Insights into the Binding of Phenyltiocarbamide (PTC) Agonist to Its Target Human TAS2R38 Bitter Receptor

Humans'' bitter taste perception is mediated by the hTAS2R subfamily of the G protein-coupled membrane receptors (GPCRs). Structural information on these receptors is currently limited. Here we identify residues involved in the binding of phenylthiocarbamide (PTC) and in receptor activation in one of the most widely studied hTAS2Rs (hTAS2R38) by means of structural bioinformatics and molecular docking. The predictions are validated by site-directed mutagenesis experiments that involve specific residues located in the putative binding site and trans-membrane (TM) helices 6 and 7 putatively involved in receptor activation. Based on our measurements, we suggest that (i) residue N103 participates actively in PTC binding, in line with previous computational studies. (ii) W99, M100 and S259 contribute to define the size and shape of the binding cavity. (iii) W99 and M100, along with F255 and V296, play a key role for receptor activation, providing insights on bitter taste receptor activation not emerging from the previously reported computational models.     

6-Methoxyflavanones as Bitter Taste Receptor Blockers for hTAS2R39

Many (dietary) bitter compounds, e.g. flavonoids, activate bitter receptor hTAS2R39 in cell-based assays. Several flavonoids, amongst which some flavanones, are known not to activate this receptor. As certain flavanones are known to mask bitter taste sensorially, flavanones might act as bitter receptor antagonists. Fourteen flavanones were investigated for their potential to reduce activation of hTAS2R39 by epicatechin gallate (ECG), one of the main bitter compounds occurring in green tea. Three flavanones showed inhibitory behavior towards the activation of hTAS2R39 by ECG: 4′-fluoro-6-methoxyflavanone, 6,3′-dimethoxyflavanone, and 6-methoxyflavanone (in order of decreasing potency). The 6-methoxyflavanones also inhibited activation of hTAS2R14 (another bitter receptor activated by ECG), though to a lesser extent. Dose-response curves of ECG at various concentrations of the full antagonist 4′-fluoro-6-methoxyflavanone and wash-out experiments indicated reversible insurmountable antagonism. The same effect was observed for the structurally different agonist denatonium benzoate.     

Structure-function relationships of the human bitter taste receptor hTAS2R1: insights from molecular modeling studies

Bitter taste receptors (T2Rs) belong to G-protein-coupled receptors (GPCRs). Despite extensive studies, the precise mechanisms of GPCR activation are still poorly understood. In this study, the models of the human bitter taste receptor hTAS2R1 alone and in complex with various ligands were constructed on the basis of template-based modeling and molecular docking. Then these models were subjected to all-atom molecular dynamics (MD) simulations in explicit lipid bilayers. The binding pocket of hTAS2R1 is mainly formed by transmembrane helix (TM) III, TM V, TM VI, and TM VII. Most of the residues contributing to ligand binding are positionally conserved comparing with other hTAS2Rs. By comparing the final conformations obtained by extensive MD simulations, we identified the changes in the transmembrane helices and the intra- and extracellular loops, which were expected to initiate the activation of the receptor. The intracellular loop II (ICL2) and TM III were found to play prominent roles in the process of activation. We proposed that a set of interactions between the aromatic Phe115 in the middle of ICL2 and three residues (Tyr103, Lys106, and Val107) at the cytoplasmic end of TM III may serve as a conformational switch of hTAS2R1 activation. All of the residues involved in the switch are highly conserved among T2Rs. This indicates that the control switch we proposed may be universal in T2Rs. Besides, our results also suggest that the formation of a short helical segment in ICL2 may be necessary for the activation of hTAS2R1.     

Structure–function relationships of the human bitter taste receptor hTAS2R1: insights from molecular modeling studies

Bitter taste receptors (T2Rs) belong to G-protein-coupled receptors (GPCRs). Despite extensive studies, the precise mechanisms of GPCR activation are still poorly understood. In this study, the models of the human bitter taste receptor hTAS2R1 alone and in complex with various ligands were constructed on the basis of template-based modeling and molecular docking. Then these models were subjected to all-atom molecular dynamics (MD) simulations in explicit lipid bilayers. The binding pocket of hTAS2R1 is mainly formed by transmembrane helix (TM) III, TM V, TM VI, and TM VII. Most of the residues contributing to ligand binding are positionally conserved comparing with other hTAS2Rs. By comparing the final conformations obtained by extensive MD simulations, we identified the changes in the transmembrane helices and the intra- and extracellular loops, which were expected to initiate the activation of the receptor. The intracellular loop II (ICL2) and TM III were found to play prominent roles in the process of activation. We proposed that a set of interactions between the aromatic Phe115 in the middle of ICL2 and three residues (Tyr103, Lys106, and Val107) at the cytoplasmic end of TM III may serve as a conformational switch of hTAS2R1 activation. All of the residues involved in the switch are highly conserved among T2Rs. This indicates that the control switch we proposed may be universal in T2Rs. Besides, our results also suggest that the formation of a short helical segment in ICL2 may be necessary for the activation of hTAS2R1.     

Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin

Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to β-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors, p-(dipropylsulfamoyl)benzoic acid (probenecid), that acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of bitter taste and as a lead for the development of broad specificity bitter blockers to improve nutrition and medical compliance.     

Computational Studies of Ligand-Receptor Interactions in Bitter Taste Receptors

Phenylthiocarbamide tastes intensely bitter to some individuals, but others find it completely tasteless. Recently, it was suggested that phenylthiocarbamide elicits bitter taste by interacting with a human G protein-coupled receptor (hTAS2R38) encoded by the PTC gene. The phenylthiocarbamide nontaster trait was linked to three single nucleotide polymorphisms occurring in the PTC gene. Using the crystal structure of bovine rhodopsin as template, we generated the 3D structure of hTAS2R38 bitter taste receptor. We were able to map on the receptor structure the amino acids affected by the genetic polymorphisms and to propose molecular functions for two of them that explained the emergence of the nontaster trait. We used molecular docking simulations to find that phenylthiocarbamide exhibited a higher affinity for the target receptor than the structurally similar molecule 6-n-propylthiouracil, in line with recent experimental studies. A 3D model was constructed for the hTAS2R16 bitter taste receptor as well, by applying the same protocol. We found that the recently published experimental ligand binding affinity data for this receptor correlated well with the binding scores obtained from our molecular docking calculations.     

Computational studies of ligand-receptor interactions in bitter taste receptors

Phenylthiocarbamide tastes intensely bitter to some individuals, but others find it completely tasteless. Recently, it was suggested that phenylthiocarbamide elicits bitter taste by interacting with a human G protein-coupled receptor (hTAS2R38) encoded by the PTC gene. The phenylthiocarbamide nontaster trait was linked to three single nucleotide polymorphisms occurring in the PTC gene. Using the crystal structure of bovine rhodopsin as template, we generated the 3D structure of hTAS2R38 bitter taste receptor. We were able to map on the receptor structure the amino acids affected by the genetic polymorphisms and to propose molecular functions for two of them that explained the emergence of the nontaster trait. We used molecular docking simulations to find that phenylthiocarbamide exhibited a higher affinity for the target receptor than the structurally similar molecule 6-n-propylthiouracil, in line with recent experimental studies. A 3D model was constructed for the hTAS2R16 bitter taste receptor as well, by applying the same protocol. We found that the recently published experimental ligand binding affinity data for this receptor correlated well with the binding scores obtained from our molecular docking calculations.     

Evaluation of the bitterness of green tea catechins by a cell-based assay with the human bitter taste receptor hTAS2R39

Catechins have a broad range of physiological functions and act as the main taste ingredient of green tea. Although catechins show a strong bitterness, the bitter taste receptor for catechins has not been fully understood. The objective of this study was to identify the receptor for the major green tea catechins such as (−)-epicatechin (EC), (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCg). By the cell-based assay using cultured cells expressing human bitter taste receptor, a clear response of hTAS2R39-expressing cells was observed to 300 μM of either ECg or EGCg, which elicit a strong bitterness in humans. The response of hTAS2R39-expressing cells to ECg was the strongest among the tested catechins, followed by EGCg. Because the cellular response to EC and EGC is much weaker than those of ECg and EGCg, galloyl groups was strongly supposed to be involved in the bitter intensity. This finding is similar to the observations of taste intensity obtained from a human sensory study. Our results suggest the participation of hTAS2R39 in the detection of catechins in humans, indicating the possibility that bitterness of tea catechins can be evaluated by using cells expressing hTAS2R39.     

Characterization of the ��-d-Glucopyranoside Binding Site of the Human Bitter Taste Receptor hTAS2R16

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and β-d-glucopyranoside and will also facilitate rational design of bitter blockers.     

Human Bitter Taste Receptors hTAS2R8 and hTAS2R39 with Differential Functions to Recognize Bitter Peptides

The strong bitter peptide, Phe-Phe-Pro-Arg, activated cultured cells expressing either of the known human bitter taste receptors, hTAS2R8 and hTAS2R39. The partial structure of Pro-Arg activated hTAS2R39, but did not activate hTAS2R8. These receptors may not indiscriminately recognize bitter peptides, but have a differential function in their recognition.     

Human bitter taste receptors hTAS2R8 and hTAS2R39 with differential functions to recognize bitter peptides

The strong bitter peptide, Phe-Phe-Pro-Arg, activated cultured cells expressing either of the known human bitter taste receptors, hTAS2R8 and hTAS2R39. The partial structure of Pro-Arg activated hTAS2R39, but did not activate hTAS2R8. These receptors may not indiscriminately recognize bitter peptides, but have a differential function in their recognition.     

Bitter taste receptor T2R1 is activated by dipeptides and tripeptides

Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, the molecular targets or receptors in humans for these bitter peptides were poorly characterized and least understood. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. In addition, we tested the ability of peptide inhibitors of the blood pressure regulatory protein, angiotensin converting enzyme (ACE) to activate T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium was measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Among the peptides examined, the bitter tri-peptide Phe-Phe-Phe (FFF), is the most potent in activating T2R1 with an EC₅₀ value in the micromolar range. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The molecular models showed that the bitter peptides bind within the same binding pocket on the receptor. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2 forming a cap like structure on the binding pocket.     

The human taste receptor hTAS2R14 responds to a variety of different bitter compounds

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, (-)-alpha-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 (-)-alpha-thujone and picrotoxinin suggests that the receptor is sufficiently sensitive to caution us against the ingestion of toxic amounts of these substances.     

The human bitter taste receptor, hTAS2R16, discriminates slight differences in the configuration of disaccharides

Sweetness and bitterness are key determinants of food acceptance and rejection, respectively. Sugars, such as sucrose and fructose, are generally recognized as sweet. However, not all sugars are sweet, and even anomers may have quite different tastes. For example, gentiobiose is bitter, whereas its anomer, isomaltose, is sweet. Despite this unique sensory character, the molecular basis of the bitterness of gentiobiose remains to be clarified. In this study, we used calcium imaging analysis of human embryonic kidney 293T cells that heterologously expressed human taste receptors to demonstrate that gentiobiose activated hTAS2R16, a bitter taste receptor, but not hT1R2/hT1R3, a sweet taste receptor. In contrast, isomaltose activated hT1R2/hT1R3. As a result, these anomers elicit different taste sensations. Mutational analysis of hTAS2R16 also indicated that gentiobiose and β-d-glucopyranosides, such as salicin share a common binding site of hTAS2R16.     

Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1 mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-β-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.     

Bitter peptides activate hTAS2Rs, the human bitter receptors

Fermented food contains numerous peptides derived from material proteins. Bitter peptides formed during the fermentation process are responsible for the bitter taste of fermented food. We investigated whether human bitter receptors (hTAS2Rs) recognize bitterness of peptides with a heterologous expression system. HEK293 cells expressing hTAS2R1, hTAS2R4, hTAS2R14, and hTAS2R16 responded to bitter casein digests. Among those cells, the hTAS2R1-expressing cell was most strongly activated by the synthesized bitter peptides Gly-Phe and Gly-Leu, and none of the cells was activated by the non-bitter dipeptide Gly-Gly. The results showed that these bitter peptides, as well as many other bitter compounds, activate hTAS2Rs, suggesting that humans utilize these hTAS2Rs to recognize and perceive the structure and bitterness of peptides.     

Members of RTP and REEP gene families influence functional bitter taste receptor expression

Functional characterization of chemosensory receptors is usually achieved by heterologous expression in mammalian cell lines. However, many chemoreceptor genes, including bitter taste receptors (TAS2Rs), show only marginal cell surface expression. Usually, these problems are circumvented by using chimeric receptors consisting of "export tags" and the receptor sequence itself. It seems likely that chemoreceptor cells express factors for cell surface targeting of native receptor molecules in vivo. For TAS2Rs, however, such factors are still unknown. The present study investigates the influence of RTP and REEP proteins on the functional expression of human TAS2Rs in heterologous cells. We expressed hTAS2Rs in HEK 293T cells and observed dramatic differences in responsiveness to agonist stimulation. By immunocytochemistry we show accumulation of the bitter beta-glucopyranoside receptor hTAS2R16 in the Golgi compartment. Coexpression of RTP and REEP proteins changed the responses of some hTAS2Rs upon agonist stimulation, which is likely due to efficient cell surface localization as demonstrated by cell surface biotinylation experiments. The coimmunoprecipitation of hTAS2R16 and RTP3 or RTP4 suggests that the mechanism by which these cofactors influence hTAS2R16 function might involve direct protein-protein interaction. Finally, expression analyses demonstrate RTP and REEP gene expression in human circumvallate papillae and testis, both of which are sites of TAS2R gene expression.     

Activation of human bitter taste receptors by polymethoxylated flavonoids

Tangeretin and nobiletin are polymethoxylated flavonoids in citrus peel. Both tangeretin and nobiletin are bitter; however, their bitterness has not been evaluated using human bitter taste receptors (hTAS2Rs). We screened 25 kinds of hTAS2Rs and found that hTAS2R14 and hTAS2R46 received both compounds.     

Functions of human bitter taste receptors depend on N-glycosylation

Human bitter taste receptors of the TAS2R gene family play a crucial role as warning sensors against the ingestion of toxic food compounds. Moreover, the genetically highly polymorphic hTAS2Rs recognize an enormous number of structurally diverse toxic and non-toxic bitter substances, and hence, may substantially influence our individual eating habits. Heterologous expression in mammalian cells is a useful tool to investigate interactions between these receptors and their agonists. However, many bitter taste receptors are poorly expressed at the cell surface of heterologous cells requiring the addition of plasma membrane export promoting epitopes to the native receptor proteins. Currently, nothing is known about amino acid motifs or other receptor-intrinsic features of TAS2Rs affecting plasma membrane association. In the present study, we analyzed the Asn-linked glycosylation of hTAS2Rs at a consensus sequence in the second extracellular loop, which is conserved among all 25 hTAS2Rs. Non-glycosylated receptors exhibit substantially lower cell surface localization and reduced association with the cellular chaperone calnexin. As the auxiliary factors receptor transporting proteins 3 and 4 are able to restore the function of non-glycosylated hTAS2R16 partially, we conclude that glycosylation is important for receptor maturation but not for its function per se .     

Plasticity of the ligand binding pocket in the bitter taste receptor T2R7

Bitter taste receptors (T2Rs) are a group of 25 G protein-coupled receptors (GPCRs) in humans. The cognate agonists and the mechanism of ligand binding to the majority of the T2Rs remain unknown. Here we report the first structure-function analysis of T2R7 and study the ability of this receptor to bind to different agonists by site-directed mutagenesis. Screening of ligands for T2R7 in calcium based assays lead to the identification of novel compounds that activate this receptor. Quinine, diphenidol, dextromethorphan and diphenhydramine showed substantial activation of T2R7. Interestingly, these bitter compounds showed different pharmacological characteristics. To investigate the structural features in T2R7 that might contribute to the observed differences in agonist specificities, molecular model guided ligand docking and site-directed mutagenesis was pursued. Amino acids D65, D86, W89, N167, T169, W170, S181, T255 and E271 in the ligand-binding pocket were replaced and the mutants characterized pharmacologically. Our results suggest D86, S181 and W170 present on the extracellular side of transmembrane 3 (TM3), TM5 and in extracellular loop 2 (ECL2) are essential for agonist binding in T2R7. Mutations of these amino acids lead to loss-of-function. We also identified gain-of-function residues that are agonist specific. These results suggest that agonists bind at an extracellular site rather than deep within the TM core involving residues present in both ECL2 and TM helices in T2R7. Similar to majority of the Class A GPCRs, ECL2 in T2R7 plays a significant role in agonist binding and activation.