Structural basis of activation of bitter taste receptor T2R1 and comparison with Class A G-protein-coupled receptors (GPCRs)

The human bitter taste receptors (T2Rs) are non-Class A members of the G-protein-coupled receptor (GPCR) superfamily, with very limited structural information. Amino acid sequence analysis reveals that most of the important motifs present in the transmembrane helices (TM1-TM7) of the well studied Class A GPCRs are absent in T2Rs, raising fundamental questions regarding the mechanisms of activation and how T2Rs recognize bitter ligands with diverse chemical structures. In this study, the bitter receptor T2R1 was used to systematically investigate the role of 15 transmembrane amino acids in T2Rs, including 13 highly conserved residues, by amino acid replacements guided by molecular modeling. Functional analysis of the mutants by calcium imaging analysis revealed that replacement of Asn-66(2.65) and the highly conserved Asn-24(1.50) resulted in greater than 90% loss of agonist-induced signaling. Our results show that Asn-24(1.50) plays a crucial role in receptor activation by mediating an hydrogen bond network connecting TM1-TM2-TM7, whereas Asn-66(2.65) is essential for binding to the agonist dextromethorphan. The interhelical hydrogen bond between Asn-24(1.50) and Arg-55(2.54) restrains T2R receptor activity because loss of this bond in I27A and R55A mutants results in hyperactive receptor. The conserved amino acids Leu-197(5.50), Ser-200(5.53), and Leu-201(5.54) form a putative LXXSL motif which performs predominantly a structural role by stabilizing the helical conformation of TM5 at the cytoplasmic end. This study provides for the first time mechanistic insights into the roles of the conserved transmembrane residues in T2Rs and allows comparison of the activation mechanisms of T2Rs with the Class A GPCRs.     

Differential expression of bitter taste receptors in non-cancerous breast epithelial and breast cancer cells

The human bitter taste receptors (T2Rs) are chemosensory receptors that belong to the G protein-coupled receptor superfamily. T2Rs are present on the surface of oral and many extra-oral cells. In humans 25 T2Rs are present, and these are activated by hundreds of chemical molecules of diverse structure. Previous studies have shown that many bitter compounds including chloroquine, quinidine, bitter melon extract and cucurbitacins B and E inhibit tumor growth and induce apoptosis in cancer cells. However, the existence of T2Rs in cancer cell is not yet elucidated. In this report using quantitative (q)-PCR and flow cytometry, we characterized the expression of T2R1, T2R4, T2R10, T2R38 and T2R49 in the highly metastatic breast cancer cell line MDA-MB-231, poorly metastatic cell line MCF-7, and non-cancerous mammary epithelial cell line MCF-10A. Among the 5 T2Rs analyzed by qPCR and flow cytometry, T2R4 is expressed at 40–70% in mammary epithelial cells in comparison to commonly used breast cancer marker proteins, estrogen receptor and E-cadherin. Interestingly, the expression of T2R4 was downregulated in breast cancer cells. An increase in intracellular calcium mobilization was observed after the application of bitter agonists, quinine, dextromethorphan, and phenylthiocarbamide that are specific for some of the 5 T2Rs. This suggests that the endogenous T2Rs expressed in these cells are functional. Taken together, our novel findings suggest that T2Rs are differentially expressed in mammary epithelial cells, with some T2Rs downregulated in breast cancer cells.     

Amino Acid Derivatives as Bitter Taste Receptor (T2R) Blockers

In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-bis(carboxymethyl)-l-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC₅₀ of 3.2 ± 0.3 μm and 59 ± 18 nm, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues.     

Functional expression of mammalian bitter taste receptors in Caenorhabditis elegans

Bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances appearing in the environment. The molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules to G protein-coupled receptors (GPCR) called T2Rs and expressed at the surface of taste receptor cells. The functional characterisation of T2R receptors is far from been completed due to the difficulty to functionally express them in heterologous systems. Taking advantage of the parallelisms between the Caenorhabditis elegans (C. elegans) and mammalian GPCR signalling pathways, we developed a C. elegans-based expression system to express functional human and rodent GPCRs of the T2R family. We generated transgenic worms expressing T2Rs in ASI chemosensory neurons and performed behavioural assays using a variety of bitter tastants. As a proof of the concept, we generated transgenic worms expressing human T2R4 or its mouse ortholog T2R8 receptors, which respond to two bitter tastants previously characterised as their functional ligands, 6-n-propyl-2-thiouracil and denatoniun. As expected, expression of human T2R4 or its mouse ortholog T2R8 in ASI neurons counteracted the water-soluble avoidance to 6-n-propyl-2-thiouracil and denatoniun observed in control wild-type worms. The expression in ASI neurons of human T2R16, the ligand of which, phenyl-beta-d-glucopyranoside, belong to a chemically different group of bitter tastants, also counteracted the water-soluble avoidance to this compound observed in wild-type worms. These results indicate that C. elegans is a suitable heterologous expression system to express functional T2Rs providing a tool to efficiently search for specific taste receptor ligands and to extend our understanding of the molecular basis of gustation.     

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.     

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.     

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.     

Coding of sweet,bitter, and umami tastes: different receptor cells sharing similar signaling pathways

Mammals can taste a wide repertoire of chemosensory stimuli. Two unrelated families of receptors (T1Rs and T2Rs) mediate responses to sweet, amino acids, and bitter compounds. Here, we demonstrate that knockouts of TRPM5, a taste TRP ion channel, or PLCbeta2, a phospholipase C selectively expressed in taste tissue, abolish sweet, amino acid, and bitter taste reception, but do not impact sour or salty tastes. Therefore, despite relying on different receptors, sweet, amino acid, and bitter transduction converge on common signaling molecules. Using PLCbeta2 taste-blind animals, we then examined a fundamental question in taste perception: how taste modalities are encoded at the cellular level. Mice engineered to rescue PLCbeta2 function exclusively in bitter-receptor expressing cells respond normally to bitter tastants but do not taste sweet or amino acid stimuli. Thus, bitter is encoded independently of sweet and amino acids, and taste receptor cells are not broadly tuned across these modalities.     

Identification of ligands for two human bitter T2R receptors

Earlier, a family of G protein-coupled receptors, termed T2Rs, was identified in the rodent and human genomes through data mining. It was suggested that these receptors mediate bitter taste perception. Analysis of the human genome revealed that the hT2R family is composed of 25 members. However, bitter ligands have been identified for only three human receptors so far. Here we report identification of two novel ligand-receptor pairs. hT2R61 is activated by 6-nitrosaccharin, a bitter derivative of saccharin. hT2R44 is activated by denatonium and 6-nitrosaccharin. Activation profiles for these receptors correlate with psychophysical data determined for the bitter compounds in human studies. Functional analysis of hT2R chimeras allowed us to identify residues in extracellular loops critical for receptor activation by ligands. The discovery of two novel bitter ligand-receptor pairs provides additional support for the hypothesis that hT2Rs mediate a bitter taste response in humans.     

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.     

T2Rs function as bitter taste receptors

Bitter taste perception provides animals with critical protection against ingestion of poisonous compounds. In the accompanying paper, we report the characterization of a large family of putative mammalian taste receptors (T2Rs). Here we use a heterologous expression system to show that specific T2Rs function as bitter taste receptors. A mouse T2R (mT2R-5) responds to the bitter tastant cycloheximide, and a human and a mouse receptor (hT2R-4 and mT2R-8) responded to denatonium and 6-n-propyl-2-thiouracil. Mice strains deficient in their ability to detect cycloheximide have amino acid substitutions in the mT2R-5 gene; these changes render the receptor significantly less responsive to cycloheximide. We also expressed mT2R-5 in insect cells and demonstrate specific tastant-dependent activation of gustducin, a G protein implicated in bitter signaling. Since a single taste receptor cell expresses a large repertoire of T2Rs, these findings provide a plausible explanation for the uniform bitter taste that is evoked by many structurally unrelated toxic compounds.     

Role of rhodopsin N-terminus in structure and function of rhodopsin-bitter taste receptor chimeras

The bitter taste receptors (T2Rs) belong to the G protein-coupled receptor (GPCR) superfamily. In humans, bitter taste sensation is mediated by 25 T2Rs. Structure–function studies on T2Rs are impeded by the low-level expression of these receptors. Different lengths of rhodopsin N-terminal sequence inserted at the N-terminal region of T2Rs are commonly used to express these receptors in heterologous systems. While the additional sequences were reported, to enhance the expression of the T2Rs, the local structural perturbations caused by these sequences and its effect on receptor function or allosteric ligand binding were not characterized. In this study, we elucidated how different lengths of rhodopsin N-terminal sequence effect the structure and function of the bitter taste receptor, T2R4. Guided by molecular models of T2R4 built using a rhodopsin crystal structure as template, we constructed chimeric T2R4 receptors containing the rhodopsin N-terminal 33 and 38 amino acids. The chimeras were functionally characterized using calcium imaging, and receptor expression was determined by flow cytometry. Our results show that rhodopsin N-terminal 33 amino acids enhance expression of T2R4 by 2.5-fold and do not cause perturbations in the receptor structure.     

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.     

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 .     

A novel family of mammalian taste receptors

In mammals, taste perception is a major mode of sensory input. We have identified a novel family of 40-80 human and rodent G protein-coupled receptors expressed in subsets of taste receptor cells of the tongue and palate epithelia. These candidate taste receptors (T2Rs) are organized in the genome in clusters and are genetically linked to loci that influence bitter perception in mice and humans. Notably, a single taste receptor cell expresses a large repertoire of T2Rs, suggesting that each cell may be capable of recognizing multiple tastants. T2Rs are exclusively expressed in taste receptor cells that contain the G protein alpha subunit gustducin, implying that they function as gustducin-linked receptors. In the accompanying paper, we demonstrate that T2Rs couple to gustducin in vitro, and respond to bitter tastants in a functional expression assay.     

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.     

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.     

Highly conserved intracellular H208 residue influences agonist selectivity in bitter taste receptor T2R14

Bitter taste receptors (T2Rs) are a specialized class of cell membrane receptors of the G protein-coupled receptor family and perform a crucial role in chemosensation. The 25 T2Rs in humans are activated by structurally diverse ligands of plant, animal and microbial origin. The mechanisms of activation of these receptors are poorly understood. Therefore, identification of structural determinants of T2Rs that regulate its efficacy could be beneficial in understanding the molecular mechanisms of T2R activation. In this work, we characterized a highly conserved histidine (H208), present at TM5-ICL3 region of T2R14 and its role in agonist-induced T2R14 signaling. Surprisingly, mutation of the conserved H208 (H208A) did not result in increased basal activity of T2R14, in contrast to similar H206A mutation in T2R4 that showed constitutive or basal activity. However, H208A mutation in T2R14 resulted in an increase in agonist-induced efficacy for Flufenamic acid (FFA). Interestingly, H208A did not affect the potency of another T2R14 agonist Diphenhydramine (DPH). The H208R compensatory mutation showed FFA response similar to wild-type T2R14. Molecular modeling suggests a FFA-induced shift in TM3 and TM5 helices of H208A, which changes the network of interactions connecting TM5-ICL3-TM6. This report identifies a crucial residue on the intracellular surface of T2Rs that is involved in bitter ligand selectivity. It also highlights the varied roles carried out by some conserved residues in different T2Rs.