to naturally occurring and synthetic amino acids, enantiomers (D- and L-forms), and achiral 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, ⁇ -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., an 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 refers to 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. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • “Conservatively 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 corresponding codons described without altering the encoded polypeptide.
  • 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
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3 rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980).
  • 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, e.g., extracellular domains, transmembrane domains, and cytoplasmic domains.
  • Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of ⁇ -sheet and ⁇ -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. Anisotropic terms are also known as energy terms.
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, as well as the complements of any such sequence. Also included are DNA, cDNA, RNA, polynucleotides, nucleotides, and the like. The term encompasses nucleic acids containing 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, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • a particular nucleic acid sequence also implicitly encompasses “splice variants.”
  • a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid.
  • “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
  • Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.
  • a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes , “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5 ⁇ SSC, and 1% SDS, incubating at 42° C., or, 5 ⁇ SSC, 1% SDS, incubating at 65° C., with wash in 0.2 ⁇ SSC, and 0.1% SDS at 65° C.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1 ⁇ SSC at 45° C. A positive hybridization is at least twice background.
  • Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology , ed. Ausubel, et al.
  • a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C. depending on primer length.
  • a temperature of about 62° C. is typical, although high stringency annealing temperatures can range from about 50° C. to about 65° C., depending on the primer length and specificity.
  • Typical cycle conditions for both high and low stringency amplifications include 30-40 cycles of the following conditions: a denaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealing phase lasting 30 sec.-2 min., and an extension phase of about 72° C. for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications (1990).
  • Antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′ 2 , a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
  • the F(ab)′ 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′ 2 dimer into an Fab′ monomer.
  • the Fab′ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.
  • antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
  • the term antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).
  • No. 4,946,778 can be adapted to produce antibodies to polypeptides of this invention.
  • transgenic mice, or other organisms such as other mammals may be used to express humanized antibodies.
  • phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).
  • a “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • the antibody is conjugated to an “effector” moiety.
  • the effector moiety can be any number of molecules, including labeling moieties such as radioactive labels or fluorescent labels, or can be a therapeutic moiety.
  • the antibody modulates the activity of the protein.
  • the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies raised to a T1R protein or a homodimeric or heterodimeric T1R3-comprising taste receptor comprising a sequence of or encoded by SEQ ID NO:1-25, polymorphic variants, alleles, orthologs, and conservatively modified variants, or splice variants, or portions thereof, can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with T1R proteins and/or homodimeric or heterodimeric T1R3-comprising taste receptors and not with other proteins.
  • the antibodies react with a homodimeric T1R3-comprising taste receptor, but not with individual protein members of the T1R family.
  • This selection may be achieved by subtracting out antibodies that cross-react with other molecules.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • 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).
  • T1R nucleic acids, polymorphic variants, orthologs, and alleles that are substantially identical to an amino acid sequences disclosed herein can be isolated using T1R nucleic acid probes and oligonucleotides under stringent hybridization conditions, by screening libraries.
  • expression libraries can be used to clone T1R protein, polymorphic variants, orthologs, and alleles by detecting expressed homologs immunologically with antisera or purified antibodies made against human T1R or portions thereof.
  • T1R RNA e.g., taste buds such as circumvallate, foliate, fungiform, and palate.
  • the mRNA is then made into cDNA using reverse transcriptase, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning.
  • Methods for making and screening cDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra).
  • the DNA is extracted from the tissue and either mechanically sheared or enzymatically digested to yield fragments of about 12-20 kb. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro. Recombinant phage are analyzed by plaque hybridization as described in Benton & Davis, Science 196:180-182 (1977). Colony hybridization is carried out as generally described in Grunstein et al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
  • T1R nucleic acid and its orthologs, alleles, mutants, polymorphic variants, and conservatively modified variants combines the use of synthetic oligonucleotide primers and amplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202 ; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)).
  • Methods such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used to amplify nucleic acid sequences of human T1R directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.
  • Degenerate oligonucleotides can be designed to amplify T1R homologs using the sequences provided herein. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of T1R encoding mRNA in physiological samples, for nucleic acid sequencing, or for other purposes. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
  • T1R Gene expression of T1R can also be analyzed by techniques known in the art, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or poly A + RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, high density polynucleotide array technology, e.g., and the like.
  • Nucleic acids encoding T1R protein can be used with high density oligonucleotide array technology (e.g., GeneChipTM) to identify T1R protein, orthologs, alleles, conservatively modified variants, and polymorphic variants in this invention (see, e.g., Gunthand et al., AIDS Res. Hum. Retroviruses 14: 869-876 (1998); Kozal et al., Nat. Med. 2:753-759 (1996); Matson et al., Anal. Biochem. 224:110-106 (1995); Lockhart et al., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al., Genome Res. 8:435-448 (1998); Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998)).
  • GeneChipTM GeneChipTM
  • the gene for T1R is typically cloned into intermediate vectors before transformation into prokaryotic or eukaryotic cells for replication and/or expression.
  • These intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors.
  • T1R nucleic acids can be co-expressed or separately expressed, preferably co-expressed on the same or a different vector.
  • Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al., and Ausubel et al, supra.
  • Bacterial expression systems for expressing the T1R protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. In one preferred embodiment, retroviral expression systems are used in the present invention.
  • the promoter used to direct expression of a heterologous nucleic acid depends on the particular application.
  • the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the T1R encoding nucleic acid in host cells.
  • a typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding T1R and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
  • the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • the particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc. Sequence tags may be included in an expression cassette for nucleic acid rescue. Markers such as fluorescent proteins, green or red fluorescent protein, ⁇ -gal, CAT, and the like can be included in the vectors as markers for vector transduction.
  • Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, retroviral vectors, and vectors derived from Epstein-Barr virus.
  • exemplary eukaryotic vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • Expression of proteins from eukaryotic vectors can be also be regulated using inducible promoters.
  • inducible promoters expression levels are tied to the concentration of inducing agents, such as tetracycline or ecdysone, by the incorporation of response elements for these agents into the promoter. Generally, high level expression is obtained from inducible promoters only in the presence of the inducing agent; basal expression levels are minimal.
  • the vectors of the invention have a regulatable promoter, e.g., tet-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, Proc. Nat'l Acad. Sci. USA 89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)).
  • a regulatable promoter e.g., tet-regulated systems and the RU-486 system
  • Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase.
  • markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase.
  • high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a T1R encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
  • the elements that are typically included in expression vectors also include a replicon that functions in E. coli , a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences.
  • the particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable.
  • the prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
  • Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of T1R protein, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification , in Methods in Enzymology , vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).
  • Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing T1R.
  • the transfected cells are cultured under conditions favoring expression of T1R, which is recovered from the culture using standard techniques identified below.
  • T1R polypeptides or T1R3-comprising receptors can be purified for use in functional assays.
  • Naturally occurring T1R proteins or T1R3-comprising receptors can be purified, e.g., from human tissue.
  • Recombinant T1R proteins or T1R3-comprising receptors can be purified from any suitable expression system.
  • T1R polypeptides are typically co-expressed in the same cell to form T1R3-comprising receptors.
  • the T1R protein or T1R3-comprising receptor may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook et al., supra).
  • T1R protein or T1R3-comprising receptor A number of procedures can be employed when recombinant T1R protein or T1R3-comprising receptor is being purified.
  • proteins having established molecular adhesion properties can be reversible fused to the T1R protein or T1R3-comprising receptor.
  • T1R protein or T1R3-comprising receptor can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity.
  • T1R protein or T1R3-comprising receptor could be purified using immunoaffinity columns.
  • Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive.
  • Promoter induction with IPTG is one example of an inducible promoter system.
  • Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein.
  • inclusion bodies Proteins expressed in bacteria may form insoluble aggregates (“inclusion bodies”).
  • purification of inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells, e.g., by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF.
  • the cell suspension can be lysed using 2-3 passages through a French Press, homogenized using a Polytron (Brinkman Instruments) or sonicated on ice. Alternate methods of lysing bacteria are apparent to those of skill in the art (see, e.g., Sambrook et al., supra; Ausubel et al., supra).
  • the inclusion bodies are solubilized, and the lysed cell suspension is typically centrifuged to remove unwanted insoluble matter. Proteins that formed the inclusion bodies may be renatured by dilution or dialysis with a compatible buffer.
  • suitable solvents include, but are not limited to urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M).
  • Some solvents which are capable of solubilizing aggregate-forming proteins are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity.
  • SDS sodium dodecyl sulfate
  • 70% formic acid are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity.
  • guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of immunologically and/or biologically active protein.
  • Other suitable buffers are known to those skilled in the art.
  • Human T1R proteins or T1R3-comprising receptors are separated from other bacterial proteins by standard separation techniques, e.g., with Ni-NTA agarose resin.
  • T1R protein or T1R3-comprising receptor from bacteria periplasm.
  • the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art.
  • the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose.
  • the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO 4 and kept in an ice bath for approximately 10 minutes.
  • the cell suspension is centrifuged and the supernatant decanted and saved.
  • the recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
  • an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest.
  • the preferred salt is ammonium sulfate.
  • Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations.
  • a typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration will precipitate the most hydrophobic of proteins.
  • the precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest.
  • the precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration.
  • Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
  • the molecular weight of the T1R proteins or T1R3-comprising receptors can be used to isolate it from proteins of greater and lesser size using ultrafiltration through membranes of different pore size (for example, Amicon or Millipore membranes).
  • membranes of different pore size for example, Amicon or Millipore membranes.
  • the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest.
  • the retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest.
  • the recombinant protein will pass through the membrane into the filtrate.
  • the filtrate can then be chromatographed as described below.
  • T1R proteins or T1R3-comprising receptors can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands.
  • antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
  • Modulation of a T1R3-comprising taste receptor can be assessed using a variety of in vitro and in vivo assays. Such assays can be used to test for inhibitors and activators of T1R3-comprising taste receptors, and, consequently, inhibitors and activators of taste. Such modulators of T1R3-comprising sweet taste receptors, which are involved in taste signal transduction. Modulators of T1R3-comprising taste receptors are tested using either recombinant or naturally occurring T1R3-comprising taste receptors, preferably human receptors.
  • the monomeric or homodimeric T1R3-comprising receptors of the invention can be used to screen for naturally occurring or artificial sweet tasting molecules, e.g., small organic molecules, amino acids, peptides, carbohydrates, lipids, polysaccharides, etc.
  • homodimeric or monomeric T1R3-comprising receptors of the invention recognize naturally occurring sweet tastants, as described below in the example section.
  • Such receptors can be used to screen for artificial sweeteners, or altered naturally occurring sweeteners, that mimic the naturally occurring sugar ligands of the homodimeric or monomeric T1R3-comprising receptor.
  • the T1R3-comprising taste receptor will have a sequence as encoded by a sequence provided herein or a conservatively modified variant thereof.
  • the T1R3-comprising taste receptor of the assay will be derived from a eukaryote and include an amino acid subsequence having substantial amino acid sequence identity to the sequences provided herein or is encoded by a nucleotide sequence that hybridizes under stringent conditions (moderate or high) to a nucleotide sequence as described herein.
  • the amino acid sequence identity will be at least 60%, preferably at least 65%, 70%, 75%, 80%, 85%, or 90%, most preferably at least 95%.
  • Measurement of sweet taste signal transduction or loss-of-sweet taste signal transduction phenotype on T1R3-comprising taste receptor or cell expressing the T1R3-comprising taste receptor, either recombinant or naturally occurring, can be performed using a variety of assays, in vitro, in vivo, and ex vivo, as described herein.
  • a suitable physical, chemical or phenotypic change that affects activity or binding can be used to assess the influence of a test compound on the polypeptide of this invention.
  • the functional effects are determined using intact cells or animals, one can also measure a variety of effects such as, in the case of signal transduction, e.g., ligand binding, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as pH changes, and changes in intracellular second messengers such as Ca2+, IP3, cGMP, or cAMP.
  • signal transduction e.g., ligand binding
  • hormone release e.g., transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots)
  • changes in cell metabolism such as pH changes
  • changes in intracellular second messengers such as Ca2+, IP3, cGMP, or cAMP.
  • Assays to identify compounds with T1R3-comprising taste receptor modulating activity can be performed in vitro.
  • Such assays can use a full length T1R3-comprising taste receptor or a variant thereof, or a fragment of a T1R3-comprising taste receptor, such as an extracellular domain, fused to a heterologous protein to form a chimera (see, e.g., WO 01/66563, WO 03/001876, WO 02/064631, and WO 03/004992).
  • Purified recombinant or naturally occurring T1R3-comprising taste receptor can be used in the in vitro methods of the invention.
  • the recombinant or naturally occurring T1R3-comprising taste receptor can be part of a cellular lysate or a cell membrane.
  • the binding assay can be either solid state or soluble.
  • the protein or membrane is bound to a solid support, either covalently or non-covalently.
  • the in vitro assays of the invention are ligand binding or ligand affinity assays, either non-competitive or competitive (with known extracellular ligands as described herein, or with a known intracellular ligand GTP).
  • Other in vitro assays include measuring changes in spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein.
  • a high throughput binding assay is performed in which the T1R3-comprising taste receptor or chimera comprising a fragment thereof is contacted with a potential modulator and incubated for a suitable amount of time.
  • the potential modulator is bound to a solid support, and the T1R3-comprising taste receptor is added.
  • the T1R3-comprising taste receptor is bound to a solid support.
  • modulators can be used, as described below, including small-organic molecules, peptides, antibodies, and T1R3-comprising taste receptor ligand analogs.
  • T1R3-comprising taste receptor-modulator binding can be used to identify T1R3-comprising taste receptor-modulator binding, including labeled protein-protein binding assays, electrophoretic mobility shifts, immunoassays, enzymatic assays such as phosphorylation assays, and the like.
  • the binding of the candidate modulator is determined through the use of competitive binding assays, where interference with binding of a known ligand is measured in the presence of a potential modulator.
  • Ligands for T1R3-comprising taste receptors are provided herein. Either the modulator or the known ligand is bound first, and then the competitor is added. After the T1R3-comprising taste receptor is washed, interference with binding, either of the potential modulator or of the known ligand, is determined. Often, either the potential modulator or the known ligand is labeled.
  • a T1R3-comprising taste receptor is expressed in a cell (e.g., by expression or co-expression one or two members of the T1R family such as T1R1 and T1R3 or T1R2 and T1R3, preferably by expression of T1R3 alone without expression of any other T1R family members), and functional, e.g., physical and chemical or phenotypic, changes are assayed to identify T1R3-comprising taste receptor taste modulators.
  • Cells expressing T1R3-comprising taste receptor can also be used in binding assays. Any suitable functional effect can be measured, as described herein.
  • ligand binding, G-protein binding, and GPCR signal transduction are all suitable assays to identify potential modulators using a cell based system.
  • Suitable cells for such cell based assays include both primary cells and cell lines, as described herein.
  • the T1R3-comprising taste receptor can be naturally occurring or recombinant.
  • chimeric T1R3-comprising taste receptors with GPCR activity can be used in cell based assays.
  • the extracellular domain of an T1R protein can be fused to the transmembrane and/or cytoplasmic domain of a heterologous protein, preferably a heterologous GPCR.
  • Such a chimeric GPCR would have GPCR activity and could be used in cell based assays of the invention.
  • cellular T1R polypeptide levels are determined by measuring the level of protein or mRNA.
  • the level of T1R protein or proteins related to T1R signal transduction are measured using immunoassays such as western blotting, ELISA and the like with an antibody that selectively binds to the T1R3-comprising taste receptor or a fragment thereof.
  • immunoassays such as western blotting, ELISA and the like with an antibody that selectively binds to the T1R3-comprising taste receptor or a fragment thereof.
  • amplification e.g., using PCR, LCR, or hybridization assays, e.g., northern hybridization, RNAse protection, dot blotting, are preferred.
  • the level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
  • T1R3-comprising receptor expression can be measured using a reporter gene system.
  • a reporter gene system can be devised using an T1R protein promoter operably linked to a reporter gene such as chloramphenicol acetyltransferase, firefly luciferase, bacterial luciferase, ⁇ -galactosidase and alkaline phosphatase.
  • the protein of interest can be used as an indirect reporter via attachment to a second reporter such as red or green fluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)).
  • the reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art.
  • a functional effect related to GPCR signal transduction can be measured.
  • An activated or inhibited T1R3-comprising G-coupled protein receptor will alter the properties of target enzymes, second messengers, channels, and other effector proteins.
  • the examples include the activation of cGMP phosphodiesterase, adenylate cyclase, phospholipase C, IP3, and modulation of diverse channels by 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.
  • Activated GPCR receptors become substrates for kinases that phosphorylate the C-terminal tail of the receptor (and possibly other sites as well).
  • activators 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.
  • IP3 inositol triphosphate
  • 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.
  • Cells expressing such G-protein coupled receptors may exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it may be desirable although not necessary to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such as EGTA, to distinguish fluorescence response resulting from calcium release from internal stores.
  • a chelating agent such as EGTA
  • T1R3-comprising taste receptor GPCR activity is measured by expressing a T1R3-comprising taste receptor 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)). Modulation of signal transduction is assayed by measuring changes in intracellular Ca 2+ levels, which change in response to modulation of the GPCR signal transduction pathway via administration of a molecule that associates with an T1R3-comprising taste receptor. Changes in Ca2+ levels are optionally measured using fluorescent Ca 2+ indicator dyes and fluorometric imaging.
  • 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 3 H-myoinositol for 48 or more hrs. The labeled cells are treated with a test compound for one hour. The treated cells are lysed and extracted in chloroform-methanol-water after which the inositol phosphates were separated by ion exchange chromatography and quantified by scintillation counting. Fold stimulation is determined by calculating the ratio of cpm in the presence of agonist to cpm in the presence of buffer control. Likewise, fold inhibition is determined by calculating the ratio of cpm in the presence of antagonist to cpm in the presence of buffer control (which may or may not contain an agonist).
  • 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.
  • adenylate cyclase e.g., adenylate cyclase
  • 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.
  • assays for G-protein coupled receptor activity include cells that are loaded with ion or voltage sensitive dyes to report receptor activity. Assays for determining activity of such receptors can also use known agonists and antagonists for other G-protein coupled receptors as negative or positive controls to assess activity of tested compounds. In assays for identifying modulatory compounds (e.g., agonists, antagonists), changes in the level of ions in the cytoplasm or membrane voltage will be monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively. Among the ion-sensitive indicators and voltage probes that may be employed are those disclosed in the Molecular Probes 1997 Catalog.
  • promiscuous G-proteins such as G ⁇ 15 and G ⁇ 16 can be used in the assay of choice (Wilkie et al., Proc. Nat'l Acad. Sci. USA 88:10049-10053 (1991)). Such promiscuous G-proteins allow coupling of a wide range of receptors.
  • Animal models of taste also find use in screening for modulators of taste, such as the T1R knockout mouse strains as described herein.
  • Transgenic animal technology including gene knockout technology, for example as a result of homologous recombination with an appropriate gene targeting vector, or gene overexpression, will result in the absence or increased expression of the T1R3-comprising receptor or components thereof.
  • tissue-specific expression or knockout of the T1R3-comprising receptors or components thereof may be necessary.
  • Transgenic animals generated by such methods find use as animal models of taste modulation and are additionally useful in screening for modulators of taste modulation.
  • the compounds tested as modulators of T1R3-comprising taste receptors can be any small organic molecule, or a biological entity, such as a protein, e.g., an antibody or peptide, an amino acid, a lipid, a fat, a sugar, e.g., a mono-, di-, or polysaccharide, a nucleic acid, e.g., an antisense oligonucleotide or a ribozyme, or a small organic molecule.
  • modulators can be genetically altered versions of a T1R3-comprising taste receptor.
  • test compounds will be small organic molecules, amino acids, peptides, lipids, and mono-, di- and polysaccharides.
  • any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
  • the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.
  • high throughput screening methods involve providing a combinatorial small organic molecule or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such “combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)).
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No.
  • WO 93/20242 random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc.
  • the invention provides soluble assays using a T1R3-comprising taste receptor, or a cell or tissue expressing a T1R3-comprising taste receptor, either naturally occurring or recombinant.
  • the invention provides solid phase based in vitro assays in a high throughput format, where the T1R3-comprising taste receptor is attached to a solid phase substrate.
  • any one of the assays described herein can be adapted for high throughput screening, e.g., ligand binding, cellular proliferation, cell surface marker flux, e.g., screening, radiolabeled GTP binding, second messenger flux, e.g., Ca 2+ , IP3, cGMP, or cAMP, cytokine production, etc.
  • high throughput screening e.g., ligand binding, cellular proliferation, cell surface marker flux, e.g., screening, radiolabeled GTP binding, second messenger flux, e.g., Ca 2+ , IP3, cGMP, or cAMP, cytokine production, etc.
  • each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
  • a single standard microtiter plate can assay about 100 (e.g., 96) modulators.
  • 1536 well plates are used, then a single plate can easily assay from about 100-about 1500 different compounds. It is possible to assay many plates per day; assay screens for up to about 6,000, 20,000, 50,000, or more than 100,000 different compounds are possible using the integrated systems of the invention.
  • the protein of interest or a fragment thereof e.g., an extracellular domain, or a cell or membrane comprising the protein of interest or a fragment thereof as part of a fusion protein can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage e.g., via a tag.
  • the tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
  • tags and tag binders can be used, based upon known molecular interactions well described in the literature.
  • a tag has a natural binder, for example, biotin, protein A, or protein G
  • tag binders avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.
  • Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).
  • any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair.
  • Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature.
  • the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody.
  • receptor-ligand interactions are also appropriate as tag and tag-binder pairs.
  • agonists and antagonists of cell membrane receptors e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherein family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993).
  • toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors e.g.
  • Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
  • linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids.
  • polypeptide sequences such as poly gly sequences of between about 5 and 200 amino acids.
  • Such flexible linkers are known to persons of skill in the art.
  • poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
  • Tag binders are fixed to solid substrates using any of a variety of methods currently available.
  • Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder.
  • groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups.
  • Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc.
  • Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
  • immunoassays In addition to the detection of T1R genes and gene expression using nucleic acid hybridization technology, one can also use immunoassays to detect T1R3-comprising taste receptors of the invention. Such assays are useful for screening for modulators of T1R3-comprising taste receptors, as well as for therapeutic and diagnostic applications. Immunoassays can be used to qualitatively or quantitatively analyze T1R3-comprising taste receptors. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).
  • Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)).
  • a number of immunogens comprising portions of T1R protein or T1R3-comprising taste receptor may be used to produce antibodies specifically reactive with T1R protein.
  • recombinant T1R protein or an antigenic fragment thereof can be isolated as described herein.
  • Recombinant protein can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above.
  • Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies.
  • a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen.
  • Naturally occurring protein may also be used either in pure or impure form.
  • the product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.
  • mice e.g., BALB/C mice
  • rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the beta subunits.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see, Harlow & Lane, supra).
  • Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse, et al., Science 246:1275-1281 (1989).
  • Monoclonal antibodies and polyclonal sera are collected and titered against the immunogen protein 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-T1R or T1R3-comprising taste receptor proteins, using a competitive binding immunoassay.
  • Specific polyclonal antisera and monoclonal antibodies will usually bind with a K d of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better.
  • Antibodies specific only for a particular T1R3-comprising taste receptor ortholog can also be made, by subtracting out other cross-reacting orthologs from a species such as a non-human mammal.
  • individual T1R proteins can be used to subtract out antibodies that bind both to the receptor and the individual T1R proteins. In this manner, antibodies that bind only to a particular receptor may be obtained.
  • the protein can be detected by a variety of immunoassay methods.
  • the antibody can be used therapeutically as a T1R3-comprising taste receptor modulators.
  • the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, supra.
  • T1R3-comprising taste receptors can be detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • immunological binding assays see also Methods in Cell Biology: Antibodies in Cell Biology , volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).
  • Immunological binding assays typically use an antibody that specifically binds to a protein or antigen of choice (in this case the T1R3-comprising taste receptor or antigenic subsequence thereof).
  • the antibody e.g., anti-T1R3-comprising taste receptor
  • the antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
  • Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen.
  • the labeling agent may itself be one of the moieties comprising the antibody/antigen complex.
  • the labeling agent may be a labeled T1R3-comprising taste receptor or a labeled anti-T1R3-comprising taste receptor antibody.
  • the labeling agent may be a third moiety, such a secondary antibody, that specifically binds to the antibody/T1R3-comprising taste receptor complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived).
  • Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent.
  • the labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin.
  • detectable moieties are well known to those skilled in the art.
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, optionally from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10° C. to 40° C.
  • Immunoassays for detecting T1R3-comprising taste receptors in samples may be either competitive or noncompetitive.
  • Noncompetitive immunoassays are assays in which the amount of antigen is directly measured.
  • the anti-T1R3-comprising taste receptor antibodies can be bound directly to a solid substrate on which they are immobilized. These immobilized antibodies then capture T1R3-comprising taste receptors present in the test sample. T1R3-comprising taste receptors thus immobilized are then bound by a labeling agent, such as a second T1R3-comprising taste receptor antibody bearing a label.
  • the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second or third antibody is typically modified with a detectable moiety, such as biotin, to which another molecule specifically binds, e.g., streptavidin, to provide a detectable moiety.
  • the amount of T1R3-comprising taste receptor present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) T1R3-comprising taste receptor displaced (competed away) from an anti-T1R3-comprising taste receptor antibody by the unknown T1R3-comprising taste receptor present in a sample.
  • a known amount of T1R3-comprising taste receptor is added to a sample and the sample is then contacted with an antibody that specifically binds to a T1R3-comprising taste receptor.
  • the amount of exogenous T1R3-comprising taste receptor bound to the antibody is inversely proportional to the concentration of T1R3-comprising taste receptor present in the sample.
  • the antibody is immobilized on a solid substrate.
  • the amount of T1R3-comprising taste receptor bound to the antibody may be determined either by measuring the amount of T1R3-comprising taste receptor present in a T1R3-comprising taste receptor/antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein.
  • the amount of T1R3-comprising taste receptor may be detected by providing a labeled T1R3-comprising taste receptor molecule.
  • a hapten inhibition assay is another preferred competitive assay.
  • the known T1R3-comprising taste receptor is immobilized on a solid substrate.
  • a known amount of anti-T1R3-comprising taste receptor antibody is added to the sample, and the sample is then contacted with the immobilized T1R3-comprising taste receptor.
  • the amount of anti-T1R3-comprising taste receptor antibody bound to the known immobilized T1R3-comprising taste receptor is inversely proportional to the amount of T1R3-comprising taste receptor present in the sample.
  • the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
  • Immunoassays in the competitive binding format can also be used for crossreactivity determinations.
  • a T1R3-comprising taste receptor can be immobilized to a solid support.
  • Proteins e.g., T1R3-comprising taste receptors and homologs
  • the ability of the added proteins to compete for binding of the antisera to the immobilized protein is compared to the ability of the T1R3-comprising taste receptor to compete with itself.
  • the percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the added proteins listed above are selected and pooled.
  • the cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the added considered proteins, e.g., distantly related homologs.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps an allele or polymorphic variant of a T1R3-comprising taste receptor, to the immunogen protein.
  • the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined.
  • the second protein required to inhibit 50% of binding is less than 10 times the amount of the T1R3-comprising taste receptor that is required to inhibit 50% of binding, then the second protein is said to specifically bind to the polyclonal antibodies generated to a T1R3-comprising taste receptor immunogen.
  • the technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind T1R3-comprising taste receptors.
  • a suitable solid support such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter
  • the anti-T1R3-comprising taste receptor antibodies specifically bind to the T1R3-comprising taste receptor on the solid support.
  • These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti-T1R3-comprising taste receptor antibodies.
  • LOA liposome immunoassays
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g., DYNABEADSTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).
  • magnetic beads e.g., DYNABEADSTM
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g., 3 H, 125 I, 35 S, 14 C, or 32 P
  • enzymes e.g., horse rad
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to another molecules (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • the ligands and their targets can be used in any suitable combination with antibodies that recognize T1R3-comprising taste receptors, or secondary antibodies that recognize anti-T1R3-comprising taste receptor.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
  • compositions of the present invention e.g., nucleic acid, oligonucleotide, amino acid, protein, peptide, small organic molecule, lipid, carbohydrate, mono-, di- or polysaccharide, particle, or transduced cell
  • suitable formulations of pharmaceutical compositions of the present invention see, e.g., Remington's Pharmaceutical Sciences, 17 th ed., 1989.
  • Administration can be in any convenient manner, e.g., by injection, oral administration, inhalation, transdermal application, or rectal administration.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • liquid solutions such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400
  • capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin
  • suspensions in an appropriate liquid such as water, saline or PEG 400
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • a flavor e.g., sucrose
  • an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • Aerosol formulations i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
  • Parenteral administration and intravenous administration are the preferred methods of administration.
  • the formulations of commends can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the dose will be determined by the efficacy of the particular vector employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient.
  • the physician evaluates circulating plasma levels of the vector, vector toxicities, progression of the disease, and the production of anti-vector antibodies.
  • the dose equivalent of a naked nucleic acid from a vector is from about 1 ⁇ g to 100 ⁇ g for a typical 70 kilogram patient, and doses of vectors which include a retroviral particle are calculated to yield an equivalent amount of therapeutic nucleic acid.
  • compounds and transduced cells of the present invention can be administered at a rate determined by the LD-50 of the inhibitor, vector, or transduced cell type, and the side-effects of the inhibitor, vector or cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses.
  • the present invention provides the nucleic acids of T1R3-comprising taste receptors for the transfection of cells in vitro and in vivo. These nucleic acids can be inserted into any of a number of well-known vectors for the transfection of target cells and organisms as described below.
  • the nucleic acids are transfected into cells, ex vivo or in vivo, through the interaction of the vector and the target cell.
  • the nucleic acid under the control of a promoter, then expresses a T1R3-comprising taste receptor of the present invention, by co-expressing two members of the T1R family, thereby mitigating the effects of absent, partial inactivation, or abnormal expression of a T1R3-comprising taste receptor.
  • the compositions are administered to a patient in an amount sufficient to elicit a therapeutic response in the patient. An amount adequate to accomplish this is defined as “therapeutically effective dose or amount.”
  • the cyclophilin homolog NinaA functions as a chaperone, forming a stable complex in vivo with its protein target rhodopsin. EMBO J. 13, 4886-4895.
  • T2Rs function as bitter taste receptors.
  • Odorant receptor localization to olfactory cilia is mediated by ODR-4, a novel membrane-associated protein.
  • Putative mammalian taste receptors a class of taste-specific GPCRs with distinct topographic selectivity. Cell 96, 541-551.
  • G-protein-coupled receptors regulatory role of receptor kinases and arrestin proteins. Cold Spring Harb. Symp. Quant. Biol. 57, 127-133.
  • Tas1r3 encoding a new candidate taste receptor, is allelic to the sweet responsiveness locus Sac. Nat. Genet. 28, 58-63.
  • G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J. Biol. Chem. 270, 15175-80.
  • T1R receptors defines three largely non-overlapping populations of taste cells in the tongue and palate: cells co-expressing T1R1 and T1R3 (T1R1+3), cells co-expressing T1R2 and T1R3 (T1R2+3), and cells expressing T1R3 alone (Nelson, G. et al., Cell, 106, 381-390 (2001)).
  • T1R1+3 cells co-expressing T1R2 and T1R3
  • T1R2+3 cells co-expressing T1R3 alone
  • T1R3 alone cells co-expressing T1R3 alone
  • T1R3 alone
  • Heterologous expression studies of T1Rs in HEK cells demonstrated that T1R1 and T1R3 combine to form a broadly tuned L-amino acid receptor, while co-expression of T1R2 and T1R3 generates a sweet taste receptor that responds to all classes of sweet-tasting compounds (Nelson, G.
  • T1R3 functions in vivo as a common component of the sweet and amino acid taste receptors, then a knockout of this GPCR should generate mice devoid of sweet and amino acid taste reception. In contrast, knockout of T1R1 or T1R2 might be expected to selectively affect a single taste modality.
  • FIG. 1 illustrates the KO strategies and shows in situ hybridization experiments demonstrating a complete lack of specific T1R staining in the corresponding homozygous KO animals.
  • This second method relies on very short exposures to tastants (5 s events over a total of 30 min versus 48 hrs for two-bottle preference assays), and therefore has the great advantage of minimizing the impact of other sensory inputs, and post-ingestive and learning effects from the assay.
  • FIG. 2 shows that knockouts of T1Rs have no significant effect either on physiological or behavioral responses to citric acid, sodium chloride, and a variety of bitter tastants.
  • T1R1+3 is the Umami Receptor
  • T1R1+3 amino acid receptor may function as the mammalian umami (glutamate) taste sensor:
  • the human and rodent T1R1+3 receptors display selectivity and sensitivity differences that mimic amino acid taste differences between rodents and humans (Nelson, G. et al., Nature, 416, 199-202 (2002); Yoshii et al., 1986).
  • Second, T1R1+3 activity is reliably enhanced by IMP and GMP, the two best known potentiators of umami taste in vivo (Nelson, G. et al., Nature, 416, 199-202 (2002); Li, X.
  • T1R1+3 is activated by psychophysically relevant concentrations of the umami agonists L-Asp and L-AP4 (Nelson, G. et al., Nature, 416, 199-202 (2002); Li, X. et al., Proc Natl Acad Sci USA, 99, 4692-4696 (2002)).
  • L-Asp the umami agonists
  • T1R3 KO mice have a dramatic loss of behavioral attraction—and a profound corresponding deficit in physiological responses to all umami tastants—including glutamate, aspartate, glutamate plus IMP, and IMP alone.
  • Damak et al independently generated T1R3 KO animals but concluded that multiple umami receptors must exist as significant MSG responses remained in their studies of KO mice (Damak, S. et al., Science, 301, 850-853 (2003)).
  • the MSG responses of the KO animals were strictly independent of IMP, a contradiction given that IMP enhancement is the hallmark of the umami modality.
  • T1R1 combines with T1R3 (T1R1+3) to generate the mammalian umami receptor
  • T1R1+3 T1R1+3
  • a knockout of T1R1 should also eliminate all umami responses.
  • FIG. 3 demonstrates that this is absolutely the case.
  • these very same tastants elicit normal, robust responses in control and in T1R2 KO animals.
  • T1R1+3 is the mammalian umami receptor.
  • T1R2+3 and T1R3 are Required for Sweet Reception and Perception
  • T1R2+3 functions in cell based assays as a heteromeric receptor for diverse chemical classes of sweet compounds including natural sugars, artificial sweeteners, Damino acids and sweet-tasting proteins (Nelson, G. et al., Cell, 106, 381-390 (2001); Li, X. et al., Proc Natl Acad Sci USA, 99, 4692-4696 (2002)).
  • sweet receptors Schottamine, Y. et al., J Neurophysiol, 81, 3087-3091 (1999)
  • FIGS. 4 and 5 demonstrate that responses to all classes of sweet tastants are dramatically impaired in T1R2 and T1R3 knockout strains.
  • T1R2 and T1R3 are linked at the distal end of chromosome 4 (Nelson, G.
  • FIGS. 4 and 5 show that T1R2, T1R3 double KO mice have lost all responses to high concentration of sugars. Together, these results illustrate the in vivo significance of the combinatorial assembly of T1Rs, and demonstrate that all sweet taste reception operates via the T1R2 and T1R3 GPCRs.
  • T1R2 or T1R3 homodimeric receptors play a significant role in sweet sensing in wild type mice? T1R2 is always expressed in cells containing T1R3 (T1R2+3 cells; Nelson, G. et al., Cell, 106, 381-390 (2001)). Therefore, even if some T1R2 were not associated with T1R3 in these cells, the much higher affinity of the T R2+3 heteromeric receptor for sweet tastants would likely dominate the cellular response. In contrast, we previously reported that T1R3 is also found in a significant fraction of cells of the tongue and palate epithelium independent of T1R1 and TR2 (T1R3 alone cells; Nelson, G.
  • T1R3 alone can function as a low affinity receptor for natural sugars
  • HEK cells stably expressing T1R3 and an optimized G protein chimera engineered to couple to T1Rs (see Experimental Procedures).
  • FIG. 6 shows that T1R3 alone in fact responds to very high concentrations of natural sugars, but not to lower concentrations ( ⁇ 300 mM), or to artificial sweeteners.
  • T1R2 Delimits Species-Specific Sweet Taste Preferences
  • T1R2 participates exclusively in sweet taste detection while T1R3 is involved in both sweet and amino acid recognition, we reasoned that T1R2 would be a particularly critical determinant of sweet taste selectivity in vivo. Therefore, we predicted that introducing the human T1R2 gene in T1R2 KO mice should both rescue and “humanize” sweet responses.
  • mice that were homozygous for the T1R2 KO allele, but instead expressed a human TR2 transgene in the native “T1R2-cells”.
  • a 12 kb genomic clone containing the T1R2 regulatory sequences was fused to a hT1R2 full length cDNA and introduced into T1R2 KO mice. Multiple independent lines were assayed for their selectivity and sensitivity to sweet tastants.
  • FIG. 7 panel a-d demonstrate that human T1R2 is selectively expressed in T1R2-expressing cells, and effectively restores sweet taste function.
  • the human transgene now confers these mice with the ability to detect and respond to several compounds that taste sweet to humans, but are not normally attractive to rodents; these include aspartame, glycyrrhizic acid and the sweet proteins thaumatin and monellin.
  • the humanized T1R2 mice still do not respond to the intensely sweet compound neohesperidin dihydrochalcone, nor do HEK cells transfected with the human T1R2 and mouse T1R3 GPCRs.
  • HEK cells transfected with the human T1R2 and mouse T1R3 they robustly respond to neohesperidin dihydrochalcone.
  • T1R2+3 receptor Activation of taste receptors trigger distinct behavioral responses in animals. For example, excitation of the T1R2+3 receptor stimulates behavioral attraction to sugars and sweet-tasting compounds in mice. Is this response a property of the receptors or the cells in which they are expressed?
  • One way to answer this question would be to express a novel receptor unrelated to the taste system in the T1R2+3 cells and examine whether its selective stimulation elicits attractive responses (Troemel, E. R. et al., Cell 91, 161-169 (1997)).
  • FIG. 7 e shows that un-induced animals, or wild type controls treated with doxycycline, are completely insensitive to the k-opioid agonist spiradoline.
  • induction of RASSL expression in the T1R2-cells generates animals that are now strongly attracted to nanomolar concentrations of spiradoline ( FIG. 7 , red trace).
  • T R2-expressing cells rather than the receptors they express, determines behavioral attraction in mice.
  • these results unequivocally show that activating a single cell type is sufficient to trigger specific taste responses; therefore a model requiring a combinatorial pattern of activity, or temporal coding, is not needed to account for attraction mediated by T1R2-expressing cells.
  • activation of these taste signaling pathways in human T1R2+3 cells regardless of the nature of the receptor, would evoke sweet taste.
  • T1Rs the receptors for sweet and umami taste in mammals are the T1Rs: umami taste is mediated by the T1R1+3 heteromeric GPCR, and sweet by the two T1R-based receptors, T1R2 and T1R3 (T1R2+3, and most likely, a homodimer of T1R3). Therefore, sweet and amino acid taste (umami)-two chemosensory inputs that trigger behavioral attraction, share a common receptor repertoire and evolutionary origin.
  • the human T1R1+3 receptor is activated by glutamate and aspartate far more effectively than by other amino acids (Li, X. et al., Proc Natl Acad Sci USA, 99, 4692-4696 (2002)).
  • the mouse T1R1+3 receptor recognizes a much broader range of L-amino acids, both in cell based assays (Nelson, G. et al., Nature, 416, 199-202 (2002)) and in vivo (this paper). If the evolutionary role of the T1R1+3 receptor was to mediate attractive responses to protein-rich foods, one may question whether the tuning of receptor selectivity in primates to just two amino acids substantially altered the ability to detect diets rich these nutrients.
  • T1Rs are the mediators of the two principal attractive taste modalities, and demonstrated that mice expressing a RASSL opioid receptor became powerfully attracted to spiradoline, a normally tasteless and nutrionally irrelevant compound, proving that to taste is to believe.
  • the discovery and functional characterization of the cells and receptors for bitter, sweet, and umami taste now provide a compelling view of how taste is encoded at the periphery: dedicated taste receptor cells mediate attractive and aversive behaviors (see, e.g., Zhang, Y. et al., Cell, 112, 293-301 (2003)).
  • T1R knockout animals The strategy used to create T1R knockout animals is shown in FIG. 1 .
  • exon 6 encoding the predicted seven transmembrane domain of the receptor was replaced by the PGK-neo r cassette.
  • Homologous recombination in R1 ES cells was detected by diagnostic Southern hybridization with probes outside the targeting construct.
  • Two targeted ES clones were injected into C57BL/6 blastocysts. Chimeric mice were bred with C57BL/6 mice and progeny backcrossed to C57BL/6 mice for two generations prior to establishing a homozygous knockout colony.
  • T1R2 For T1R2, a similar approach deleted exons 5 and 6 (see FIG. 1 ). Chimeric animals were bred with C57BL/6 mice and progeny backcrossed to C57BL/6 mice for four generations.
  • the T1R3 taster(C57) and non-taster (129) alleles were identified based on an EcoRI polymorphism ⁇ 12 kb upstream of the starting ATG of T1R3. All of the T1R2 knockout animals used in this study carried a taster allele of T1R3.
  • T1R2 KO mice homozygous for the non-taster T1R3 allele produced qualitatively similar results (data not shown).
  • T1R3 KO knockout animals we replaced exons 1 to 5 encoding the N-terminal extracellular domain with the PGK-neor cassette (see FIG. 1 ). Chimeric mice were bred with C57BL/6 mice and progeny backcrossed to C57BL/6 mice for two generations.
  • T1R knockouts have normal viability, body weight, overall anatomy and general behavior. Similarly, taste receptor cells appear normal morphologically and numerically in all knockout backgrounds.
  • Double-label fluorescent detection used fluorescein (full-length hT1R2) and digoxigenin (full-length mT1R2) probes at high stringency (hybridization, 5 ⁇ SSC, 50% formamide, 65-72° C.; washing, 0.2 ⁇ SSC, 72° C.). Hybridization was detected with distinct fluorescent substrates (Adler, E. et al., Cel,l 100, 693-702 (2000)) and specificity of labeling was checked using T1R2-knockout and non transgenic controls.
  • T1R2 An approx. 12 kb genomic fragment upstream of mouse T1R2 was fused to a human T R2 cDNA and to a reverse-tetracycline dependent transactivator (rtTA) construct (Gossen, M. et al., Curr Opin Biotechnol, 5, 516-520 (1994)).
  • rtTA reverse-tetracycline dependent transactivator
  • Transgenic lines were produced by pronuclear injection of zygotes from FVB/N mice.
  • Three independent human T1R2 transgenic lines displayed behavioral attraction to aspartame (10 mM). One line was crossed into the T1R2 knockout background, and assayed for taste responses and transgene expression. No expression outside T1R2-cells was detected.
  • T1R2-rtTA transgenic lines were crossed with tetO-Ro1/tetO-lacZ transgenic animals (Redfern, C. H. et al., Nat Biotechnol, 17, 165-169 (1999)).
  • Doubleheterozygous progeny were induced by doxycycline treatment (6 gm/kg) (Bio-Serv) for 3 days (Gogos, J. A. et al., Cel,l 103, 609-620 (2000)) and examined for ⁇ -galactosidase activity (Zack, D. J. et al., Neuron 6, 187-199 (1991)) and RASSL expression in the tongue and palate.
  • a line displaying appropriate ⁇ -galactosidase staining and RASSL expression pattern was selected for behavioral assays.
  • Taste behavior was assayed using a short term assay that directly measures taste preferences by counting immediate licking responses in a multi-channel gustometer (Davis MS 160-Mouse gustometer; DiLog Instruments, Tallahassee, Fla.). Mice were trained and tested as described previously (Zhang, Y. et al., Cell, 112, 293-301 (2003)). Individual mice were placed in the gustometer for 30 minutes, and stimuli were presented in random order for 5s trials that were initiated by the mouse licking the stimulus spout. For sodium saccharin, glutamate and aspartate, 100 ⁇ M amiloride was added to all solutions (including the control) to minimize effects of salt taste.
  • Data points represent the mean rate that mice licked a tastant relative to their sampling of an appropriate control tastant (ratio defined as lick rate relative to control); lick suppression is defined as 1 minus the lick rate relative to control.
  • the control tastant was water but for amino acids +1 mM IMP, 200 mM MSG and 10 mM IMP the controls were 1 mM IMP, 200 mM sodium gluconate and 10 mM CMP, respectively.
  • mice carrying T1R2-rtTA and tetO-Ro1/tetO-lacZ transgenes expression was induced by doxycycline treatment 3 days prior to, and during the behavioral testing.
  • mice appear to “learn” to identify solutions containing very high concentrations of natural sugars (>500 mM); successive exposure resulted in decreased detection threshold and increased preference ratios. Because mice are repeatedly exposed to test compounds for 48 hrs in standard twobottle assays, they may use other sensory inputs like texture or smell to distinguish tastant from water. If not properly controlled, this could be easily misunderstood as behavioral attraction via taste pathways. To avoid this problem, we used either short term immediate lick response assays (see above) or two-bottle assays with naive knockout mice (i.e. never exposed to such tastants during either training or testing).
  • Neural signals were amplified (5,000 ⁇ ) with a Grass P511 AC amplifier (Astro-Med), digitized with a Digidata 1200B A/D converter (Axon Instruments), and integrated (r.m.s. voltage) with a time constant of 0.5 s.
  • Taste stimuli were presented at a constant flow rate of 4 ml min ⁇ 1 for 20 s intervals interspersed by 2 min rinses with artificial saliva (Danilova, V., and Hellekant, G., BMC Neurosci, 4, 5. (2003)) between presentations. All data analyses used the integrated response over a 25 s period immediately after the application of the stimulus. Each experimental series consisted of the application of 6 tastants bracketed by presentations of 0.1 M citric acid to ensure the stability of the recording. The mean response to 0.1 M citric acid was used to normalize responses to each experimental series.
  • Tastants used for nerve recordings were: sucrose, glucose, maltose (600 mM); sodium saccharin (40 mM); AceK (60 mM); Citric Acid (100 mM); NaCl (100 mM); NH 4 Cl (100 mM); 6-n-propyl thiouracil (10 mM), quinine (10 mM); cycloheximide (1 mM); L-Ser, L-Ala, (30 mM with 0.5 mM IMP added) MSG and MPG (300 mM with or without 0.5 mM IMP); D-Ala, D-Phe, and D-Trp (100 mM). Amiloride (50 uM) was added to reduce sodium responses as indicated in the figure legends.
  • HEK-293 cells (PEAK rapid cells; Edge BioSystems, MD) were grown, transfected with T1Rs and promiscuous G-proteins and assayed for functional responses to tastants by Ca-imaging essentially as described previously (Nelson, G. et al., Cell, 106, 381-390 (2001)). Minor differences in FURA-2 loading and Ca-imaging included using 199(H) Medium (Biosource) containing 0.1% BSA, 100 ⁇ M EGTA and 200 ⁇ M CaCl 2 as assay buffer as well as reducing the time allowed for FURA-AM ester cleavage to 10 minutes.
  • the imaging system was an Olympus IX50 microscope equipped with a 10 ⁇ /0.5 N.A. fluor objective (Zeiss), the TILL imaging system (TILL Photonics GmbH), and a cooled CCD camera. Acquisition and analysis of fluorescence images used TILL-Vision software.
  • a chimera containing the C-terminal 25 residues of gustducin proved particularly effective at mediating responses of mouse T1R2+3 and T1R1+3 in transient transfection assays, and was used for further studies.
  • Cell lines stably expressing T1R3 and G gust-25 were established using puromycin and Zeocin (Invitrogen) selection. Three independent lines expressing T1R3 and G gust-25 were used to examine the specificity and dose response of the T1R3 receptor.
  • Sucrose and maltose (>300 mM) elicited dose dependent responses that were T1R3 and G gust-25 dependent, but attempts to use high concentrations of several other sugars (glucose, fructose, trehalose and galactose) proved impractical because they induced significant receptor independent rises in [Ca 2+ ]i.

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WO2005033125A2 (fr) 2005-04-14
US7459277B2 (en) 2008-12-02
JP2007507544A (ja) 2007-03-29
AU2004278425A1 (en) 2005-04-14
WO2005033125A3 (fr) 2006-03-23
US20090155819A1 (en) 2009-06-18
EP1684789A2 (fr) 2006-08-02
EP1684789A4 (fr) 2006-12-27
CA2540837A1 (fr) 2005-04-14
US20070105159A1 (en) 2007-05-10

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