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The human taste receptor hTAS2R14 responds to a variety of different bitter compounds
BBRC Biochemical and Biophysical Research Communications 319 (2004) 479–485 www.elsevier.com/locate/ybbrc
The human taste receptor hTAS2R14 responds to a variety of different bitter compounds Maik Behrens, Anne Brockhoff, Christina Kuhn, Bernd Bufe, Marcel Winnig, and Wolfgang Meyerhof * Department of Molecular Genetics, German Institute of Human Nutrition, Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany Received 21 April 2004 Available online 18 May 2004
Abstract The recent advances in the functional expression of TAS2Rs in heterologous systems resulted in the identification of bitter tastants that specifically activate receptors of this family. All bitter taste receptors reported to date exhibit a pronounced selectivity for single substances or structurally related bitter compounds. In the present study we demonstrate the expression of the hTAS2R14 gene by RT-PCR analyses and in situ hybridisation in human circumvallate papillae. By functional expression in HEK-293T cells we show that hTAS2R14 displays a, so far, unique broad tuning towards a variety of structurally diverse bitter compounds, including the potent neurotoxins, ())-a-thujone, the pharmacologically active component of absinthe, and picrotoxinin, a poisonous substance of fishberries. The observed activation of heterologously expressed hTAS2R14 by low concentrations of ())-a-thujone and picrotoxinin suggests that the receptor is sufficiently sensitive to caution us against the ingestion of toxic amounts of these substances. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Bitter taste; hTAS2R; In situ hybridisation; Heterologous expression; ())-a-Thujone; Picrotoxinin
The sense of taste is important for the evaluation of the quality of food components. Of the five main taste modalities [1] salty, sour, sweet, bitter, and umami, the bitter taste is clearly the most complex process given the number of structurally divergent bitter compounds being detected. It is generally believed that the main function of bitter taste is to avoid intoxication by the ingestion of poisonous substances, although the correlation between toxicity and bitter taste thresholds of compounds can vary depending on the nutritional habits of the studied animals [2]. In humans, bitter taste is mediated by approximately 30 G protein-coupled receptors belonging to the TAS2R gene family [3–5]. In rodents, many bitter taste receptors are co-expressed within the same subset of taste receptor cells [6] that share the same set of signal transduction components * Corresponding author. Fax: +49-33200-88-384. E-mail address: [email protected] (W. Meyerhof).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.05.019
[7]. This may explain the uniquely perceived bitter taste of structurally diverse compounds. Humans are able to detect thousands of different bitter compounds with a limited genetic repertoire of about 30 receptor genes. Since their discovery in the year 2000 [6,8,9] only few mammalian TAS2Rs have been deorphanised. The murine mTAS2R5 [8] and the rat rTAS2R9 [3] respond to the toxic bitter substance cycloheximide, the mouse mTAS2R8 and the human hTAS2R4 respond to high doses of denatonium and, to a lesser extent, to 6-n-propyl-2-thiouracil [8], the human hTAS2R10 and hTAS2R16 respond selectively to strychnine and bitter b-glucopyranosides, respectively [3]. Although for some TAS2Rs a limited promiscuity (mTAS2R8, hTAS2R4) or specificity for a group of chemically related compounds (hTAS2R16) was reported, the relative selectivity of ligand recognition by the receptors published to date does, by far, not explain the enormous number of bitter tastants recognised by
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the mammalian gustatory system. There are several possible mechanisms conceivable to increase the number of tastants recognised by a limited number of taste receptor genes. The simplest way would be to have receptors which exhibit a broad tuning to a great number of structurally divergent ligands. The present report shows the functional characterisation of the human bitter receptor hTAS2R14. We provide evidence for its expression within human vallate papillae and a broad tuning towards a variety of bitter compounds. Among the compounds activating hTAS2R14 are two very potent neurotoxic substances, ())-a-thujone and picrotoxinin. Both substances are naturally occurring plant metabolites and activate hTAS2R14 at low concentrations.
Materials and methods RT-PCR analysis of human tissues. Specimens of human vallate papillae and papillae-free lingual epithelium were obtained with the written consent of volunteers and approved by the local ethical committee. Total RNA was extracted using TRIzol reagent (Invitrogen). Total RNAs from human cerebellum, salivary glands, kidney, and testis were purchased from BD Biosciences Clontech. RNAs were subjected to digestion with RNAse-free DNAse I (Invitrogen) and cDNA synthesis using the Smart cDNA synthesis kit (Clontech). For amplification of hTAS2R14 cDNA oligonucleotides R14_for 50 -GGCCAAT TGGAATTCATGGGTGGTGTCATAAAGAGCATATTTACA-30 and R14_rev 50 -TCCTCAATTGTCATCAGCGGCCGCCAGATGA TT CTCTAAATTCTTTGTGACCTGAG-30 were used. For controls the cDNA of GAPDH was amplified with oligonucleotides GAPDH_ for 50 -ACCACAGTCCATGCCATCAC-30 and GAPDH_rev 50 -TC CCACCACCCTGTTGCTGTA-30 . For negative controls reverse transcriptase was omitted during the cDNA synthesis. The PCR conditions for hTAS2R14 were: 5 min 94 °C predenaturation, 1 min 64 °C, 1.5 min 72 °C, 0.5 min 94 °C for 3 cycles, followed by 35 cycles of 2.5 min 72 °C, 0.5 min 94 °C, and 15 min 72 °C for polishing of PCR products. For GAPDH amplification the following protocol was used: 5 min 94 °C, 28 cycles; 45 s 58 °C, 45 s 72 °C, 30 s 94 °C, and 5 min 58 °C, 10 min 72 °C. In situ hybridization of human vallate papilla. In situ hybridization was mainly done as before [10]. Briefly, 20 lm cross-sections of circumvallate papillae of human tongues were processed and thawmounted onto positively charged glass slides. Prior to hybridisation the sections were postfixated, permeabilised, and acetylated. Prehybridisation was done at 50 °C for 5 h, followed by hybridisation overnight at 50 °C. After hybridisation the slides were washed several times at low stringency, followed by RNAse A treatment and high stringency washes using 0.4 SSC buffer at 50 °C. Hybridised riboprobes were detected using an anti-digoxigenin antibody and colourimetry. Photomicrographs were taken with a CCD camera (RT slider, Diagnostic Instruments) mounted to a Zeiss Axioplan microscope. Immunocytochemistry. Transfection into HEK-293/15 cells, surface labelling, and immunological detection of hTAS2R14 were performed as before [3]. The coding region of hTAS2R14 cloned into the vector pcDNA5FRT (Invitrogen) was amino terminally tagged with 45 amino acids of the amino terminus of the rat SSTR 3 to facilitate plasma membrane targeting [11] and carboxy terminally with the herpes simplex virus glycoprotein D tag (HSV) for immunological detection. Functional expression of hTAS2R14. The cDNA of hTAS2R14 supplemented with an amino terminal export tag corresponding to amino acids 1–45 of rat somatostatin receptor 3 and a carboxy terminal HSV-tag was transiently transfected into HEK-293T cells stably
expressing the chimeric G-protein subunit Ga16gust44 [12] using Lipofectamine 2000 (Invitrogen). Ligand screening using an automated fluorometric imaging plate reader (Molecular Devices) was done 24–32 h after transfection as described [3]. Chemical compounds (Sigma) were dissolved in a suitable mixture of solution C1 and DMSO and administered in solution C1 (130 mM NaCl, 5 mM KCl, 10 mM Hepes, 2 mM CaCl2 , and 10 mM glucose (pH 7.4)) not exceeding a final DMSO concentration of 1% (v/v). All substances were tested in three different concentrations spanning 2 orders of magnitude. Data were collected from two independent experiments carried out in triplicate. Some substances used for the experiments elicited artefact signals on mock-transfected cells at high doses. Data obtained from those concentrations were not incorporated into Fig. 4. The dose–response curves were corrected for background fluorescence and normalised to the maximal response observed.
Results Expression of hTAS2R14 mRNA in human tissues Expression of the hTAS2R14 gene in taste-related and control tissues was analysed by RT-PCR (Fig. 1). A PCR product of 991 bp specific for hTAS2R14 was only detected in lingual tissue containing vallate papillae (A, lane 1). Adjacent tongue tissue devoid of taste papillae does not express hTAS2R14 mRNA as demonstrated by the absence of a specific PCR product (A, lane 2). Cerebellum, salivary glands, kidney, and testis contain no hTAS2R14 mRNA indicating its specificity for gustatory tissues (A, lanes 3–6). The presence of cDNA is demonstrated in all tissues by the amplification of cDNA for GAPDH (B, lanes 1–6). PCRs using DNAse I-digested total RNA without the following reverse transcription were performed to demonstrate the absence of contaminating genomic DNA (A and B, lanes 7–13).
Fig. 1. RT-PCR analysis of hTAS2R14 gene expression in different human tissues. (A) PCR products specific for hTAS2R14, (B) presence of cDNA as detected by GAPDH-specific oligonucleotides used as positive controls. Lane M, molecular weight standard; lane 1, circumvallate papilla; lane 2, lingual epithelium containing no taste papillae; lane 3, salivary gland; lane 4, cerebellum; lane 5, kidney; lane 6, testis; lane 7, circumvallate papillae )RT (reverse transcriptase omitted to assess contaminating genomic DNA); lane 8, lingual epithelium )RT; lane 9, salivary gland )RT; lane 10, cerebellum )RT; lane 11, kidney )RT; lane 12, testis )RT; and lane 13, H2 O control.
M. Behrens et al. / Biochemical and Biophysical Research Communications 319 (2004) 479–485
In situ hybridisation of human vallate papillae In order to show specific expression of hTAS2R14 mRNA within taste receptor cells of the vallate papillae in situ hybridisation using digoxigenin-labelled riboprobes were performed. Detection with the hTAS2R14 antisense probe resulted in labelling of a subset of taste receptor cells within taste buds typical for the expression pattern of TAS2Rs (Fig. 2A). No staining was observed using the sense probe demonstrating the specificity of the procedure (Fig. 2B). Immunocytochemical detection of hTAS2R14 In order to demonstrate expression at plasma membrane level we transfected hTAS2R14 tagged with an export sequence of rat SSTR3 and the HSV-epitope into HEK-293/15 cells (Fig. 3). Detection of the HSV-tag (green fluorescence, A) shows staining at the level of the plasma membrane as well as intracellularly
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located hTAS2R14 protein. The cell surface is visualised by concanavalin A (red fluorescence, B), a plant lectin binding to glycoproteins. The overlay of the green and red fluorescences results in yellow colour demonstrating co-localisation at the plasma membrane (Fig. 3C). Numerous ligands with different affinities activate functionally expressed hTAS2R14 Initially, a panel of 33 structurally diverse putative bitter compounds was screened for activation of hTAS2R14 in transiently transfected HEK-293T cells stably expressing the chimeric G protein Ga16gust44 (Fig. 4). Surprisingly, of this group of compounds eight substances were agonists, comprising almost a quarter of the tested substances. Based on their potency at hTAS2R14 the ligands fall into three groups. The tastants with the highest potency (63 lM) are 1-naphthoic acid, picrotoxinin, and ())-a-thujone. Moderate
Fig. 2. In situ hybridisation of hTAS2R14 mRNA in human circumvallate papillae. Twenty micrometer cryostat cross-sections of human circumvallate papillae were hybridised with digoxigenin-labelled antisense (A) or sense (B) riboprobes. Taste buds are circled. Scale bar ¼ 50 lm.
Fig. 3. Immunocytochemical detection of hTAS2R14 at the cell surface of cells. (A) Detection of the HSV-epitope by an anti-HSV primary antibody and an Alexa488-conjugated secondary antibody. (B) Cell surface labelling as detected by biotin-conjugated concanavalin A and avidin-conjugated Texas Red. (C) Overlay of (A) and (B). Scale bar ¼ 10 lm.
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Fig. 4. Compounds tested for hTAS2R14 activation. The substances used for the ligand screening are identified by their names (row 1) and their chemical structure (row 2). The concentrations tested for hTAS2R14 activation are listed in row 3. The fluorescence traces monitored as a result of an increase of the intracellular calcium concentration after stimulation with the indicated different concentrations of compounds are shown in row 4. In row 5 responses of mock-transfected cells to the corresponding concentrations of compounds are depicted as negative controls. Vertical scale is 8000 counts, horizontal scale is 10 min. n.r. ¼ no response. (A) Compounds activating hTAS2R14 and (B) compounds tested negative.
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potency (630 lM) ligands are: 1,8-naphthalaldehydic acid, 1-nitronaphthalene, and picrotin. Piperonylic acid and sodium benzoate are low potency ligands (6100 lM). There appears to be no obvious common structural motif shared by all substances tested positive for hTAS2R14 responses except that they contain one or several ring systems and at least one electronegative side chain. The molecular sizes of the ligands vary considerably, suggesting that the size of the binding pocket of hTAS2R14 was not a limiting parameter for the substances tested. This observation is further supported by the fact that the high potency ligands ())-a-thujone, 1naphthoic acid, and picrotoxinin contain 1, 2, and 3 ring structures, respectively, whereas the low potency ligand sodium benzoate is the smallest molecule activating hTAS2R14. However, the large fraction of tastants which stimulate hTAS2R14, and their structural and chemical diversity do not predict a low specificity in general as small changes of the molecular structures of ligands have a profound influence on ligand/receptor interactions. The relatively small difference within a single side-chain between picrotin and picrotoxinin results in a significant change in the potency for the two compounds. This is further exemplified by comparing the high potency ligand 1-naphthoic acid with the structurally similar, moderate ligands 1-nitronaphthalene and 1,8-naphthalaldehydic acid, respectively. Among the 25 substances that did not activate hTAS2R14, cycloheximide, 6-n-propyl-2-thiouracil (PROP), D -())-salicin, and strychnine were shown previously to activate other members of the TAS2R gene family. So far, the ligand profiles of the identified human bitter receptors, hTAS2R4 (denatonium, PROP), hTAS2R10 (strychnine), hTAS2R16 (b-glucopyranosides), and hTAS2R14, show no overlap indicating a low degree of functional redundancy. It is important to note that all of these receptors are only distantly related with each other (for a dendrogram see [3]), for more closely related hTAS2Rs a partially overlapping ligand profile appears conceivable. Comparison of the ligand profiles of the identified rodent receptors, mTAS2R5 (cycloheximide), mTAS2R8 (denatonium, PROP), and rTAS2R9 (cycloheximide), with the group of hTAS2R14 agonists demonstrates that hTAS2R14 is not an orthologue of one of the rodent receptors.
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Fig. 5. Dose–response curves of 1-naphthoic acid, picrotoxinin, and ())-a-thujone. Different concentrations of 1-naphthoic acid (A), picrotoxinin (B), and ())-a-thujone (C) were administered by bath application to hTAS2R14-transfected cells. Dose–response curves and the corresponding EC50 values of the effects of these three substances were calculated using sigma plot.
hTAS2R14 by increasing concentrations of 1-naphthoic acid is illustrated by a sigmoidal curve (Fig. 5A) where half-maximal activation (EC50 ) is reached at 36 lM and maximum activation at several hundred micromolars. Picrotoxinin (Fig. 5B) reaches EC50 already at 18 lM and exhibits signal saturation at concentrations around several hundred micromolars. ())-a-Thujone (Fig. 5C) exhibits the lowest EC50 at 15 lM.
Activation of hTAS2R14 is concentration dependent In order to confirm the concentration dependency of hTAS2R14 activation by tastants we determined dose– response curves for three ligands showing the highest potency during our initial screening, 1-naphthoic acid, picrotoxinin, and ())-a-thujone. As shown already in Fig. 4 these substances activate the receptor already at low micromolar concentrations. Activation of
Discussion The recent identification of bitter taste receptors and the functional expression in heterologous systems allowed the characterisation of the ligand profiles of several rodent and human TAS2Rs. All TAS2Rs reported to date exhibit high selectivity for their corresponding
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ligands (mTAS2R5, rTAS2R9), limited promiscuity for few ligands (hTAS2R4, mTAS2R8) or for a family of structurally related compounds (hTAS2R16). The present study on the characterisation of hTAS2R14 demonstrates its broad tuning towards a large variety of putative bitter tastants that are structurally very divergent. This, so far, unique feature of hTAS2R14 might explain how the limited number of bitter taste receptors enables mammals to recognise thousands of different bitter compounds with a limited set of TAS2Rs. It is tempting to speculate that the TAS2R-family consists of members exhibiting high selectivity towards a limited number of ligands, perhaps the most important or abundant toxic plant metabolites present in a given habitat, whereas other members, like hTAS2R14, show a broad tuning in order to facilitate detection of all potentially harmful bitter substances encountered in nature. However, since not all bitter substances are toxic and some, e.g., salicin and quinine, show even beneficial effects, one should not link bitter taste solely with the detection of toxic substances. It has been reported that the bitter rejection response of different animal phyla is not linked to the toxicity of a given bitter substance but rather to the different nutritional habits of the animal [2] and that the rejection of bitter food in some circumstances might even be causally linked to the activity of detoxifying enzymes [13]. Two of the substances we have demonstrated to activate hTAS2R14 are powerful antagonists of GABAA -receptors leading to neurotoxic effects including convulsant action in human and rodents. The IC50 for ())-a-thujone during bath application on rat dorsal root ganglion neurons was reported to be 21 lM [14], the IC50 for picrotoxinin on rat GABAA -receptors expressed in Xenopus laevis oocytes is 1 lM [15]. The high sensitivity of hTAS2R14 for these two substances we observed in our cell culture experiments which have been demonstrated for other TAS2Rs to resemble animal and human psychophysical studies closely [3,8] suggests that this receptor indeed is a functional warning sensor as the concentrations within food containing those substances need to be much higher to lead to pharmacologically active systemic concentrations. One major requirement to allow the binding of structurally highly divergent substances is that the size of the binding pocket should not be limiting for larger molecules. For hTAS2R16 it has been demonstrated that this receptor can accommodate ligand molecules varying considerably in size. This has been attributed to the fact that only the b-glucopyranoside group of the diverse ligands is an important determinant for receptor activations whereas the aglycons seem to influence binding by hTAS2R16 to a lesser extent [3]. This might also be the case for hTAS2R14. Perhaps this receptor recognises only a small, frequently oc-
curring part of bitter tastant molecules allowing for the observed promiscuity. On the other hand, the ligand binding domain of hTAS2R14 might contain several contact sites for different molecular structures of potential ligand molecules which bind tastants with nonoverlapping chemical structures with different affinities. This would imply that there may be even more and bigger ligand molecules than those we used for our ligand profiling. These substances might activate the receptor at even lower concentrations because interaction occurs at multiple contact sites resulting in additive effects of ligand–receptor interactions. Given the large fraction of ligands vs non-ligands we obtained during our screening procedure and the structural diversity of bitter compounds activating hTAS2R14, it appears likely that a considerable number of potential ligand molecules with structures differing from those we identified remain to be discovered. Based on our panel of hTAS2R14 compounds it is very difficult to identify the molecular substructure(s) that are important determinants for the observed differences with regard to hTAS2R14 activation. It is likely that the recognition of the ligands depends on the exact threedimensional presentation of the interacting moieties within the binding pocket of the receptor in addition to other characteristics like electrostatic forces, formation of hydrogen bonds, and occurrence/absence of steric hindrance by parts of the molecules not directly involved in binding. A more detailed study on hTAS2R14/ligand interaction is necessary to answer these questions. Interestingly, although we screened non-toxic and toxic and synthetic as well as natural compounds for their interaction with hTAS2R14, all agonists are natural plant metabolites or derivatives thereof. Picrotoxinin, a GABAA -receptor antagonist [16], is found as a mixture called picrotoxin with the non-toxic picrotin in fishberries (Cocculus indicus) the seeds of the plant Anamirta paniculata. Piperonylic acid is a potent inhibitor for a plant P450 enzyme, the cinnamate 4-hydroxylase [17]. The same enzyme is a target for the equally potent substance 2-hydroxy-1-naphthoic acid [17], which is chemically very similar to the three naphthalene-based compounds we used for our screening (1-naphthoic acid, 1-nitronaphthalene, and 1,8-naphthalaldehydic acid). ())-a-Thujone is the psychotropic component of absinthe, a liqueur made from extracts of wormwood (Artemisia absinthium). It has been shown that ())-a-thujone inhibits both, GABAA - and 5-HT(3) receptor activity [18]. Sodium benzoate is a compound found naturally in many plants and is widely used as an anti-microbial food preservative and for the treatment of hyperammonemia [19]. In conclusion our study demonstrates the specific expression of hTAS2R14 in human taste receptor cells and its functional expression in a heterologous system.
M. Behrens et al. / Biochemical and Biophysical Research Communications 319 (2004) 479–485
We screened a panel of structurally diverse compounds for activation of hTAS2R14 and found that about onequarter of the substances used elicited specific responses. This demonstrates a unique broad tuning of the receptor for structurally highly divergent bitter substances. Among the substances activating hTAS2R14 ())-a-thujone and picrotoxinin are pharmacologically potent plant metabolites implicating that their bitter taste is mediated by hTAS2R14 and serves a warning function against the ingestion of these toxic components.
Acknowledgments We thank Ms. Ulrike Lerner for her excellent technical assistance. This work was supported by a grant from the German Science Foundation (DFG, Me1024/2).
References [1] R.E. Stewart, J.A. DeSimone, D.L. Hill, New perspectives in gustatory physiology: transduction, development, and plasticity, Am. J. Physiol. Cell Physiol. 41 (1997) C1–C26. [2] J.I. Glendinning, Is the bitter rejection response always adaptive, Physiol. Behav. 56 (1994) 1217–1227. [3] B. Bufe, T. Hofmann, D. Krautwurst, J.D. Raguse, W. Meyerhof, The human TAS2R16 receptor mediates bitter taste in response to beta-glucopyranosides, Nat. Genet. 32 (2002) 397–401. [4] C. Conte, M. Ebeling, A. Marcuz, P. Nef, P.J. Andres-Barquin, Identification and characterization of human taste receptor genes belonging to the TAS2R family, Cytogenet. Genome Res. 98 (2002) 45–53. [5] P. Shi, J.Z. Zhang, H. Yang, Y.P. Zhang, Adaptive diversification of bitter taste receptor genes in mammalian evolution, Mol. Biol. Evol. 20 (2003) 805–814. [6] E. Adler, M.A. Hoon, K.L. Mueller, J. Chandrashekar, N.J.P. Ryba, C.S. Zuker, A novel family of mammalian taste receptors, Cell 100 (2000) 693–702. [7] R.F. Margolskee, Molecular mechanisms of bitter and sweet taste transduction, J. Biol. Chem. 277 (2002) 1–4.
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[8] J. Chandrashekar, K.L. Mueller, M.A. Hoon, E. Adler, L.X. Feng, W. Guo, C.S. Zuker, N.J.P. Ryba, T2Rs function as bitter taste receptors, Cell 100 (2000) 703–711. [9] H. Matsunami, J.P. Montmayeur, L.B. Buck, A family of candidate taste receptors in human and mouse, Nature 404 (2000) 601–604. [10] M. Behrens, G. Venkatraman, R.M. Gronostajski, R.R. Reed, F.L. Margolis, NFI in the development of the olfactory neuroepithelium and the regulation of olfactory marker protein gene expression, Eur. J. Neurosci. 12 (2000) 1372–1384. [11] C. Ammon, J. Schafer, O.J. Kreuzer, W. Meyerhof, Presence of a plasma membrane targeting sequence in the amino-terminal region of the rat somatostatin receptor 3, Arch. Physiol. Biochem. 110 (2002) 137–145. [12] T. Ueda, S. Ugawa, H. Yamamura, Y. Imaizumi, S. Shimada, Functional interaction between T2R taste receptors and G-protein alpha subunits expressed in taste receptor cells, J. Neurosci. 23 (2003) 7376–7380. [13] M.J. Snyder, J.I. Glendinning, Causal connection between detoxification enzyme activity and consumption of a toxic plant compound, J. Comp. Physiol. A 179 (1996) 255–261. [14] K.M. Hold, N.S. Sirisoma, T. Ikeda, T. Narahashi, J.E. Casida, Alpha-thujone (the active component of absinthe): gamma-aminobutyric acid type A receptor modulation and metabolic detoxification, Proc. Natl. Acad. Sci. USA 97 (2000) 3826–3831. [15] P. Perret, X. Sarda, M. Wolff, T.T. Wu, D. Bushey, M. Goeldner, Interaction of non-competitive blockers within the gamma-aminobutyric acid type A chloride channel using chemically reactive probes as chemical sensors for cysteine mutants, J. Biol. Chem. 274 (1999) 25350–25354. [16] K.W. Yoon, D.F. Covey, S.M. Rothman, Multiple mechanisms of picrotoxin block of gaba-induced currents in rat hippocampalneurons, J. Physiol. (London) 464 (1993) 423–439. [17] M. Schalk, F. Cabello-Hurtado, M.A. Pierrel, R. Atanossova, P. Saindrenan, D. Werck-Reichhart, Piperonylic acid, a selective, mechanism-based inactivator of the trans-cinnamate 4-hydroxylase: a new tool to control the flux of metabolites in the phenylpropanoid pathway, Plant Physiol. 118 (1998) 209–218. [18] T. Deiml, R. Haseneder, W. Zieglgansberger, G. Rammes, B. Eisensamer, R. Rupprecht, G. Hapfelmeier, Alpha-thujone reduces 5-HT3 receptor activity by an effect on the agonist-induced desensitization, Neuropharmacology 46 (2004) 192–201. [19] B.A. Borshop, J. Breuer, J. Holm, J. Leslie, W.L. Nyhan, Excretion of hippuric acid during sodium benzoate therapy in patients with hyperglycinemia or hyperammonemia, J. Inherit. Metab. Dis. 12 (1989) 72–79.
The human taste receptor hTAS2R14 responds to a variety of different bitter compounds Maik Behrens, Anne Brockhoff, Christina Kuhn, Bernd Bufe, Marcel Winnig, and Wolfgang Meyerhof * Department of Molecular Genetics, German Institute of Human Nutrition, Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany Received 21 April 2004 Available online 18 May 2004
Abstract The recent advances in the functional expression of TAS2Rs in heterologous systems resulted in the identification of bitter tastants that specifically activate receptors of this family. All bitter taste receptors reported to date exhibit a pronounced selectivity for single substances or structurally related bitter compounds. In the present study we demonstrate the expression of the hTAS2R14 gene by RT-PCR analyses and in situ hybridisation in human circumvallate papillae. By functional expression in HEK-293T cells we show that hTAS2R14 displays a, so far, unique broad tuning towards a variety of structurally diverse bitter compounds, including the potent neurotoxins, ())-a-thujone, the pharmacologically active component of absinthe, and picrotoxinin, a poisonous substance of fishberries. The observed activation of heterologously expressed hTAS2R14 by low concentrations of ())-a-thujone and picrotoxinin suggests that the receptor is sufficiently sensitive to caution us against the ingestion of toxic amounts of these substances. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Bitter taste; hTAS2R; In situ hybridisation; Heterologous expression; ())-a-Thujone; Picrotoxinin
The sense of taste is important for the evaluation of the quality of food components. Of the five main taste modalities [1] salty, sour, sweet, bitter, and umami, the bitter taste is clearly the most complex process given the number of structurally divergent bitter compounds being detected. It is generally believed that the main function of bitter taste is to avoid intoxication by the ingestion of poisonous substances, although the correlation between toxicity and bitter taste thresholds of compounds can vary depending on the nutritional habits of the studied animals [2]. In humans, bitter taste is mediated by approximately 30 G protein-coupled receptors belonging to the TAS2R gene family [3–5]. In rodents, many bitter taste receptors are co-expressed within the same subset of taste receptor cells [6] that share the same set of signal transduction components * Corresponding author. Fax: +49-33200-88-384. E-mail address: [email protected] (W. Meyerhof).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.05.019
[7]. This may explain the uniquely perceived bitter taste of structurally diverse compounds. Humans are able to detect thousands of different bitter compounds with a limited genetic repertoire of about 30 receptor genes. Since their discovery in the year 2000 [6,8,9] only few mammalian TAS2Rs have been deorphanised. The murine mTAS2R5 [8] and the rat rTAS2R9 [3] respond to the toxic bitter substance cycloheximide, the mouse mTAS2R8 and the human hTAS2R4 respond to high doses of denatonium and, to a lesser extent, to 6-n-propyl-2-thiouracil [8], the human hTAS2R10 and hTAS2R16 respond selectively to strychnine and bitter b-glucopyranosides, respectively [3]. Although for some TAS2Rs a limited promiscuity (mTAS2R8, hTAS2R4) or specificity for a group of chemically related compounds (hTAS2R16) was reported, the relative selectivity of ligand recognition by the receptors published to date does, by far, not explain the enormous number of bitter tastants recognised by
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the mammalian gustatory system. There are several possible mechanisms conceivable to increase the number of tastants recognised by a limited number of taste receptor genes. The simplest way would be to have receptors which exhibit a broad tuning to a great number of structurally divergent ligands. The present report shows the functional characterisation of the human bitter receptor hTAS2R14. We provide evidence for its expression within human vallate papillae and a broad tuning towards a variety of bitter compounds. Among the compounds activating hTAS2R14 are two very potent neurotoxic substances, ())-a-thujone and picrotoxinin. Both substances are naturally occurring plant metabolites and activate hTAS2R14 at low concentrations.
Materials and methods RT-PCR analysis of human tissues. Specimens of human vallate papillae and papillae-free lingual epithelium were obtained with the written consent of volunteers and approved by the local ethical committee. Total RNA was extracted using TRIzol reagent (Invitrogen). Total RNAs from human cerebellum, salivary glands, kidney, and testis were purchased from BD Biosciences Clontech. RNAs were subjected to digestion with RNAse-free DNAse I (Invitrogen) and cDNA synthesis using the Smart cDNA synthesis kit (Clontech). For amplification of hTAS2R14 cDNA oligonucleotides R14_for 50 -GGCCAAT TGGAATTCATGGGTGGTGTCATAAAGAGCATATTTACA-30 and R14_rev 50 -TCCTCAATTGTCATCAGCGGCCGCCAGATGA TT CTCTAAATTCTTTGTGACCTGAG-30 were used. For controls the cDNA of GAPDH was amplified with oligonucleotides GAPDH_ for 50 -ACCACAGTCCATGCCATCAC-30 and GAPDH_rev 50 -TC CCACCACCCTGTTGCTGTA-30 . For negative controls reverse transcriptase was omitted during the cDNA synthesis. The PCR conditions for hTAS2R14 were: 5 min 94 °C predenaturation, 1 min 64 °C, 1.5 min 72 °C, 0.5 min 94 °C for 3 cycles, followed by 35 cycles of 2.5 min 72 °C, 0.5 min 94 °C, and 15 min 72 °C for polishing of PCR products. For GAPDH amplification the following protocol was used: 5 min 94 °C, 28 cycles; 45 s 58 °C, 45 s 72 °C, 30 s 94 °C, and 5 min 58 °C, 10 min 72 °C. In situ hybridization of human vallate papilla. In situ hybridization was mainly done as before [10]. Briefly, 20 lm cross-sections of circumvallate papillae of human tongues were processed and thawmounted onto positively charged glass slides. Prior to hybridisation the sections were postfixated, permeabilised, and acetylated. Prehybridisation was done at 50 °C for 5 h, followed by hybridisation overnight at 50 °C. After hybridisation the slides were washed several times at low stringency, followed by RNAse A treatment and high stringency washes using 0.4 SSC buffer at 50 °C. Hybridised riboprobes were detected using an anti-digoxigenin antibody and colourimetry. Photomicrographs were taken with a CCD camera (RT slider, Diagnostic Instruments) mounted to a Zeiss Axioplan microscope. Immunocytochemistry. Transfection into HEK-293/15 cells, surface labelling, and immunological detection of hTAS2R14 were performed as before [3]. The coding region of hTAS2R14 cloned into the vector pcDNA5FRT (Invitrogen) was amino terminally tagged with 45 amino acids of the amino terminus of the rat SSTR 3 to facilitate plasma membrane targeting [11] and carboxy terminally with the herpes simplex virus glycoprotein D tag (HSV) for immunological detection. Functional expression of hTAS2R14. The cDNA of hTAS2R14 supplemented with an amino terminal export tag corresponding to amino acids 1–45 of rat somatostatin receptor 3 and a carboxy terminal HSV-tag was transiently transfected into HEK-293T cells stably
expressing the chimeric G-protein subunit Ga16gust44 [12] using Lipofectamine 2000 (Invitrogen). Ligand screening using an automated fluorometric imaging plate reader (Molecular Devices) was done 24–32 h after transfection as described [3]. Chemical compounds (Sigma) were dissolved in a suitable mixture of solution C1 and DMSO and administered in solution C1 (130 mM NaCl, 5 mM KCl, 10 mM Hepes, 2 mM CaCl2 , and 10 mM glucose (pH 7.4)) not exceeding a final DMSO concentration of 1% (v/v). All substances were tested in three different concentrations spanning 2 orders of magnitude. Data were collected from two independent experiments carried out in triplicate. Some substances used for the experiments elicited artefact signals on mock-transfected cells at high doses. Data obtained from those concentrations were not incorporated into Fig. 4. The dose–response curves were corrected for background fluorescence and normalised to the maximal response observed.
Results Expression of hTAS2R14 mRNA in human tissues Expression of the hTAS2R14 gene in taste-related and control tissues was analysed by RT-PCR (Fig. 1). A PCR product of 991 bp specific for hTAS2R14 was only detected in lingual tissue containing vallate papillae (A, lane 1). Adjacent tongue tissue devoid of taste papillae does not express hTAS2R14 mRNA as demonstrated by the absence of a specific PCR product (A, lane 2). Cerebellum, salivary glands, kidney, and testis contain no hTAS2R14 mRNA indicating its specificity for gustatory tissues (A, lanes 3–6). The presence of cDNA is demonstrated in all tissues by the amplification of cDNA for GAPDH (B, lanes 1–6). PCRs using DNAse I-digested total RNA without the following reverse transcription were performed to demonstrate the absence of contaminating genomic DNA (A and B, lanes 7–13).
Fig. 1. RT-PCR analysis of hTAS2R14 gene expression in different human tissues. (A) PCR products specific for hTAS2R14, (B) presence of cDNA as detected by GAPDH-specific oligonucleotides used as positive controls. Lane M, molecular weight standard; lane 1, circumvallate papilla; lane 2, lingual epithelium containing no taste papillae; lane 3, salivary gland; lane 4, cerebellum; lane 5, kidney; lane 6, testis; lane 7, circumvallate papillae )RT (reverse transcriptase omitted to assess contaminating genomic DNA); lane 8, lingual epithelium )RT; lane 9, salivary gland )RT; lane 10, cerebellum )RT; lane 11, kidney )RT; lane 12, testis )RT; and lane 13, H2 O control.
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In situ hybridisation of human vallate papillae In order to show specific expression of hTAS2R14 mRNA within taste receptor cells of the vallate papillae in situ hybridisation using digoxigenin-labelled riboprobes were performed. Detection with the hTAS2R14 antisense probe resulted in labelling of a subset of taste receptor cells within taste buds typical for the expression pattern of TAS2Rs (Fig. 2A). No staining was observed using the sense probe demonstrating the specificity of the procedure (Fig. 2B). Immunocytochemical detection of hTAS2R14 In order to demonstrate expression at plasma membrane level we transfected hTAS2R14 tagged with an export sequence of rat SSTR3 and the HSV-epitope into HEK-293/15 cells (Fig. 3). Detection of the HSV-tag (green fluorescence, A) shows staining at the level of the plasma membrane as well as intracellularly
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located hTAS2R14 protein. The cell surface is visualised by concanavalin A (red fluorescence, B), a plant lectin binding to glycoproteins. The overlay of the green and red fluorescences results in yellow colour demonstrating co-localisation at the plasma membrane (Fig. 3C). Numerous ligands with different affinities activate functionally expressed hTAS2R14 Initially, a panel of 33 structurally diverse putative bitter compounds was screened for activation of hTAS2R14 in transiently transfected HEK-293T cells stably expressing the chimeric G protein Ga16gust44 (Fig. 4). Surprisingly, of this group of compounds eight substances were agonists, comprising almost a quarter of the tested substances. Based on their potency at hTAS2R14 the ligands fall into three groups. The tastants with the highest potency (63 lM) are 1-naphthoic acid, picrotoxinin, and ())-a-thujone. Moderate
Fig. 2. In situ hybridisation of hTAS2R14 mRNA in human circumvallate papillae. Twenty micrometer cryostat cross-sections of human circumvallate papillae were hybridised with digoxigenin-labelled antisense (A) or sense (B) riboprobes. Taste buds are circled. Scale bar ¼ 50 lm.
Fig. 3. Immunocytochemical detection of hTAS2R14 at the cell surface of cells. (A) Detection of the HSV-epitope by an anti-HSV primary antibody and an Alexa488-conjugated secondary antibody. (B) Cell surface labelling as detected by biotin-conjugated concanavalin A and avidin-conjugated Texas Red. (C) Overlay of (A) and (B). Scale bar ¼ 10 lm.
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Fig. 4. Compounds tested for hTAS2R14 activation. The substances used for the ligand screening are identified by their names (row 1) and their chemical structure (row 2). The concentrations tested for hTAS2R14 activation are listed in row 3. The fluorescence traces monitored as a result of an increase of the intracellular calcium concentration after stimulation with the indicated different concentrations of compounds are shown in row 4. In row 5 responses of mock-transfected cells to the corresponding concentrations of compounds are depicted as negative controls. Vertical scale is 8000 counts, horizontal scale is 10 min. n.r. ¼ no response. (A) Compounds activating hTAS2R14 and (B) compounds tested negative.
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potency (630 lM) ligands are: 1,8-naphthalaldehydic acid, 1-nitronaphthalene, and picrotin. Piperonylic acid and sodium benzoate are low potency ligands (6100 lM). There appears to be no obvious common structural motif shared by all substances tested positive for hTAS2R14 responses except that they contain one or several ring systems and at least one electronegative side chain. The molecular sizes of the ligands vary considerably, suggesting that the size of the binding pocket of hTAS2R14 was not a limiting parameter for the substances tested. This observation is further supported by the fact that the high potency ligands ())-a-thujone, 1naphthoic acid, and picrotoxinin contain 1, 2, and 3 ring structures, respectively, whereas the low potency ligand sodium benzoate is the smallest molecule activating hTAS2R14. However, the large fraction of tastants which stimulate hTAS2R14, and their structural and chemical diversity do not predict a low specificity in general as small changes of the molecular structures of ligands have a profound influence on ligand/receptor interactions. The relatively small difference within a single side-chain between picrotin and picrotoxinin results in a significant change in the potency for the two compounds. This is further exemplified by comparing the high potency ligand 1-naphthoic acid with the structurally similar, moderate ligands 1-nitronaphthalene and 1,8-naphthalaldehydic acid, respectively. Among the 25 substances that did not activate hTAS2R14, cycloheximide, 6-n-propyl-2-thiouracil (PROP), D -())-salicin, and strychnine were shown previously to activate other members of the TAS2R gene family. So far, the ligand profiles of the identified human bitter receptors, hTAS2R4 (denatonium, PROP), hTAS2R10 (strychnine), hTAS2R16 (b-glucopyranosides), and hTAS2R14, show no overlap indicating a low degree of functional redundancy. It is important to note that all of these receptors are only distantly related with each other (for a dendrogram see [3]), for more closely related hTAS2Rs a partially overlapping ligand profile appears conceivable. Comparison of the ligand profiles of the identified rodent receptors, mTAS2R5 (cycloheximide), mTAS2R8 (denatonium, PROP), and rTAS2R9 (cycloheximide), with the group of hTAS2R14 agonists demonstrates that hTAS2R14 is not an orthologue of one of the rodent receptors.
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Fig. 5. Dose–response curves of 1-naphthoic acid, picrotoxinin, and ())-a-thujone. Different concentrations of 1-naphthoic acid (A), picrotoxinin (B), and ())-a-thujone (C) were administered by bath application to hTAS2R14-transfected cells. Dose–response curves and the corresponding EC50 values of the effects of these three substances were calculated using sigma plot.
hTAS2R14 by increasing concentrations of 1-naphthoic acid is illustrated by a sigmoidal curve (Fig. 5A) where half-maximal activation (EC50 ) is reached at 36 lM and maximum activation at several hundred micromolars. Picrotoxinin (Fig. 5B) reaches EC50 already at 18 lM and exhibits signal saturation at concentrations around several hundred micromolars. ())-a-Thujone (Fig. 5C) exhibits the lowest EC50 at 15 lM.
Activation of hTAS2R14 is concentration dependent In order to confirm the concentration dependency of hTAS2R14 activation by tastants we determined dose– response curves for three ligands showing the highest potency during our initial screening, 1-naphthoic acid, picrotoxinin, and ())-a-thujone. As shown already in Fig. 4 these substances activate the receptor already at low micromolar concentrations. Activation of
Discussion The recent identification of bitter taste receptors and the functional expression in heterologous systems allowed the characterisation of the ligand profiles of several rodent and human TAS2Rs. All TAS2Rs reported to date exhibit high selectivity for their corresponding
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ligands (mTAS2R5, rTAS2R9), limited promiscuity for few ligands (hTAS2R4, mTAS2R8) or for a family of structurally related compounds (hTAS2R16). The present study on the characterisation of hTAS2R14 demonstrates its broad tuning towards a large variety of putative bitter tastants that are structurally very divergent. This, so far, unique feature of hTAS2R14 might explain how the limited number of bitter taste receptors enables mammals to recognise thousands of different bitter compounds with a limited set of TAS2Rs. It is tempting to speculate that the TAS2R-family consists of members exhibiting high selectivity towards a limited number of ligands, perhaps the most important or abundant toxic plant metabolites present in a given habitat, whereas other members, like hTAS2R14, show a broad tuning in order to facilitate detection of all potentially harmful bitter substances encountered in nature. However, since not all bitter substances are toxic and some, e.g., salicin and quinine, show even beneficial effects, one should not link bitter taste solely with the detection of toxic substances. It has been reported that the bitter rejection response of different animal phyla is not linked to the toxicity of a given bitter substance but rather to the different nutritional habits of the animal [2] and that the rejection of bitter food in some circumstances might even be causally linked to the activity of detoxifying enzymes [13]. Two of the substances we have demonstrated to activate hTAS2R14 are powerful antagonists of GABAA -receptors leading to neurotoxic effects including convulsant action in human and rodents. The IC50 for ())-a-thujone during bath application on rat dorsal root ganglion neurons was reported to be 21 lM [14], the IC50 for picrotoxinin on rat GABAA -receptors expressed in Xenopus laevis oocytes is 1 lM [15]. The high sensitivity of hTAS2R14 for these two substances we observed in our cell culture experiments which have been demonstrated for other TAS2Rs to resemble animal and human psychophysical studies closely [3,8] suggests that this receptor indeed is a functional warning sensor as the concentrations within food containing those substances need to be much higher to lead to pharmacologically active systemic concentrations. One major requirement to allow the binding of structurally highly divergent substances is that the size of the binding pocket should not be limiting for larger molecules. For hTAS2R16 it has been demonstrated that this receptor can accommodate ligand molecules varying considerably in size. This has been attributed to the fact that only the b-glucopyranoside group of the diverse ligands is an important determinant for receptor activations whereas the aglycons seem to influence binding by hTAS2R16 to a lesser extent [3]. This might also be the case for hTAS2R14. Perhaps this receptor recognises only a small, frequently oc-
curring part of bitter tastant molecules allowing for the observed promiscuity. On the other hand, the ligand binding domain of hTAS2R14 might contain several contact sites for different molecular structures of potential ligand molecules which bind tastants with nonoverlapping chemical structures with different affinities. This would imply that there may be even more and bigger ligand molecules than those we used for our ligand profiling. These substances might activate the receptor at even lower concentrations because interaction occurs at multiple contact sites resulting in additive effects of ligand–receptor interactions. Given the large fraction of ligands vs non-ligands we obtained during our screening procedure and the structural diversity of bitter compounds activating hTAS2R14, it appears likely that a considerable number of potential ligand molecules with structures differing from those we identified remain to be discovered. Based on our panel of hTAS2R14 compounds it is very difficult to identify the molecular substructure(s) that are important determinants for the observed differences with regard to hTAS2R14 activation. It is likely that the recognition of the ligands depends on the exact threedimensional presentation of the interacting moieties within the binding pocket of the receptor in addition to other characteristics like electrostatic forces, formation of hydrogen bonds, and occurrence/absence of steric hindrance by parts of the molecules not directly involved in binding. A more detailed study on hTAS2R14/ligand interaction is necessary to answer these questions. Interestingly, although we screened non-toxic and toxic and synthetic as well as natural compounds for their interaction with hTAS2R14, all agonists are natural plant metabolites or derivatives thereof. Picrotoxinin, a GABAA -receptor antagonist [16], is found as a mixture called picrotoxin with the non-toxic picrotin in fishberries (Cocculus indicus) the seeds of the plant Anamirta paniculata. Piperonylic acid is a potent inhibitor for a plant P450 enzyme, the cinnamate 4-hydroxylase [17]. The same enzyme is a target for the equally potent substance 2-hydroxy-1-naphthoic acid [17], which is chemically very similar to the three naphthalene-based compounds we used for our screening (1-naphthoic acid, 1-nitronaphthalene, and 1,8-naphthalaldehydic acid). ())-a-Thujone is the psychotropic component of absinthe, a liqueur made from extracts of wormwood (Artemisia absinthium). It has been shown that ())-a-thujone inhibits both, GABAA - and 5-HT(3) receptor activity [18]. Sodium benzoate is a compound found naturally in many plants and is widely used as an anti-microbial food preservative and for the treatment of hyperammonemia [19]. In conclusion our study demonstrates the specific expression of hTAS2R14 in human taste receptor cells and its functional expression in a heterologous system.
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We screened a panel of structurally diverse compounds for activation of hTAS2R14 and found that about onequarter of the substances used elicited specific responses. This demonstrates a unique broad tuning of the receptor for structurally highly divergent bitter substances. Among the substances activating hTAS2R14 ())-a-thujone and picrotoxinin are pharmacologically potent plant metabolites implicating that their bitter taste is mediated by hTAS2R14 and serves a warning function against the ingestion of these toxic components.
Acknowledgments We thank Ms. Ulrike Lerner for her excellent technical assistance. This work was supported by a grant from the German Science Foundation (DFG, Me1024/2).
References [1] R.E. Stewart, J.A. DeSimone, D.L. Hill, New perspectives in gustatory physiology: transduction, development, and plasticity, Am. J. Physiol. Cell Physiol. 41 (1997) C1–C26. [2] J.I. Glendinning, Is the bitter rejection response always adaptive, Physiol. Behav. 56 (1994) 1217–1227. [3] B. Bufe, T. Hofmann, D. Krautwurst, J.D. Raguse, W. Meyerhof, The human TAS2R16 receptor mediates bitter taste in response to beta-glucopyranosides, Nat. Genet. 32 (2002) 397–401. [4] C. Conte, M. Ebeling, A. Marcuz, P. Nef, P.J. Andres-Barquin, Identification and characterization of human taste receptor genes belonging to the TAS2R family, Cytogenet. Genome Res. 98 (2002) 45–53. [5] P. Shi, J.Z. Zhang, H. Yang, Y.P. Zhang, Adaptive diversification of bitter taste receptor genes in mammalian evolution, Mol. Biol. Evol. 20 (2003) 805–814. [6] E. Adler, M.A. Hoon, K.L. Mueller, J. Chandrashekar, N.J.P. Ryba, C.S. Zuker, A novel family of mammalian taste receptors, Cell 100 (2000) 693–702. [7] R.F. Margolskee, Molecular mechanisms of bitter and sweet taste transduction, J. Biol. Chem. 277 (2002) 1–4.
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[8] J. Chandrashekar, K.L. Mueller, M.A. Hoon, E. Adler, L.X. Feng, W. Guo, C.S. Zuker, N.J.P. Ryba, T2Rs function as bitter taste receptors, Cell 100 (2000) 703–711. [9] H. Matsunami, J.P. Montmayeur, L.B. Buck, A family of candidate taste receptors in human and mouse, Nature 404 (2000) 601–604. [10] M. Behrens, G. Venkatraman, R.M. Gronostajski, R.R. Reed, F.L. Margolis, NFI in the development of the olfactory neuroepithelium and the regulation of olfactory marker protein gene expression, Eur. J. Neurosci. 12 (2000) 1372–1384. [11] C. Ammon, J. Schafer, O.J. Kreuzer, W. Meyerhof, Presence of a plasma membrane targeting sequence in the amino-terminal region of the rat somatostatin receptor 3, Arch. Physiol. Biochem. 110 (2002) 137–145. [12] T. Ueda, S. Ugawa, H. Yamamura, Y. Imaizumi, S. Shimada, Functional interaction between T2R taste receptors and G-protein alpha subunits expressed in taste receptor cells, J. Neurosci. 23 (2003) 7376–7380. [13] M.J. Snyder, J.I. Glendinning, Causal connection between detoxification enzyme activity and consumption of a toxic plant compound, J. Comp. Physiol. A 179 (1996) 255–261. [14] K.M. Hold, N.S. Sirisoma, T. Ikeda, T. Narahashi, J.E. Casida, Alpha-thujone (the active component of absinthe): gamma-aminobutyric acid type A receptor modulation and metabolic detoxification, Proc. Natl. Acad. Sci. USA 97 (2000) 3826–3831. [15] P. Perret, X. Sarda, M. Wolff, T.T. Wu, D. Bushey, M. Goeldner, Interaction of non-competitive blockers within the gamma-aminobutyric acid type A chloride channel using chemically reactive probes as chemical sensors for cysteine mutants, J. Biol. Chem. 274 (1999) 25350–25354. [16] K.W. Yoon, D.F. Covey, S.M. Rothman, Multiple mechanisms of picrotoxin block of gaba-induced currents in rat hippocampalneurons, J. Physiol. (London) 464 (1993) 423–439. [17] M. Schalk, F. Cabello-Hurtado, M.A. Pierrel, R. Atanossova, P. Saindrenan, D. Werck-Reichhart, Piperonylic acid, a selective, mechanism-based inactivator of the trans-cinnamate 4-hydroxylase: a new tool to control the flux of metabolites in the phenylpropanoid pathway, Plant Physiol. 118 (1998) 209–218. [18] T. Deiml, R. Haseneder, W. Zieglgansberger, G. Rammes, B. Eisensamer, R. Rupprecht, G. Hapfelmeier, Alpha-thujone reduces 5-HT3 receptor activity by an effect on the agonist-induced desensitization, Neuropharmacology 46 (2004) 192–201. [19] B.A. Borshop, J. Breuer, J. Holm, J. Leslie, W.L. Nyhan, Excretion of hippuric acid during sodium benzoate therapy in patients with hyperglycinemia or hyperammonemia, J. Inherit. Metab. Dis. 12 (1989) 72–79.