The Sequence Listing submitted Aug. 8, 2012, as a text file named “10031014US1_ST25.txt,” created on Aug. 7, 2012, and having a size of 13.6 kilobytes is hereby incorporated by reference.
Numerous tastants are utilized in consumables. Additionally, agents can modulate bitter taste, for example by decreasing bitter taste in consumables, such as foods, beverages and medicines. Means for screening agents to identify tastants and to identify modulators of bitter receptors are thus useful.
This disclosure relates to cell lines and assays that can be utilized to identify taste receptor modulators. For example, provided herein is a method for identifying a bitter taste modulator comprising contacting a cell with a bitter tastant and a test compound, wherein the cell is derived from airway tissue and endogenously expresses bitter taste receptor, and measuring bitter taste receptor activity. Optionally, the cell can endogenously express RGS21. A change in bitter taste receptor activity by the bitter tastant in the presence of the test compound indicates modulation of the bitter taste receptor by the test compound, thus identifying a bitter taste modulator.
Further provided is a method for identifying a bitter tastant comprising, contacting a cell, wherein the cell is derived from airway tissue and endogenously expresses a bitter taste receptor, with a test compound and measuring bitter taste receptor activity. Optionally, the cell can endogenously express RGS21. An increase in bitter taste receptor activity indicates that the test compound is a bitter tastant.
Further provided is an isolated, relatively pure population of airway cells that express a bitter taste receptor. The receptor is optionally endogenously expressed by the airway cell, but the airway cell can be genetically modified to express one or more bitter taste receptors or to overexpress one or more bitter taste receptors. Optionally, the cell can endogenously express RGS21 but can also be genetically modified to express RGS21 or to overexpress RGS21.
Also provided is a method for identifying a bitter tastant or modulator comprising contacting a cell with a test compound and measuring taste receptor activity, wherein the cell is a 16HBE cell or a derivative thereof that is derived from airway tissue and endogenously expresses a taste receptor.
Uses for Cell Lines Comprising Bitter Taste Receptors
Provided herein is a method for identifying a bitter taste modulator comprising contacting a cell with a bitter tastant and a test compound, wherein the cell is derived from airway tissue and endogenously expresses a bitter taste receptor, and measuring bitter taste receptor activity. Optionally, the cell can endogenously express RGS21. A change in bitter taste receptor activity by the bitter tastant in the presence of the test compound indicates modulation of the bitter taste receptor by the test compound, thus identifying a bitter taste modulator.
As used throughout, a bitter taste modulator is a compound that modulates bitter taste receptor activity, for example, by inhibiting or blocking bitter taste receptor activation by a bitter tastant, or by enhancing bitter taste receptor activation by a bitter tastant. In one example, the methods of identifying bitter taste modulators identify compounds that modulate, preferably block or inhibit, the activation of a bitter taste receptor by a bitter tastant. As used throughout, such blockers or inhibitors act directly on the receptor but can optionally act upstream or downstream of the receptor.
Any cell derived from airway tissue that endogenously expresses a bitter taste receptor can be utilized in the methods set forth herein. For example, lung or bronchial cells, such as lung or bronchial epithelial cells can be utilized. Known human airway cell lines can optionally be utilized. Examples of airway cells that can be utilized include, but are not limited to, 16HBE cells and cells derived from 16HBE cells wherein the cells express a bitter taste receptor.
In the methods set forth herein, the bitter taste receptor responds to at least one bitter tastant or bitterant. Bitter tastants include, but are not limited to, acesulfame K, acetaminophen, 2-acetyl pyrazine, aloin, amino-2-norbornane-carboxylic acid, amygadalin, andrographolide, arbutin, aristolochic acid, atropine, brucine, 4-benzylpiperidine, caffeine, chloramphenicol, chloroquine, cinchonine, ciprofloxacin, clarithromycin, clindamycin, cycloheximide, cyclooctanone, denatonium benzoate, dexamethasone, diltiazem hydrochloride, diisobutylamine, dimethylbiguanide, 2,6-dimethylpiperidine, doxepin, enalapril maleate, edrophonium, enoxacin, (−)-epicatechin, (−)-erythromycin, ethylpyrazine, famotidine, gabapentin, ginkgolide A, goitrin, guaicol glyceryl ether, labetalol-HCl, linamarin, lomefloxacin, (−)-lupinine, N-methylthiourea, 1-methy-2-quinolinone, methylprednisolone, nitrophthalene, nitrosaccharin, ofloxacin, oleuropein, omeprazole, oxybutynin chloride, oxyphenonium HBr, peptide-LPFNQL (SEQ ID NO:1), peptide-LPFSQL (SEQ ID NO: 2), peptide-YQEPVLGPVRGPFPIIV (SEQ ID NO: 3), peptide-PVLGPVRGPFPIIV (SEQ ID NO: 4), peptide-PVRGPFPIIV (SEQ ID NO: 5), peptide-RGPFPIIV (SEQ ID NO: 6), N-ethyl-N′-phenylurea, 2-picoline, picric acid, pirenzepine dihydrochloride, phenylthiocarbamide, prednisone, procainamide, 6-n-propyl-2-thiouracil, quassin, quinacrine, quinine, ranitidine, saccharin, D-(−)-salicin, spartein sulfate pentahydrate, sucrose octaacetate, strychnine, sulfamethoxazole, theobromine, thioacetanilide, thiocarbanilide, tolazoline, tolylurea, trapidil, trimethoprim, and L-tryptophan.
As used throughout, a test compound can be a naturally occurring compound, a protein, a peptide, a polysaccharide, a chemical, a small molecule or a polynucleotide (for example, a cDNA, an aptamer, a morpholino, a triple helix molecule, an siRNA, a shRNA, an miRNA, an antisense RNA, an LNA, a ribozyme or any other polynucleotide now known or identified in the future). In the methods set forth herein, the compound can be in a library. The libraries can comprise natural products or synthetic compounds. Therefore, provided herein are methods for screening libraries of compounds in order to identify a bitter taste modulator or a bitter tastant. RGS21 is also known as a regulator of G-protein signaling 21 and is capable of binding to or inhibiting Gαi class proteins or other Gα proteins. As set forth above, the airway cells utilized in the present methods can optionally endogenously express RGS21. RGS21 can be encoded by a nucleotide sequence comprising the human sequence set forth in GenBank Accession No. AY643711.1 (SEQ ID NO: 7) This nucleotide sequence encodes the protein sequence set forth in GenBank Accession No. NP—001034241.1 (SEQ ID NO: 8). Airway cells from human or other species comprising an RGS21 nucleotide sequence or an RGS21 protein sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97%, 98%, 99% or more identical to the sequence set forth in GenBank Accession No. AY643711.1 or the sequence set forth in GenBank Accession No. NP—001034241.1, respectively, can also be utilized in the methods described herein. Optionally, the protein sequence comprises one or more conservative amino acid substitutions as compared to the provided sequence. In particular, cells comprising an RGS21 sequence, wherein the RGS21 retains at least one activity of RGS21, for example, interaction with a Gα protein can be utilized in the methods set forth herein.
The cells described herein can be genetically modified to express or overexpress RGS21. For example, an airway cell described herein can be genetically modified by introducing an exogenous nucleic acid comprising a nucleotide sequence encoding RGS21. The nucleic acid can be stably or transiently introduced into the cell. A cell that is genetically modified includes a cell wherein the introduced nucleic acid is also endogenous to the cell. The exogenous nucleic acid can be in a construct or vector that comprises a promoter that is operably linked to the nucleotide sequence encoding RGS21. The promoter can be a constitutive promoter or an inducible promoter. Exemplary inducible promoters include tissue-specific promoters and promoters responsive or unresponsive to a particular stimulus (such as light, oxygen or chemical concentration, for example, a tetracycline inducible promoter).
As utilized throughout, Gα proteins include all members of the Gαi class now known or later discovered, including but not limited to, Gαi1, Gαi2, and Gαi3, gustducin, transducin, Gαo, Gαtr, Gαg, Gαtr, Gαtc and Gαz. Also included are all members of the Gq class now known or later discovered, including but not limited to, Gαq Gα11Gα14, Gα15 and Gα16. The cells described herein can comprise one or more types of Gαi that are endogenously or recombinantly expressed in the cells. The cells can also comprise chimeric Gα proteins, for example Gαq-Gustducin or Gα16-gustducin 44 as described in U.S. Patent Publication No. 20090311686, incorporated in its entirety by this reference.
The bitter taste receptor can be selected from any bitter taste receptor, including, for example, T2R46 or T2R38. T2R46 is also known as taste receptor type 2, member 46 of the G protein-coupled receptor family and mediates the perception of bitterness through a G protein-coupled second messenger pathway. An example of a nucleotide sequence encoding T2R46 is the human sequence set forth in GenBank Accession No. NM—176887.2 (SEQ ID NO: 9). This sequence encodes the protein sequence set forth in GenBank Accession No. NP—795368.2 (SEQ ID NO: 10). Airway cells from human or other species endogenously comprising a T2R46 nucleotide sequence or a T2R46 protein sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97%, 98%, 99% or more identical to the sequence set forth in GenBank Accession No. NM—176887.2, or GenBank Accession No. NP—795368.2, can be utilized in the methods set forth herein. Optionally, the protein sequence comprises one or more conservative amino acid substitutions as compared to the provided sequence. In particular, cells comprising a T2R46 sequence, wherein the T2R46 receptor retains the ability to respond to at least one bitter tastant, can be used in the methods described herein.
T2R38 is also known as taste receptor type 2, member 38 of the G protein-coupled receptor family and also mediates the perception of bitterness through a G protein-coupled second messenger pathway. An example of a nucleotide sequence encoding T2R38 is the human sequence set forth in GenBank Accession No. NM—176817.4 (SEQ ID NO: 11). This sequence encodes the protein sequences set forth in GenBank Accession No. NP—789787.4 (SEQ ID NO: 12). Airway cells from human or other species endogenously comprising a T2R38 nucleotide sequence or a T2R38 protein sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97%, 98%, 99% or more identical to the sequence set forth in GenBank Accession No. NM—176817.4 or GenBank Accession No. NP—789787.4, can be utilized in the methods set forth herein. Optionally, the protein sequence comprises one or more conservative amino acid substitutions as compared to the provided sequence. In particular, cells comprising a T2R38 sequence, wherein the T2R38 receptor retains the ability to respond to at least one bitter tastant, can be used in the methods described herein.
The cells described herein can be genetically modified to express or overexpress the bitter taste receptor. For example, an airway cell described herein can be genetically modified by introducing an exogenous nucleic acid comprising a nucleotide sequence encoding T2R46 or T2R38. The nucleic acid can be stably or transiently introduced into the cell. A cell that is genetically modified includes a cell wherein the introduced nucleic acid is also endogenous to the cell. The exogenous nucleic acid can be in a construct or vector that comprises a promoter that is operably linked to the nucleotide sequence encoding T2R46 or T2R38. The promoter can be a constitutive promoter or an inducible promoter. Exemplary inducible promoters include tissue-specific promoters and promoters responsive or unresponsive to a particular stimulus (such as light, oxygen or chemical concentration, for example, a tetracycline inducible promoter).
In the methods described herein, the cell(s) can be grown on an appropriate substrate, such as a multi-well plate, a tissue culture dish, a flask, etc. The cell can be in a population of cells. This population can be an isolated, relatively pure population of airway cells. One of skill in the art would know how to select the appropriate growth conditions and medium for a given cell type. The methods described herein can further comprise contacting the cell with a dye, substrate, assay medium or any other composition necessary to assess the output from a signaling pathway. For example, the method can comprise loading the cells with calcium-sensitive fluorescent dye in order to measure changes in cytoplasmic calcium levels. The incubation periods necessary to effect bitter taste activation and subsequent assessment of bitter taste receptor activity will vary by cell type but can be empirically determined by one of skill in the art. The cell(s) can be contacted with a test compound before, during or after contacting the cells with the bitter tastant. Screening methods can optionally be performed in vivo. Therefore, the cell can be in a subject.
In the methods described throughout, taste receptor activity can be measured by any means standard in the art. Any suitable physiological change that is a consequence of G protein-coupled receptor activity can be used to assess the effect of a test compound on a taste receptor. Methods for assaying G protein coupled receptor activity are available in the art (see Williams and Hill “GPCR signaling: understanding the pathway to successful drug discovery,” Methods Mol Biol. 2009; 552:39-50 (2009); and De los Frailes and Diez “Screening technologies for G protein-coupled receptors: from HTS to uHTS,” Methods Mol Biol. 552:15-37 (2009)).
One of skill in the art can measure changes in the level of a second messenger in the cell. Examples of second messengers include, cAMP, cGMP, diacylglycerol (DAG), Phosphatidylinositol 4,5-bisphosphate (PIP2), inositol 1,4,5-trisphosphate (IP3) and intracellular calcium. For example, changes in intracellular cAMP or cGMP can be measured using immunoassays. The method described in Offermanns & Simon, J. Bio. Chem., 270:15175-15180 (1995), can be used to determine the level of cAMP. Also, the method described in Felley-Bosco et al., Am. J. Resp. Cell and Mol. Biol., 11:159-164 (1994), can be used to determine the level of cGMP. Further, an assay kit for measuring cAMP and/or cGMP is described in U.S. Pat. No. 4,115,538, incorporated herein by this reference.
Activation of some G protein-coupled receptors stimulates the formation of inositol triphosphate (IP3) through phospholipase C-mediated hydrolysis of phosphatidylinositol. IP3 stimulates the release of intracellular calcium ions. Thus, 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. Increased cytoplasmic calcium levels can result from the release of intracellular calcium stores as well as from extracellular calcium entry via plasma membrane ion channels. Methods for measuring changes in cytoplasmic calcium levels are available to those of skill in the art. For example, calcium levels can be measured fluorescent Ca2+ indicator dyes and fluorimetric imaging (See Liu et al. “A multiplex calcium assay for identification of GPCR agonists and antagonists,” Assay Drug Dev Technol. June; 8(3):367-79 (2010); and Liu et al. “Comparison on functional assays for Gq-coupled GPCRs by measuring inositol monophospate-1 and intracellular calcium in 1536-well plate format,” Curr Chem Genomics. 2008 Jul. 11; 1:70-8 (2008)).
RGS21 GTPase activating protein (GAP) activity can also be measured to assess receptor activity. For example, one of skill in the art can measure a change in the interaction between RGS21 and a G protein, for example, a Gα protein. This interaction can be measured by fluorescence resonance energy transfer, immunoassay or any other means for measuring the interaction between two proteins. Also, radiolabelled (or fluorescent) GTPγS binding to isolated membrane preps from cells expressing the appropriate endogenous tastant receptor can be measured (See, for example, Cooper et al. “[35S]GTPgammaS binding G protein-coupled receptor assays” Methods Mol. Biol. 552:143-151 (2009)). In these methods, activation of the receptor leads to guanine nucleotide exchange on the heterotrimeric G-protein, leading the G-alpha subunit to bind (irreversibly) to the radiolabeled (or fluorescent) GTPγS.
Binding activity can also be used to measure taste receptor activity, for example, via competitive binding assay or surface plasmon resonance (see Salamon et al. “Chapter 6. Plasmon resonance methods in membrane protein biology applications to GPCR signaling,” Methods Enzymol. 2009; 461:123-46 (2009); and Harding et al. “Direct analysis of a GPCR-agonist interaction by surface plasmon resonance,” Eur Biophys J. October; 35(8):709-12 (2006).
Receptor internalization and/or receptor desensitization can also be measured (see, for example, Kershaw et al. “Analysis of chemokine receptor endocytosis and intracellular trafficking,” Methods Enzymol. 460:357-77(2009); and Di Certo et al. “Delayed internalization and lack of recycling in a beta2-adrenergic receptor fused to the G protein alpha-subunit,” BMC Cell Biol. October 7; 9:56(2008)). Receptor-dependent activation of gene transcription can also be measured to assess taste receptor activity. The amount of transcription may be measured by using any method known to those of skill in the art. For example, mRNA expression of the protein of interest may be detected using PCR techniques, microarray or Northern blot. The amount of a polypeptide produced by an mRNA can be determined by methods standard in the art for quantitating proteins in a cell, such as Western blotting, ELISA, ELISPOT, immunoprecipitation, immunofluorescence (e.g., FACS), immunohistochemistry, immunocytochemistry, etc., as well as any other method now known or later developed for quantitating protein in or produced by a cell.
Beta-arrestin recruitment and/or receptor desensitization is optionally measured. See, for example, Bohn et al., “Seeking Ligand Bias: Assessing GPCR Coupling to Beta-Arrestins for Drug Discovery. Drug Discov Today Technol. Spring; 7(1):e37-e42 (2010).
Taste receptor dependent physical changes to a cell can also be measured, for example, by microscopically assessing size, shape, density or any other physical change mediated by taste receptor activation. Flow cytometry can also be utilized to assess physical changes and/or determine the presence or absence of cellular markers.
This method can further comprise contacting the cell with a second bitter tastant, after contacting the cell with the test compound and the first bitter tastant and prior to measuring bitter taste receptor activity. The first bitter tastant and the second bitter tastant can be the same or different.
When measuring a change in bitter taste receptor activity, bitter receptor activity in a cell contacted with a test compound and a bitter tastant can be compared to bitter receptor activity in a control cell contacted with a bitter tastant, but not contacted with the test compound. Bitter taste receptor activity can also be compared to bitter taste receptor activity in the same cell prior to addition of the test compound or after the effect of the test compound has subsided. For example, decreased concentration of cAMP can occur upon bitter receptor activation. If an increase in cAMP concentration is measured in a cell contacted with a test compound and a bitter tastant as compared to a cell contacted with the bitter tastant, the test compound is a bitter taste modulator that inhibits activation of a bitter taste receptor by the bitter tastant. If a decrease in cAMP concentration is measured in a cell contacted with a test compound and a bitter tastant as compared to a cell contacted with the bitter tastant, the test compound is a bitter taste modulator that enhances activation of a bitter taste receptor by the bitter tastant. In another example, increased release of intracellular calcium can occur upon bitter receptor activation. If a decrease in intracellular calcium is measured in a cell contacted with a test compound and a bitter tastant as compared to a cell contacted with the bitter tastant, the test compound is a bitter taste modulator that inhibits activation of a bitter taste receptor by the bitter tastant. If an increase in intracellular concentration is measured in a cell contacted with a test compound and a bitter tastant as compared to a cell contacted with the bitter tastant, the test compound is a bitter taste modulator that enhances activation of a bitter taste receptor by the bitter tastant. These examples are merely exemplary as any parameter described herein can be measured and compared to appropriate control cells to measure changes in bitter taste receptor activity affected by test compounds.
This method can further comprise measuring the effect of the identified bitter taste modulator in a human or other taste tests in order to evaluate the effect of the bitter taste modulator on bitter taste. Any of the bitter taste modulators identified via the methods described herein can be used in foods, beverages and medicines as flavor or taste modulators in order to inhibit the bitter taste associated with beverages, foods or medicines.
As utilized throughout, consumables include all food products, including but not limited to, cereal products, rice products, tapioca products, sago products, baker's products, biscuit products, pastry products, bread products, confectionery products, dessert products, gums, chewing gums, chocolates, ices, honey products, treacle products, yeast products, baking-powder, salt and spice products, savory products, mustard products, vinegar products, sauces (condiments), tobacco products, cigars, cigarettes, processed foods, cooked fruits and vegetable products, meat and meat products, jellies, jams, fruit sauces, egg products, milk and dairy products, yoghurts, cheese products, butter and butter substitute products, milk substitute products, soy products, edible oils and fat products, medicaments, beverages, carbonated beverages, alcoholic drinks, beers, soft drinks, mineral and aerated waters and other non-alcoholic drinks, fruit drinks, fruit juices, coffee, artificial coffee, tea, cocoa, including forms requiring reconstitution, food extracts, plant extracts, meat extracts, condiments, sweeteners, nutraceuticals, gelatins, pharmaceutical and non-pharmaceutical gums, tablets, lozenges, drops, emulsions, elixirs, syrups and other preparations for making beverages, and combinations thereof.
This method can further comprise comparing bitter receptor activity in a cell contacted with an identified bitter taste modulator and a known bitter tastant with bitter receptor activity in a control cell contacted with a known bitter taste modulator and a known bitter tastant. One of skill in the art would know that known bitter taste modulators have established potencies or activity levels. By comparing bitter taste modulators identified by the methods described herein with known bitter taste modulators, potencies can be established for the identified bitter taste modulators. Depending on the amount of bitter taste receptor activity necessary for a particular food, beverage, medicine or process, one of skill in the art can select one or more of the bitter taste modulators identified by the methods set forth herein based on its potency. The bitter taste modulators identified by the methods set forth herein can be combined with known bitter tastants, sweeteners, umami tastants, bitter taste modulators, sweet taste modulators, umami taste modulators or any combination thereof.
Further provided is a method for identifying a bitter tastant comprising contacting a cell, wherein the cell is derived from airway tissue and endogenously expresses a bitter taste receptor with a test compound, and measuring bitter taste receptor activity. Optionally, the cell endogenously expresses RGS21. An increase in bitter taste receptor activity indicates that the test compound is a bitter tastant.
When measuring bitter taste receptor activity, bitter receptor activity in a cell contacted with a test compound can be compared to bitter receptor activity in a control cell not contacted with the test compound. Bitter taste receptor activity can also be compared to bitter taste receptor activity in the same cell prior to addition of the test compound or after the effect of the test compound has subsided. For example, decreased concentration of cAMP can occur upon bitter receptor activation. If a decrease in cAMP concentration is measured in a cell contacted with a test compound as compared to a control cell not contacted with the test compound, the test compound is a bitter tastant. In another example, increased release of intracellular calcium can occur upon bitter receptor activation. If an increase in intracellular calcium concentration is measured in a cell contacted with a test compound as compared to a control cell not contacted with the test compound, the test compound is a bitter tastant. These examples are merely exemplary as any parameter described herein can be measured and compared to appropriate control cells to measure bitter taste receptor activity effected by test compounds.
This method can further comprise measuring the effect of the identified bitter tastant in a human or other taste tests in order to evaluate the effect of the bitter tastant on bitter taste. Any of the bitter tastants identified via the methods described herein can be used in consumables such as foods, beverages and medicines in order to increase bitterness associated with beverages, foods or medicines. Alternatively, any of the bitter tastants identified via the methods described herein can be selectively removed from beverages, foods or medicines or the processes utilized to make beverages, food and medicines in order to reduce bitterness.
This method can further comprise comparing bitter receptor activity in a cell contacted with an identified bitter tastant with bitter receptor activity in a control cell contacted with a known bitter tastant. One of skill in the art would know that known bitter tastants have established potencies or activity levels. By comparing bitter tastants identified by the methods described herein with known bitter tastants, potencies can be established for the identified bitter tastants. Depending on the amount of bitter taste receptor activity necessary for a particular food, beverage, medicine or process, one of skill in the art can select one or more of the bitter tastants identified by the methods set forth herein based on its potency. The bitter tastants identified by the methods set forth herein can be combined with known bitter tastants, sweeteners or umami tastants.
Cell Lines
Further provided is an isolated, relatively pure population of airway cells that express a bitter taste receptor. The bitter taste receptor can be T2R46 or T2R38. Optionally, the cells endogenously express the bitter taste receptor and RGS21 but endogenously expressing cells can be modified to overexpress bitter taste receptors.
Also provided is a population of cells that can be utilized to assess bitter taste receptor activity for a test compound. For example, a population of airway cells that endogenously express a bitter taste receptor can be contacted with the test compound. Taste receptor activity can be measured in the cells as described herein. The test compound can then be identified as a bitter tastant. Optionally, at the initial screening stage, a combined population of airway cells in which a subset expresses one receptor and another subset expresses a different receptor could be used. If this combined population provides a positive effect, subsequent tests can be performed with cell populations expressing only one type of receptor for more specific analysis.
Similarly, a population of airway cells that endogenously express a bitter taste receptor can be contacted with a bitter tastant and the test compound. Taste receptor activity can be measured in the cells as described herein. The test compound can be then be identified as a bitter taste modulator.
For example, a population of 16HBE cells can be utilized to assess bitter taste receptor activity. These examples are not meant to be limiting as the cell can be any airway cell that endogenously expresses a bitter taste receptor. The cell can optionally express RGS21.
As used herein, the terms isolated and relatively pure refer to a state of purification greater than that which occurs naturally. In particular, isolated populations of cells described herein are substantially free from the materials with which the cells are normally associated in nature. By relatively pure is meant in a percentage of purity that exceeds nature, including for example 80% to 100% pure or any value in between.
As used in the specification and the appended claims, the singular forms “a, an and the” include plural referents unless the context clearly dictates otherwise. The term or refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, comprises means includes. Thus, comprising A or B, means “including A, B, or A and B, without excluding additional elements.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention except as and to the extent that they are included in the accompanying claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for.
Cell lines were selected that endogenously express a bitter taste receptor. These cells lines include, but are not limited to, 16HBE. 16HBE cells express RGS21 as well as bitter taste receptors T2R46 and T2R38 (
Functional Assay Using Tastants
Transient Gene Overexpression
16-HBE cells were seeded onto 6 well plates at a density of 3×105 cells per well and incubated in DMEM supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37° C. in a 5% CO2/95% air atmosphere. After 24 hours, media was replaced with fresh media. Plasmid DNA (1.5 μg) and FuGENE 6 (Roche; Indianapolis, Ind.) were complexed and added dropwise to each well, per the manufacturer's instructions. Cell monolayers were incubated an additional 24 hours prior to use in the FLIPR assay for transient intracellular calcium mobilization.
Stable Gene Underexpression
Stable 16-HBE cell lines were generated via lentiviral infection. pLK0.1 plasmids encoding human RGS21-directed shRNA (Oligo IDs TRCN0000036859, TRCN0000036861, and TRCN0000036863; generated by The RNAi Consortium and purchased from Open Biosystems as catalog # RH53979-9604267, RH53979-98492449, and RH53979-9604271) were prepared from bacterial stocks via maxiprep (Qiagen; Valencia, Calif.) and packaged into a lentiviral vector by the UNC Lineberger Comprehensive Cancer Center Lenti-shRNA Core Facility. A control empty lentiviral vector (Open Biosystems; Huntsville, Ala. (catalog #RHS4080)) was also packaged to establish the negative control cell line. These viruses were used to infect separate 16-HBE cell cultures seeded onto 100 mm dishes at 50% confluency. Stably-transfected cell lines were selected with puromycin (Cellgro; Manassas, Va.) and maintained in standard media supplemented with puromycin for several weeks prior to use in the FLIPR assay.
GloSensor cAMP Assays
Twenty-four hours post-transfection, cells were re-plated on poly-D-lysine-treated, clear-bottom, white 384-well plates at a density of 15,000 cells/well. Forty-eight hours post-transfection, culture medium was aspirated and cells were washed once with assay medium (DMEM (without FBS or phenol), 15 mM HEPES pH 7.4) before being incubated for 2 hours with 20 ill/well of equilibration medium (assay medium with 4% GloSensor™ substrate (Promega; Madison, Wis.)). After two hours, 10 μl of 3× final concentration denatonium benzoate (diluted in 3 μM forskolin-containing assay medium) was added to each well and allowed to incubate for 10 minutes before GloSensor™ emission was read on a MicroBeta Plate Counter (PerkinElmer; Waltham, Mass.). Before plotting, luminescence counts were normalized to 100% maximal response for each condition to account for variability in GloSensor™ expression, transfection efficiency, and the exact number of cells per well.
Fluorescence Imaging Plate Reader (FLIPR) calcium Flux Assays
Calcium flux assays were performed as previously described in Strachan et al., “Ribosomal S6 kinase 2 directly phosphorylates the 5-hydroxytryptamine 2A (5-HT2A) serotonin receptor, thereby modulating 5-HT2A signaling,” J Biol Chem 284:5557-5573 (2009). 16-HBE cells were trypsinized, counted, and seeded onto clear-bottomed 96 well plates (Greiner Bio-One; Monroe, N.C.) pre-coated with poly-D-lysine, at a density of 7.5×105 cells per well. After a 24 hour incubation, media was removed and replaced with a Ca2+ assay buffer (20 mM HEPES, lx HBSS, 2.5 mM probenecid, and Ca2+ assay dye, pH 7.4) (FLIPR® Calcium Assay Kit; Molecular Device Corp, Sunnyvale, Calif.). After a 1-hour incubation at 37° C., during which the cells were allowed to take up the dye, fluorescence responses of cells were measured with a FLIPRTETRA (Molecular Device Corp; Sunnyvale, Calif.) device upon the addition of variable concentrations of tastant, or vehicle, in the presence of assay buffer (20 mM HEPES, pH 7.4, 1× Hanks Balanced Salt [Invitrogen; Carlsbad, Calif.] and 2.5 mM probenecid). After data acquisition, a subsequent addition of 5 mM thapsigargin was injected into each well, and fluorescence was measured again. Net peak responses to tastants were normalized to net peak responses to thapsigargin. Responses were compared with that of wild-type control 16-HBE cells. Statistical and graphical analyses were performed using Prism v. 5.0b (GraphPad Software; La Jolla, Calif.).
Results
16HBE cells were selected for bitter taste stimulation. The cells were loaded with fluorescent calcium-sensitive dye, treated with a variety of tastants, and monitored for intracellular calcium release with a FLIPR imaging device.
As shown in
These results show that 16HBE cells can be used for the cell-based detection of bitterants, or bitter tastants.
This application claims benefit of and priority to U.S. Provisional Application No. 61/521,129 filed Aug. 8, 2011, the contents of which are hereby incorporated by reference in their entirety.
Number | Name | Date | Kind |
---|---|---|---|
4115538 | Satoh et al. | Sep 1978 | A |
4132770 | Barth | Jan 1979 | A |
4146501 | Henkin | Mar 1979 | A |
4586625 | Garrett | May 1986 | A |
4677126 | Janusz et al. | Jun 1987 | A |
4692512 | Janusz | Sep 1987 | A |
4692513 | Blum et al. | Sep 1987 | A |
4883888 | Gardlik | Nov 1989 | A |
4896303 | Leslie et al. | Jan 1990 | A |
4918103 | Park et al. | Apr 1990 | A |
4940056 | Heck et al. | Jul 1990 | A |
5233988 | Raghuprasad | Aug 1993 | A |
5242693 | Kurihara et al. | Sep 1993 | A |
5275823 | France et al. | Jan 1994 | A |
5326284 | Bohbot et al. | Jul 1994 | A |
5447869 | Okahata | Sep 1995 | A |
5482855 | Yamafuji et al. | Jan 1996 | A |
5536491 | Asai et al. | Jul 1996 | A |
5631038 | Kurtz et al. | May 1997 | A |
5631231 | Kurtz et al. | May 1997 | A |
5631232 | Kurtz et al. | May 1997 | A |
5631240 | Kurtz et al. | May 1997 | A |
5631252 | Kurtz et al. | May 1997 | A |
5631272 | Kurtz et al. | May 1997 | A |
5631292 | Kurtz et al. | May 1997 | A |
5631294 | Kurtz et al. | May 1997 | A |
5631295 | Kurtz et al. | May 1997 | A |
5631299 | Kurtz et al. | May 1997 | A |
5637618 | Kurtz et al. | Jun 1997 | A |
5639788 | Kurtz et al. | Jun 1997 | A |
5641795 | Kurtz et al. | Jun 1997 | A |
5641799 | Kurtz et al. | Jun 1997 | A |
5641811 | Kurtz et al. | Jun 1997 | A |
5641812 | Kurtz et al. | Jun 1997 | A |
5643894 | Kurtz et al. | Jul 1997 | A |
5643941 | Kurtz et al. | Jul 1997 | A |
5643945 | Kurtz et al. | Jul 1997 | A |
5643955 | Kurtz et al. | Jul 1997 | A |
5643956 | Kurtz et al. | Jul 1997 | A |
5646122 | Kurtz et al. | Jul 1997 | A |
5650403 | Kurtz et al. | Jul 1997 | A |
5654311 | Kurtz et al. | Aug 1997 | A |
5665755 | Kurtz et al. | Sep 1997 | A |
5688662 | Margolskee | Nov 1997 | A |
5693756 | Li et al. | Dec 1997 | A |
5700792 | Kurtz et al. | Dec 1997 | A |
5703053 | Kurtz et al. | Dec 1997 | A |
5766622 | Nelson | Jun 1998 | A |
5785984 | Kurihara et al. | Jul 1998 | A |
5817759 | Margoiskee | Oct 1998 | A |
5866608 | Kurtz et al. | Feb 1999 | A |
5891646 | Barak et al. | Apr 1999 | A |
5910508 | Thoreau et al. | Jun 1999 | A |
6008000 | Margolskee | Dec 1999 | A |
6008250 | Kurtz et al. | Dec 1999 | A |
6015792 | Kurtz et al. | Jan 2000 | A |
6019851 | Pittet et al. | Feb 2000 | A |
6048999 | Pajor et al. | Apr 2000 | A |
6086920 | Frisbee et al. | Jul 2000 | A |
6110693 | Barak et al. | Aug 2000 | A |
6153220 | Cumming et al. | Nov 2000 | A |
6166076 | Gilbertson | Dec 2000 | A |
6242029 | Pittet et al. | Jun 2001 | B1 |
6245376 | Pittet et al. | Jun 2001 | B1 |
6251193 | Rossy et al. | Jun 2001 | B1 |
6251463 | Rossy et al. | Jun 2001 | B1 |
6270807 | Danielson et al. | Aug 2001 | B1 |
6273719 | Whitman | Aug 2001 | B1 |
6306372 | Stier et al. | Oct 2001 | B1 |
6352851 | Nielsen et al. | Mar 2002 | B1 |
6383778 | Zuker et al. | May 2002 | B1 |
6420527 | Bolen et al. | Jul 2002 | B1 |
6461658 | Merkel et al. | Oct 2002 | B1 |
6475510 | Venkatesh et al. | Nov 2002 | B1 |
6540978 | Margolskee et al. | Apr 2003 | B1 |
6558910 | Zuker et al. | May 2003 | B2 |
6608176 | Chaudhari et al. | Aug 2003 | B2 |
6623939 | Zuker et al. | Sep 2003 | B1 |
6815176 | Zuker et al. | Nov 2004 | B1 |
6818747 | Yao et al. | Nov 2004 | B2 |
6875574 | Zuker | Apr 2005 | B1 |
6893827 | Palmer et al. | May 2005 | B1 |
6913906 | Bolen et al. | Jul 2005 | B2 |
6929925 | Zuker et al. | Aug 2005 | B1 |
6942874 | McGregor et al. | Sep 2005 | B2 |
6955887 | Adler et al. | Oct 2005 | B2 |
6998144 | Merkel et al. | Feb 2006 | B2 |
7000718 | Adachi et al. | Feb 2006 | B2 |
7018812 | Oakley et al. | Mar 2006 | B2 |
7022488 | Servant et al. | Apr 2006 | B2 |
7030630 | Haas et al. | Apr 2006 | B2 |
7041457 | Yao et al. | May 2006 | B2 |
7052857 | Zoller et al. | May 2006 | B2 |
7087394 | Johnson et al. | Aug 2006 | B2 |
7105650 | Adler | Sep 2006 | B2 |
7122365 | Nielsen et al. | Oct 2006 | B2 |
7125564 | Chen et al. | Oct 2006 | B2 |
7144733 | Miesenbock et al. | Dec 2006 | B2 |
7179614 | Margolskee et al. | Feb 2007 | B2 |
7186819 | San Gabriel et al. | Mar 2007 | B2 |
7196190 | Ning et al. | Mar 2007 | B2 |
7208290 | Li et al. | Apr 2007 | B2 |
7223551 | Adler et al. | May 2007 | B2 |
7227302 | Iida et al. | Jun 2007 | B2 |
7241880 | Adler et al. | Jul 2007 | B2 |
7244584 | Zuker et al. | Jul 2007 | B2 |
7244835 | Adler et al. | Jul 2007 | B2 |
7270967 | Zuker et al. | Sep 2007 | B2 |
7279338 | Kim et al. | Oct 2007 | B2 |
7282557 | Zuker et al. | Oct 2007 | B2 |
7287557 | Bunke et al. | Oct 2007 | B2 |
7291485 | Yao et al. | Nov 2007 | B2 |
7294474 | Zoller et al. | Nov 2007 | B2 |
7297543 | Zoller et al. | Nov 2007 | B2 |
7297772 | Zoller et al. | Nov 2007 | B2 |
7301009 | Zoller et al. | Nov 2007 | B2 |
7303886 | Zoller et al. | Dec 2007 | B2 |
7309577 | Zoller et al. | Dec 2007 | B2 |
7314716 | Huang et al. | Jan 2008 | B2 |
7314725 | Drayna et al. | Jan 2008 | B2 |
7332292 | Oakley et al. | Feb 2008 | B2 |
7335479 | Zuker | Feb 2008 | B2 |
7335487 | Liao et al. | Feb 2008 | B2 |
7338771 | Pronin et al. | Mar 2008 | B2 |
7341842 | Margolskee et al. | Mar 2008 | B2 |
7344845 | Han et al. | Mar 2008 | B2 |
7344859 | Zoller et al. | Mar 2008 | B2 |
7344882 | Lee et al. | Mar 2008 | B2 |
7354753 | Nielsen et al. | Apr 2008 | B2 |
7364867 | Margolskee et al. | Apr 2008 | B2 |
7364903 | Zoller et al. | Apr 2008 | B2 |
7368285 | Zoller et al. | May 2008 | B2 |
7371546 | Svendsen | May 2008 | B2 |
7374878 | Stryer et al. | May 2008 | B2 |
7393654 | Adler | Jul 2008 | B2 |
7396651 | Adler | Jul 2008 | B2 |
7396663 | Burford et al. | Jul 2008 | B2 |
7399601 | Adler | Jul 2008 | B2 |
7402400 | Zuker et al. | Jul 2008 | B2 |
7402415 | Nair et al. | Jul 2008 | B2 |
7405283 | Sampson et al. | Jul 2008 | B2 |
7407765 | Li et al. | Aug 2008 | B2 |
7407769 | Zuker et al. | Aug 2008 | B2 |
7413867 | Bufe et al. | Aug 2008 | B2 |
7419791 | Adler et al. | Sep 2008 | B2 |
7435552 | Adler et al. | Oct 2008 | B2 |
7452685 | Adler et al. | Nov 2008 | B2 |
7452694 | Zuker et al. | Nov 2008 | B2 |
RE40594 | Margolskee et al. | Dec 2008 | E |
7459277 | Erienbach et al. | Dec 2008 | B2 |
7459532 | Lee et al. | Dec 2008 | B2 |
7465550 | Zuker et al. | Dec 2008 | B2 |
7476399 | Tachdjian et al. | Jan 2009 | B2 |
7479373 | Zuker et al. | Jan 2009 | B2 |
7488599 | Rawson et al. | Feb 2009 | B2 |
7504481 | Lee et al. | Mar 2009 | B2 |
7507544 | Adler et al. | Mar 2009 | B2 |
7507793 | Zuker et al. | Mar 2009 | B2 |
7517972 | Adler et al. | Apr 2009 | B2 |
7524637 | Adler et al. | Apr 2009 | B2 |
7527944 | Li et al. | May 2009 | B2 |
7534577 | Adler et al. | May 2009 | B2 |
7541158 | Li et al. | Jun 2009 | B2 |
7542162 | Muratani | Jun 2009 | B2 |
7579453 | Drayna et al. | Aug 2009 | B2 |
7588900 | Zuker et al. | Sep 2009 | B2 |
7588916 | Adler et al. | Sep 2009 | B2 |
7595166 | Zuker et al. | Sep 2009 | B2 |
7598430 | Weeks et al. | Oct 2009 | B2 |
7601513 | Zoller et al. | Oct 2009 | B2 |
7601883 | Zuker et al. | Oct 2009 | B2 |
7611848 | Gaven et al. | Nov 2009 | B2 |
7622258 | Sampson et al. | Nov 2009 | B2 |
7629134 | Matsunami et al. | Dec 2009 | B2 |
7638289 | Adler | Dec 2009 | B2 |
7655422 | Adler et al. | Feb 2010 | B2 |
7704698 | Adler | Apr 2010 | B2 |
7705121 | Adler et al. | Apr 2010 | B2 |
7718383 | Adler | May 2010 | B2 |
7723051 | Adler | May 2010 | B2 |
7723481 | Adler | May 2010 | B2 |
7736862 | Adler | Jun 2010 | B2 |
7737254 | Adler et al. | Jun 2010 | B2 |
7745601 | Zuker et al. | Jun 2010 | B2 |
7763431 | Zoller et al. | Jul 2010 | B1 |
7772385 | Adler et al. | Aug 2010 | B2 |
7776561 | Pronin et al. | Aug 2010 | B2 |
20020119526 | Zuker et al. | Aug 2002 | A1 |
20020160424 | Adler et al. | Oct 2002 | A1 |
20020164645 | Zuker et al. | Nov 2002 | A1 |
20030008344 | Adler et al. | Jan 2003 | A1 |
20030022288 | Zuker et al. | Jan 2003 | A1 |
20030036630 | Zuker et al. | Feb 2003 | A1 |
20030040045 | Zuker et al. | Feb 2003 | A1 |
20030054448 | Adler et al. | Mar 2003 | A1 |
20030101001 | Huang | May 2003 | A1 |
20030148448 | Liao et al. | Aug 2003 | A1 |
20030166137 | Zuker et al. | Sep 2003 | A1 |
20030220479 | Li et al. | Nov 2003 | A1 |
20030232407 | Zoller et al. | Dec 2003 | A1 |
20040108149 | Adachi et al. | Jun 2004 | A1 |
20040132075 | Elliot et al. | Jul 2004 | A1 |
20040171042 | Adler et al. | Sep 2004 | A1 |
20040175792 | Zoller et al. | Sep 2004 | A1 |
20040175793 | Zoller et al. | Sep 2004 | A1 |
20040185469 | Zoller et al. | Sep 2004 | A1 |
20040191805 | Adler et al. | Sep 2004 | A1 |
20040191862 | Zoller et al. | Sep 2004 | A1 |
20040209286 | Adler et al. | Oct 2004 | A1 |
20040214239 | Servant et al. | Oct 2004 | A1 |
20040219632 | Margolskee et al. | Nov 2004 | A1 |
20040229239 | Adler et al. | Nov 2004 | A1 |
20050032158 | Adler et al. | Feb 2005 | A1 |
20050048508 | Ariyasu et al. | Mar 2005 | A1 |
20050084506 | Tachdjian et al. | Apr 2005 | A1 |
20050084932 | Zoller et al. | Apr 2005 | A1 |
20050085625 | Li et al. | Apr 2005 | A1 |
20050106571 | Erlenbach et al. | May 2005 | A1 |
20050164310 | Zuker et al. | Jul 2005 | A1 |
20050177886 | Margolskee et al. | Aug 2005 | A1 |
20050181461 | Zuker | Aug 2005 | A1 |
20050244810 | Egan et al. | Nov 2005 | A1 |
20050260599 | Ryba et al. | Nov 2005 | A1 |
20050287517 | Adler et al. | Dec 2005 | A1 |
20060014208 | Zoller et al. | Jan 2006 | A1 |
20060040255 | Ookura et al. | Feb 2006 | A1 |
20060045953 | Tachdjian et al. | Mar 2006 | A1 |
20060127977 | Zoller et al. | Jun 2006 | A1 |
20060134693 | Servant et al. | Jun 2006 | A1 |
20060160176 | Zoller et al. | Jul 2006 | A1 |
20060257543 | Tachdjian et al. | Nov 2006 | A1 |
20060275765 | Slack et al. | Dec 2006 | A1 |
20070003680 | Tachdjian et al. | Jan 2007 | A1 |
20070037134 | Servant et al. | Feb 2007 | A1 |
20070042435 | Zuker et al. | Feb 2007 | A1 |
20070054266 | Sato et al. | Mar 2007 | A1 |
20070065884 | Zuker et al. | Mar 2007 | A1 |
20070104709 | Li et al. | May 2007 | A1 |
20070105159 | Erlenbach et al. | May 2007 | A1 |
20070161053 | Li et al. | Jul 2007 | A1 |
20070166728 | Abramson | Jul 2007 | A1 |
20070185312 | Zuker et al. | Aug 2007 | A1 |
20080039534 | Radhakrishna et al. | Feb 2008 | A1 |
20080187936 | Li et al. | Aug 2008 | A1 |
20090311686 | Slack et al. | Dec 2009 | A1 |
20130131108 | Liggett et al. | May 2013 | A1 |
Number | Date | Country |
---|---|---|
WO0006593 | Feb 2000 | WO |
Entry |
---|
Robinett KS, et al. Am. J. Respir. Cell Mol. Biol. doi: 10.1165/rcmb.2011-0061OC. Epub Jun. 3, 2011. |
Doggrell SA, Expert Opin Ther Targets. Jul. 2011;15(7):899-902. doi: 10.1517/14728222.2011.580279. Epub Apr. 27, 2011. |
Tizzano M, et al. BMC Pulmonary Medicine 2011, 11:3 doi:10.1186/1471-2466-11-3. |
Despande DA et al., Nat Med. Nov. 2010;16(11):1299-1304. doi: 10.1038/nm.2237. Epub Oct. 24, 2010. |
Dehkordi O, et al. Life Sci. Feb. 13, 2010;86(7-8):281-8. doi: 10.1016/j.lfs.2009.12.016. Epub Jan. 10, 2010. |
Deshpande DA, et al. Nature Medicine, 16(11):1299-1305, Nov. 2010. |
Cohen SP, et al. J. Biol. Chem. 287(50):41706-41719, Dec. 7, 2012. |
White SR, et al. Am. J. Physiol. 259(4 Pt 1):L294-L303, Oct. 1990. |
Aaronson et al., Human Sarcoma Cells in Culture, Experimental Cell Research, 61:1-5 (1970). |
Akabas et al., A Bitter Substance Induces a Rise in Intracellular Calcium in a Subpopulation of Rat Taste Cells, Science, 242:1047-1050 (1988). |
Bachmanov et al., Taste Receptor Genes, Annu. Rev. Nutr., 27:389-414 (2007). |
Behrens et al., Gustatory Expression Pattern of the Human TAS2R Bitter Receptor Gene Family Reveals a Heterogenous Population of Bitter Responsive Taste Receptor Cells, J. Neuroscience, 27(46):12630-12640 (2007). |
Bohn et al., Seeking Ligand Bias: Assessing GPCR Coupling to Beta-Arrestins for Drug Discovery, Drug Discov. Today Technol., 7(1):e37-e42 (2010). |
Chandrashekar et al., The Receptors and Cells for Mammalian Taste, Nature, 444:288-294 (2006). |
Cooper et al., [35S]GTPYS binding G protein-coupled receptor assays, Methods Mol. Biol., 552:143-151 (2009). |
Danilova et al., Comparison of the Responses of the Chorda Tympani and glossopharyngeal nerves to taste stimuli in C57BL/6J Mice, BMC Neuroscience, 4:5 (Mar. 2003). |
Danilova et al., Sense of Taste in a New World Monkey, the Common Marmoset: Recordings from the Chorda Tympani and Glossopharyngeal Nerves, J. Neurophysiol., 88:579-594 (2002). |
Danilova et al., Responses of Single Taste Fibers and Whole Chorda Tympani and Glossopharyngeal Nerve in the Domestic Pig, Chem. Senses, 24:301-316 (1999). |
Danilova et al., Gustatory Responses of the Hamster Mesocricetus auratus to Various Compounds Considered Sweet by Humans, J. Neurophysiol., 80:2102-2112 (1998). |
De Los Frailes et al., Screening technologies for G protein-coupled receptors: from HTS to uHTS, Methods Mol. Biol., 552:15-37 (2009). |
Di Certo et al., Delayed internalization and lack of recycling in a beta2-adrenergic receptor fused to the G protein alpha-subunit, BMC Cell Biol., 9:56 (2008). |
Farbman et al., Evidence for a Novel Mechanism of Binding and Release of Stimuli in the Primate Taste Bud, J. Neuroscience, 9(10):3522-3528 (1989). |
Felley-Bosco et al., Constitutive expression of inducible nitric oxide synthase in human bronchial epithelial cells induces c-fos and stimulates the cGMP pathway, Am. J. Resp. Cell and Mol. Biol., 11:159-164 (1994). |
Fujiyama et al., Intracellular free calcium concentration in human taste bud cells increases in response to taste stimuli, FEBS Letters, 434:47-50 (1998). |
Harding et al., Direct analysis of a GPCR-agonist interaction by surface plasmon resonance, Eur. Biophys. J., 35(8):709-712 (2006). |
Hellekant et al., Primate Sense of Taste: Behavioral and Single Chorda Tympani and Glossopharyngeal Nerve Fiber Recordings in the Rhesus Monkey, Macaca mulatta, J. Neurophysiol., 77:978-993 (1997). |
Kershaw et al., Analysis of Chemokine Receptor Endocytosis and Intracellular Trafficking, Methods Enzymol., 460:357-377(2009). |
Liu et al., A multiplex calcium assay for identification of GPCR agonists and antagonists, Assay Drug Dev. Technol., 8(3):367-379 (2010). |
Liu et al., Comparison on functional assays for Gq-coupled GPCRs by measuring inositol monophospate-1 and intracellular calcium in 1536-well plate format, Curr. Chem. Genomics, 1:70-78 (2008). |
Offermanns et al., Gα15 and Gα16 Couple with a Wide Variety of Receptors to Phospholipase C, J. Bio. Chem., 270:15175-15180 (1995). |
Ogura et al., Bitter Taste Transduction of Denatonium in the Mudpuppy Necturus maculosus, J. Neuroscience, 17(10):3580-3587 (1997). |
Orola et al., Intracellular Free Calcium Concentration in Single Taste Receptor Cells in the Guinea Pig, Acta Otolaryngol (Stockh),112:120-127 (1992). |
Salamon et al., Chapter 6. Plasmon Resonance Methods in Membrane Protein Biology Applications to GPCR Signaling, Methods Enzymol., 461:123-146 (2009). |
Shah et al., Motile Cilia of Human Airway Epithelia are Chemosensory, Science, 325(5944):1131-1134 (2009). |
Strachan et al., Ribosomal S6 kinase 2 directly phosphorylates the 5-hydroxytryptamine 2A (5-HT2A) serotonin receptor, thereby modulating 5-HT2A signaling, J. Biol. Chem., 284:5557-5573 (2009). |
Williams et al., GPCR signaling: understanding the pathway to successful drug discovery, Methods Mol. Biol., 552:39-50 (2009). |
Number | Date | Country | |
---|---|---|---|
20130040316 A1 | Feb 2013 | US |
Number | Date | Country | |
---|---|---|---|
61521129 | Aug 2011 | US |