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1.
Chem Senses ; 40(8): 577-86, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26377607

ABSTRACT

The sweet taste receptor, a heterodimeric G protein-coupled receptor comprised of T1R2 and T1R3, binds sugars, small molecule sweeteners, and sweet proteins to multiple binding sites. The dipeptide sweetener, aspartame binds in the Venus Flytrap Module (VFTM) of T1R2. We developed homology models of the open and closed forms of human T1R2 and human T1R3 VFTMs and their dimers and then docked aspartame into the closed form of T1R2's VFTM. To test and refine the predictions of our model, we mutated various T1R2 VFTM residues, assayed activity of the mutants and identified 11 critical residues (S40, Y103, D142, S144, S165, S168, Y215, D278, E302, D307, and R383) in and proximal to the binding pocket of the sweet taste receptor that are important for ligand recognition and activity of aspartame. Furthermore, we propose that binding is dependent on 2 water molecules situated in the ligand pocket that bridge 2 carbonyl groups of aspartame to residues D142 and L279. These results shed light on the activation mechanism and how signal transmission arising from the extracellular domain of the T1R2 monomer of the sweet receptor leads to the perception of sweet taste.


Subject(s)
Aspartame/metabolism , Receptors, G-Protein-Coupled/metabolism , Sweetening Agents/metabolism , Animals , Aspartame/chemistry , Binding Sites , Dimerization , HEK293 Cells , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Mice , Molecular Docking Simulation , Mutagenesis, Site-Directed , Protein Binding , Protein Structure, Quaternary , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics , Sweetening Agents/chemistry , Taste/physiology
2.
Methods Mol Biol ; 914: 47-63, 2012.
Article in English | MEDLINE | ID: mdl-22976022

ABSTRACT

Detection of weak ligand binding to membrane-spanning proteins, such as receptor proteins at low physiological concentrations, poses serious experimental challenges. Saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy offers an excellent way to surmount these problems. As the name suggests, magnetization transferred from the receptor to its bound ligand is measured by directly observing NMR signals from the ligand itself. Low-power irradiation is applied to a (1)H NMR spectral region containing protein signals but no ligand signals. This irradiation spreads quickly throughout the membrane protein by the process of spin diffusion and saturates all protein (1)H NMR signals. (1)H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the (1)H NMR signals measured from the pool of free ligand. The experiment is repeated with the irradiation pulse placed outside the spectral region of protein and ligand, a condition that does not lead to saturation transfer to the ligand. The two resulting spectra are subtracted to yield the difference spectrum. As an illustration of the methodology, we review here STD-NMR experiments designed to investigate binding of ligands to the human sweet taste receptor, a member of the large family of G-protein-coupled receptors. Sweetener molecules bind to the sweet receptor with low affinity but high specificity and lead to a variety of physiological responses.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Membrane Proteins/metabolism , Animals , Glucose/metabolism , HEK293 Cells , Humans , Ligands , Mice , Models, Biological , Protein Binding , Receptors, Cell Surface/metabolism , Statistics as Topic
3.
Chem Senses ; 36(9): 821-30, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21765060

ABSTRACT

Brazzein, a 54 residue sweet-tasting protein, is thought to participate in a multipoint binding interaction with the sweet taste receptor. Proposed sites for interaction with the receptor include 2 surface loops and the disulfide bond that connects the N- and C-termini. However, the importance of each site is not well understood. To characterize the structural role of the termini in the sweetness of brazzein, the position of the disulfide bond connecting the N- and C-termini was shifted by substituting K3-C4-K5 with C3-K4-R5. The apparent affinity and V(max) of the C3-K4-R5-brazzein (CKR-brazzein) variant were only modestly decreased compared with the wild-type (WT) brazzein. We determined a high-resolution structure of CKR-brazzein by nuclear magnetic resonance spectroscopy (backbone root mean square deviation of 0.39 Å). Comparing the structure of CKR-brazzein with that of WT-brazzein revealed that the terminal ß-strands of the variant display extended ß-structure and increased dynamics relative to WT-brazzein. These results support previous mutagenesis studies and further suggest that, whereas interactions involving the termini are necessary for full function of brazzein, the termini do not constitute the primary site of interaction between brazzein and the sweet taste receptor.


Subject(s)
Disulfides/chemistry , Models, Molecular , Plant Proteins/chemistry , Sweetening Agents/chemistry , Taste , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Molecular Structure , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Sweetening Agents/isolation & purification , Sweetening Agents/metabolism
4.
J Mol Biol ; 398(4): 584-99, 2010 May 14.
Article in English | MEDLINE | ID: mdl-20302879

ABSTRACT

The sweet protein brazzein [recombinant protein with sequence identical with the native protein lacking the N-terminal pyroglutamate (the numbering system used has Asp2 as the N-terminal residue)] activates the human sweet receptor, a heterodimeric G-protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (T1R3). In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: site 1 (Loop43), site 2 (N- and C-termini and adjacent Glu36, Loop33), and site 3 (Loop9-19). Basic residues in site 1 and acidic residues in site 2 were essential for positive responses from each assay. Mutation of Y39A (site 1) greatly reduced positive responses. A bulky side chain at position 54 (site 2), rather than a side chain with hydrogen-bonding potential, was required for positive responses, as was the presence of the native disulfide bond in Loop9-19 (site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus flytrap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in brazzein response. The exception, hT1R2 (human T1R2 subunit of the sweet receptor):R217A/hT1R3 (human T1R3 subunit of the sweet receptor), which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site is involved in subunit-subunit interaction rather than in direct brazzein binding. Results from this study support a multi-point interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.


Subject(s)
Plant Proteins/metabolism , Protein Interaction Mapping , Receptors, G-Protein-Coupled/metabolism , Amino Acid Substitution/genetics , Cell Line , Humans , Models, Biological , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins/genetics , Mutant Proteins/metabolism , Plant Proteins/genetics , Protein Binding , Protein Structure, Quaternary , Receptors, G-Protein-Coupled/genetics
5.
Biochim Biophys Acta ; 1798(2): 82-6, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19664591

ABSTRACT

The sweet receptor is a member of the G-protein coupled receptor family C that detects a wide variety of chemically and structurally diverse sweet-tasting molecules. We recently used saturation transfer difference spectroscopy (STD) to monitor the direct binding of a set of sweet agonists and antagonists to the human taste receptor in membranes prepared from human embryonic kidney (HEK293) cells transfected with and expressing the sweet receptor [F.M. Assadi-Porter, M. Tonelli, E. Maillet, K. Hallenga, O. Benard, M. Max, J.L. Markley, J. Am. Chem. Soc. 130 (2008) 7212-7213]. Here we review this work and related studies, discuss the procedures involved, and expand on their potential for identifying specific binding interactions of ligands to the membrane spanning and extracellular regions of the full heterodimeric sweet taste receptor. Whereas activity assays are unable to distinguish mutations that alter ligand-binding sites from those that alter signal transduction downstream of the binding site, STD NMR now allows us to make this distinction.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Plant Proteins/chemistry , Receptors, G-Protein-Coupled/chemistry , Sweetening Agents/chemistry , Animals , Binding Sites/physiology , Cell Line , Humans , Ligands , Mice , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Binding/physiology , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Sweetening Agents/metabolism
6.
J Am Chem Soc ; 130(23): 7212-3, 2008 Jun 11.
Article in English | MEDLINE | ID: mdl-18481853

ABSTRACT

We present a robust method for monitoring the binding of ligands to the heterodimeric (T1R2+T1R3) human sweet receptor (a family 3 GPCR receptor). The approach utilizes saturation transfer difference (STD) NMR spectroscopy with receptor proteins expressed on the surface of human epithelial kidney cells. The preparation investigated by NMR can contain either live cells or membranes isolated from these cells containing the receptor. We have used this approach to confirm the noncompetitive binding of alitame and cyclamate to the receptor and to determine that greatly reduced receptor binding affinity compared to wild-type brazzein explains the lack of sweetness of brazzein mutant A16C17. This approach opens new avenues for research on the mechanism of action of the sweet receptor and for the design of new noncalorigenic sweeteners.


Subject(s)
Receptors, G-Protein-Coupled/chemistry , Sweetening Agents/chemistry , Humans , Kinetics , Ligands , Nuclear Magnetic Resonance, Biomolecular/methods , Plant Proteins/chemistry , Plant Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Sweetening Agents/metabolism
7.
J Struct Biol ; 162(1): 50-62, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18308584

ABSTRACT

The crystal structure of a sweet protein Mabinlin II (Mab II) isolated from the mature seeds of Capparis masaikai Levl. grown in Southern China has been determined at 1.7A resolution by the SIRAS method. The Mab II 3D structure features in an "all alpha" fold mode consisting of A- and B-chains crosslinked by four disulfide bridges, which is distinct from all known sweet protein structures. The Mabinlin II molecule shows an amphiphilic surface, a cationic face (Face A) and a neutral face (Face B). A unique structural motif consisting of B54-B64 was found in Face B, which adopts a special sequence, NL-P-NI-C-NI-P-NI, featuring four [Asn-Leu/Ile] units connected by three conformational-constrained residues, thus is called the [NL/I] tetralet motif. The experiments for testing the possible interactions of separated A-chain and B-chain and the native Mabinlin II to the sweet-taste receptor were performed through the calcium imaging experiments with the HEK293E cells coexpressed hT1R2/T1R3. The result shows that hT1R2/T1R3 responds to both the integrated Mabinlin II and the individual B-chain in the same scale, but not to A-chain. The sweetness evaluation further identified that the separated B-chain can elicit the sweetness alone, but A-chain does not. All data in combination revealed that the sweet protein Mabinlin II can interact with the sweet-taste receptor hT1R2/T1R3 to elicit its sweet taste, and the B-chain with a unique [NL/I] tetralet motif is the essential structural element for the interaction with sweet-taste receptor to elicit the sweetness, while the A-chain may play a role in gaining a long aftertaste for the integrate Mabinlin II. The findings reported in this paper will be advantage for understanding the diversity of sweet proteins and engineering research for development of a unique sweetener for the food and agriculture based on the Mabinlin II structure as a native model.


Subject(s)
Plant Proteins/chemistry , Sweetening Agents/chemistry , Amino Acid Motifs , Crystallography, X-Ray/methods , Models, Molecular , Plant Proteins/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sweetening Agents/metabolism
8.
Curr Pharm Des ; 12(35): 4591-600, 2006.
Article in English | MEDLINE | ID: mdl-17168764

ABSTRACT

The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.


Subject(s)
Ligands , Receptors, G-Protein-Coupled/metabolism , Sweetening Agents/metabolism , Taste Buds/metabolism , Amino Acid Sequence , Benzene Derivatives/pharmacology , Binding Sites , Cysteine/metabolism , Dimerization , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary , Protein Subunits/metabolism , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/chemistry , Sweetening Agents/chemistry , Taste Buds/drug effects
9.
J Biol Chem ; 280(40): 34296-305, 2005 Oct 07.
Article in English | MEDLINE | ID: mdl-16076846

ABSTRACT

The artificial sweetener cyclamate tastes sweet to humans, but not to mice. When expressed in vitro, the human sweet receptor (a heterodimer of two taste receptor subunits: hT1R2 + hT1R3) responds to cyclamate, but the mouse receptor (mT1R2 + mT1R3) does not. Using mixed-species pairings of human and mouse sweet receptor subunits, we determined that responsiveness to cyclamate requires the human form of T1R3. Using chimeras, we determined that it is the transmembrane domain of hT1R3 that is required for the sweet receptor to respond to cyclamate. Using directed mutagenesis, we identified several amino acid residues within the transmembrane domain of T1R3 that determine differential responsiveness to cyclamate of the human versus mouse sweet receptors. Alanine-scanning mutagenesis of residues predicted to line a transmembrane domain binding pocket in hT1R3 identified six residues specifically involved in responsiveness to cyclamate. Using molecular modeling, we docked cyclamate within the transmembrane domain of T1R3. Our model predicts substantial overlap in the hT1R3 binding pockets for the agonist cyclamate and the inverse agonist lactisole. The transmembrane domain of T1R3 is likely to play a critical role in the interconversion of the sweet receptor from the ground state to the active state.


Subject(s)
Receptors, G-Protein-Coupled/physiology , Alanine , Animals , Binding Sites/physiology , Chimera , Cyclamates , Humans , Mice , Models, Molecular , Mutagenesis, Site-Directed , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics
10.
J Biol Chem ; 280(15): 15238-46, 2005 Apr 15.
Article in English | MEDLINE | ID: mdl-15668251

ABSTRACT

The detection of sweet-tasting compounds is mediated in large part by a heterodimeric receptor comprised of T1R2+T1R3. Lactisole, a broad-acting sweet antagonist, suppresses the sweet taste of sugars, protein sweeteners, and artificial sweeteners. Lactisole's inhibitory effect is specific to humans and other primates; lactisole does not affect responses to sweet compounds in rodents. By heterologously expressing interspecies combinations of T1R2+T1R3, we have determined that the target for lactisole's action is human T1R3. From studies with mouse/human chimeras of T1R3, we determined that the molecular basis for sensitivity to lactisole depends on only a few residues within the transmembrane region of human T1R3. Alanine substitution of residues in the transmembrane region of human T1R3 revealed 4 key residues required for sensitivity to lactisole. In our model of T1R3's seven transmembrane helices, lactisole is predicted to dock to a binding pocket within the transmembrane region that includes these 4 key residues.


Subject(s)
Benzene Derivatives/pharmacology , Receptors, G-Protein-Coupled/chemistry , Alanine/chemistry , Animals , Benzene Derivatives/chemistry , Binding Sites , Cell Line , Cell Membrane/metabolism , Cysteine/chemistry , Dose-Response Relationship, Drug , Humans , Ligands , Mice , Models, Chemical , Models, Molecular , Mutation , Point Mutation , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/metabolism , Recombinant Fusion Proteins/chemistry , Taste , Tryptophan/chemistry
11.
J Biol Chem ; 279(43): 45068-75, 2004 Oct 22.
Article in English | MEDLINE | ID: mdl-15299024

ABSTRACT

A wide variety of chemically diverse compounds taste sweet, including natural sugars such as glucose, fructose, sucrose, and sugar alcohols, small molecule artificial sweeteners such as saccharin and acesulfame K, and proteins such as monellin and thaumatin. Brazzein, like monellin and thaumatin, is a naturally occurring plant protein that humans, apes, and Old World monkeys perceive as tasting sweet but that is not perceived as sweet by other species including New World monkeys, mouse, and rat. It has been shown that heterologous expression of T1R2 plus T1R3 together yields a receptor responsive to many of the above-mentioned sweet tasting ligands. We have determined that the molecular basis for species-specific sensitivity to brazzein sweetness depends on a site within the cysteine-rich region of human T1R3. Other mutations in this region of T1R3 affected receptor activity toward monellin, and in some cases, overall efficacy to multiple sweet compounds, implicating this region as a previously unrecognized important determinant of sweet receptor function.


Subject(s)
Cysteine/chemistry , Plant Proteins/chemistry , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Taste , Alanine/chemistry , Animals , Cell Line , Dose-Response Relationship, Drug , Fluorescence Resonance Energy Transfer , Humans , Mice , Mutation , Phenylalanine/chemistry , Point Mutation , Protein Binding , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/chemistry , Recombinant Fusion Proteins/chemistry , Taste Buds/metabolism , Transfection
12.
J Comp Neurol ; 455(1): 1-10, 2003 Jan 01.
Article in English | MEDLINE | ID: mdl-12454992

ABSTRACT

We investigated the expression of Ggamma13, a recently discovered G protein subunit, and a selection of Gbeta subunits in retinal bipolar cells, by using a transgenic mouse strain in which green fluorescent protein is strongly expressed in a single type of cone bipolar cell. The cells have ON morphology, and patch-clamp recordings in slices confirmed that they are of the physiological ON type. Immunohistochemistry showed that Ggamma13 is expressed in rod bipolar cells and ON cone bipolar cells, where it is colocalized in the dendrites with Galphaomicron. ON and OFF cone bipolar cells and rod bipolar cells were identified among dissociated cells by their green fluorescence and/or distinct morphology. Hybridization of single-cell polymerase chain reaction products with cDNA probes for G protein subunits Gbeta1 to 5 showed that Gbeta3, Gbeta4, and Ggamma13 are coexpressed in ON bipolar cells but not present in OFF bipolar cells. Gbeta1, 2, and 5 are expressed in partially overlapping subpopulations of cone bipolar cells. Ggamma13 and Gbeta3 and/or Gbeta4, thus, seem selectively to participate in signal transduction by ON bipolar cells.


Subject(s)
Heterotrimeric GTP-Binding Proteins/biosynthesis , Protein Subunits/biosynthesis , Retina/metabolism , Animals , GTP-Binding Protein alpha Subunits, G12-G13 , GTP-Binding Protein alpha Subunits, Gi-Go , Gene Expression Regulation/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Retina/chemistry , Retinal Cone Photoreceptor Cells/chemistry , Retinal Cone Photoreceptor Cells/metabolism , Retinal Rod Photoreceptor Cells/chemistry , Retinal Rod Photoreceptor Cells/metabolism
13.
Nat Neurosci ; 5(11): 1169-76, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12368808

ABSTRACT

We used differential screening of cDNAs from individual taste receptor cells to identify candidate taste transduction elements in mice. Among the differentially expressed clones, one encoded Trpm5, a member of the mammalian family of transient receptor potential (TRP) channels. We found Trpm5 to be expressed in a restricted manner, with particularly high levels in taste tissue. In taste cells, Trpm5 was coexpressed with taste-signaling molecules such as alpha-gustducin, Ggamma13, phospholipase C-beta2 (PLC-beta2) and inositol 1,4,5-trisphosphate receptor type III (IP3R3). Our heterologous expression studies of Trpm5 indicate that it functions as a cationic channel that is gated when internal calcium stores are depleted. Trpm5 may be responsible for capacitative calcium entry in taste receptor cells that respond to bitter and/or sweet compounds.


Subject(s)
Calcium Channels/genetics , Calcium Channels/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Taste Buds/physiology , Taste/physiology , Animals , CHO Cells , Calcium/metabolism , Cloning, Molecular , Cricetinae , Gene Expression , Inositol 1,4,5-Trisphosphate Receptors , Isoenzymes/metabolism , Mice , Oocytes/physiology , Phospholipase C beta , RNA, Messenger/analysis , Receptors, Cytoplasmic and Nuclear/metabolism , TRPM Cation Channels , Transducin/genetics , Type C Phospholipases/metabolism , Xenopus laevis
14.
Chem Senses ; 27(8): 719-27, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12379596

ABSTRACT

The transduction of responses to bitter and sweet compounds utilizes guanine nucleotide binding proteins (G proteins) and their coupled receptors. Alpha-gustducin, a transducin-like G protein alpha-subunit, and rod alpha-transducin are expressed in taste receptor cells. Alpha-gustducin knockout mice have profoundly diminished behavioral and electrophysiological responses to many bitter and sweet compounds, although these mice retain residual responses to these compounds. Alpha-gustducin and rod alpha-transducin are biochemically indistinguishable in their in vitro interactions with retinal phosphodiesterase, rhodopsin and G protein betagamma-subunits. To determine if alpha-transducin can function in taste receptor cells and to compare the function of alpha-gustducin versus alpha-transducin in taste transduction in vivo, we generated transgenic mice that express alpha-transducin under the control of the alpha-gustducin promoter in the alpha-gustducin null background. Immunohistochemistry showed that the alpha-transducin transgene was expressed in about two-thirds of the alpha-gustducin lineage of taste receptor cells. Two-bottle preference tests showed that transgenic expression of rod alpha-transducin partly rescued responses to denatonium benzoate, sucrose and the artificial sweetener SC45647, but not to quinine sulfate. Gustatory nerve recordings showed a partial rescue by the transgene of the response to sucrose, SC45647 and quinine, but not to denatonium. These results demonstrate that alpha-transducin can function in taste receptor cells and transduce some taste cell responses. Our results also suggest that alpha-transducin and alpha-gustducin may differ, at least in part, in their function in these cells, although this conclusion must be qualified because of the limited fidelity of the transgene expression.


Subject(s)
Taste/physiology , Transducin/physiology , Analgesics, Non-Narcotic/pharmacology , Animals , Dose-Response Relationship, Drug , Green Fluorescent Proteins , Guanidines/pharmacology , Immunohistochemistry , Luminescent Proteins/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Microscopy, Fluorescence , Polymerase Chain Reaction , Promoter Regions, Genetic , Quaternary Ammonium Compounds/pharmacology , Quinine/pharmacology , Sucrose/pharmacology , Sweetening Agents/pharmacology , Taste/genetics , Taste Buds/physiology , Transducin/genetics , Transgenes
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