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The vertebrate sense of taste allows rapid assessment of the nutritional quality and potential presence of harmful substances prior to ingestion. Among the five basic taste qualities, salty, sour, sweet, umami, and bitter, bitterness is associated with the presence of putative toxic substances and elicits rejection behaviors in a wide range of animals including humans. However, not all bitter substances are harmful, some are thought to be health-beneficial and nutritious. Among those compound classes that elicit a bitter taste although being non-toxic and partly even essential for humans are bitter peptides and l-amino acids. Using functional heterologous expression assays, we observed that the 5 dominant human bitter taste receptors responsive to bitter peptides and amino acids are activated by bile acids, which are notorious for their extreme bitterness. We further demonstrate that the cross-reactivity of bitter taste receptors for these two different compound classes is evolutionary conserved and can be traced back to the amphibian lineage. Moreover, we show that the cross-detection by some receptors relies on "structural mimicry" between the very bitter peptide l-Trp-Trp-Trp and bile acids, whereas other receptors exhibit a phylogenetic conservation of this trait. As some bile acid-sensitive bitter taste receptor genes fulfill dual-roles in gustatory and non-gustatory systems, we suggest that the phylogenetic conservation of the rather surprising cross-detection of the two substance classes could rely on a gene-sharing-like mechanism in which the non-gustatory function accounts for the bitter taste response to amino acids and peptides. [ABSTRACT FROM AUTHOR]

Bitterness from phenylthiocarbamide and 6-n-propylthiouracil (PROP) varies with polymorphisms in the TAS2R38 gene. Three SNPs form two common (AVI, PAV) and four rare haplotypes (AAI, AAV, PVI, and PAI). AVI homozygotes exhibit higher detection thresholds and lower suprathreshold bitterness for PROP compared to PAV homozygotes and heterozygotes, and these differences may influence alcohol and vegetable intake. Within a diplotype, substantial variation in suprathreshold bitterness persists, and some AVI homozygotes report moderate bitterness at high concentrations. A second receptor encoded by a gene containing a functional polymorphism may explain this. Early work has suggested that PROP might activate TAS2R4 in vitro, but later work did not replicate this. Here, we identify three TAS2R4 SNPs that result in three diplotypes—SLN/SLN, FVS/SLN, and FVS/FVS—which make up 25.1%, 44.9%, and 23.9% of our sample. These TAS2R4 haplotypes show minimal linkage disequilibrium with TAS2R38, so we examined the suprathreshold bitterness as a function of both. The participants (n = 243) rated five PROP concentrations in duplicate, interleaved with other stimuli. As expected, the TAS2R38 haplotypes explained ~29% (p < 0.0001) of the variation in the bitterness ratings, with substantial variation within the haplotypes (AVI/AVI, PAV/AVI, and PAV/PAV). Notably, the TAS2R4 diplotypes (independent of the TAS2R38 haplotypes) explained ~7–8% of the variation in the bitterness ratings (p = 0.0001). Given this, we revisited if PROP could activate heterologously expressed TAS2R4 in HEK293T cells, and calcium imaging indicated 3 mM PROP is a weak TAS2R4 agonist. In sum, our data are consistent with the second receptor hypothesis and may explain the recovery of the PROP tasting phenotype in some AVI homozygotes; further, this finding may potentially help explain the conflicting results on the TAS2R38 diplotype and food intake. [ABSTRACT FROM AUTHOR]

Many animal and plant species synthesize toxic compounds as deterrent; thus, detection of these compounds is of vital importance to avoid their ingestion. Often, such compounds are recognized by taste 2 receptors that mediate bitter taste in humans. Until now, bitter taste receptors have only been found in bony vertebrates, where they occur as a large family already in coelacanth, a "living fossil" and the earliest-diverging extant lobe-finned fish. Here, we have revisited the evolutionary origin of taste 2 receptors (T2Rs) making use of a multitude of recently available cartilaginous fish genomes. We have identified a singular T2R in 12 cartilaginous fish species (9 sharks, 1 sawfish, and 2 skates), which represents a sister clade to all bony fish T2Rs. We have examined its ligands for two shark species, a catshark and a bamboo shark. The ligand repertoire of bamboo shark represents a subset of that of the catshark, with roughly similar thresholds. Amarogentin, one of the most bitter natural substances for humans, also elicited the highest signal amplitudes with both shark receptors. Other subsets of ligands are shared with basal bony fish T2Rs indicating an astonishing degree of functional conservation over nearly 500 mya of separate evolution. Both shark receptors respond to endogenous steroids as well as xenobiotic compounds, whereas separate receptors exist for xenobiotics both in early-and late-derived bony vertebrates (coelacanth, zebrafish, and human), consistent with the shark T2R reflecting the original ligand repertoire of the ancestral bitter taste receptor at the evolutionary origin of this family. [ABSTRACT FROM AUTHOR]

Despite the important role of bitter taste for the rejection of potentially harmful food sources, birds have long been suspected to exhibit inferior bitter tasting abilities. Although more recent reports on the bitter recognition spectra of several bird species have cast doubt about the validity of this assumption, the bitter taste of avian species is still an understudied field. Previously, we reported the bitter activation profiles of three zebra finch receptors Tas2r5, -r6, and -r7, which represent orthologs of a single chicken bitter taste receptor, Tas2r1. In order to get a better understanding of the bitter tasting capabilities of zebra finches, we selected another Tas2r gene of this species that is similar to another chicken Tas2r. Using functional calcium mobilization experiments, we screened zebra finch Tas2r1 with 72 bitter compounds and observed responses for 7 substances. Interestingly, all but one of the newly identified bitter agonists were different from those previously identified for Tas2r5, -r6, and -r7 suggesting that the newly investigated receptor fills important gaps in the zebra finch bitter recognition profile. The most potent bitter agonist found in our study is cucurbitacin I, a highly toxic natural bitter substance. We conclude that zebra finch exhibits an exquisitely developed bitter taste with pronounced cucurbitacin I sensitivity suggesting a prominent ecological role of this compound for zebra finch. [ABSTRACT FROM AUTHOR]

Beside the oral cavity, bitter taste receptors are expressed in several non-gustatory tissues. Whether extra-oral bitter taste receptors function as sensors for endogenous agonists is unknown. To address this question, we devised functional experiments combined with molecular modeling approaches to investigate human and mouse receptors using a variety of bile acids as candidate agonists. We show that five human and six mouse receptors are responsive to an array of bile acids. Moreover, their activation threshold concentrations match published data of bile acid concentrations in human body fluids, suggesting a putative physiological activation of non-gustatory bitter receptors. We conclude that these receptors could serve as sensors for endogenous bile acid levels. These results also indicate that bitter receptor evolution may not be driven solely by foodstuff or xenobiotic stimuli, but also depend on endogenous ligands. The determined bitter receptor activation profiles of bile acids now enable detailed physiological model studies. Using a combined experimental-computational approach, the activation profiles of human and mouse bitter taste receptors, TAS2Rs and Tas2rs, by bile acids is described, suggesting a physiological role of bile acids for taste receptors in non-gustatory tissues. [ABSTRACT FROM AUTHOR]

Bitterness is an aversive cue elicited by thousands of chemically diverse compounds. Bitter taste may prevent consumption of foods and jeopardize drug compliance. The G protein-coupled receptors for bitter taste, TAS2Rs, have species-dependent number of subtypes and varying expression levels in extraoral tissues. Molecular recognition by TAS2R subtypes is physiologically important, and presents a challenging case study for ligand-receptor matchmaking. Inspired by hybrid recommendation systems, we developed a new set of similarity features, and created the BitterMatch algorithm that predicts associations of ligands to receptors with ~ 80% precision at ~ 50% recall. Associations for several compounds were tested in-vitro, resulting in 80% precision and 42% recall. The encouraging performance was achieved by including receptor properties and integrating experimentally determined ligand-receptor associations with chemical ligand-to-ligand similarities. BitterMatch can predict off-targets for bitter drugs, identify novel ligands and guide flavor design. The novel features capture information regarding the molecules and their receptors, which could inform various chemoinformatic tasks. Inclusion of neighbor-informed similarities improves as experimental data mounts, and provides a generalizable framework for molecule-biotarget matching. [ABSTRACT FROM AUTHOR]

Understanding individual responses to nutrition and medicine is of growing interest and importance. There is evidence that differences in bitter taste receptor (TAS2R) genes which give rise to two frequent haplotypes, TAS2R38-PAV (functional) and TAS2R38-AVI (non-functional), may impact inter-individual differences in health status. We here analyzed the relevance of the TAS2R38 receptor in the regulation of the human immune response using the TAS2R38 agonist allyl isothiocyanate (AITC) from Brassica plants. A differential response in calcium mobilization upon AITC treatment in leucocytes from healthy humans confirmed a relevance of TAS2R38 functionality, independent from cation channel TRPV1 or TRPA1 activation. We further identified a TAS2R38-dependence of MAPK and AKT signaling activity, bactericidal (toxicity against E. coli) and anti-inflammatory activity (TNF-alpha inhibition upon cell stimulation). These in vitro results were derived at relevant human plasma levels in the low micro molar range as shown here in a human intervention trial with AITC-containing food. [ABSTRACT FROM AUTHOR]

Hydrogen to deuterium isotopic substitution has only a minor effect on physical and chemical properties of water and, as such, is not supposed to influence its neutral taste. Here we conclusively demonstrate that humans are, nevertheless, able to distinguish D2O from H2O by taste. Indeed, highly purified heavy water has a distinctly sweeter taste than same-purity normal water and can add to perceived sweetness of sweeteners. In contrast, mice do not prefer D2O over H2O, indicating that they are not likely to perceive heavy water as sweet. HEK 293T cells transfected with the TAS1R2/TAS1R3 heterodimer and chimeric G-proteins are activated by D2O but not by H2O. Lactisole, which is a known sweetness inhibitor acting via the TAS1R3 monomer of the TAS1R2/TAS1R3, suppresses the sweetness of D2O in human sensory tests, as well as the calcium release elicited by D2O in sweet taste receptor-expressing cells. The present multifaceted experimental study, complemented by homology modelling and molecular dynamics simulations, resolves a long-standing controversy about the taste of heavy water, shows that its sweet taste is mediated by the human TAS1R2/TAS1R3 taste receptor, and opens way to future studies of the detailed mechanism of action. Ben Abu, Mason and colleagues use molecular dynamics, cell-based experiments, mouse models, and human subjects to determine that, unlike ordinary water, heavy water tastes sweet to humans, but not mice. Mechanistically, this effect is mediated by the human TAS1R/TAS1R3 sweet taste receptor. [ABSTRACT FROM AUTHOR]

The bitter taste sensation is important to warn mammals of the ingestion of potentially toxic food compounds. For mammals, whose nutrition relies on highly specific food sources, such as blood in the case of vampire bats, it is unknown if bitter sensing is involved in prey selection. By contrast to other bat species, vampire bats exhibit numerous bitter taste receptor pseudogenes, which could point to a decreased importance of bitter taste. However, electrophysiological and behavioural studies suggest the existence of functional bitter taste transmission. To determine the agonist spectra of the three bitter taste receptors that are conserved in all three vampire bat species, we investigated the in vitro activation of Desmodus rotundus T2R1, T2R4 and T2R7. Using a set of 57 natural and synthetic bitter compounds, we were able to identify agonists for all three receptors. Hence, we confirmed a persisting functionality and, consequently, a putative biological role of bitter taste receptors in vampire bats. Furthermore, the activation of the human TAS2R7 by metal ions is shown to be conserved in D. rotundus. [ABSTRACT FROM AUTHOR]

Bitter taste perception plays vital roles in animal behavior and fitness. By signaling the presence of toxins in foods, particularly noxious defense compounds found in plants, it enables animals to avoid exposure. In vertebrates, bitter perception is initiated by TAS2Rs, a family of G protein-coupled receptors expressed on the surface of taste buds. There, oriented toward the interior of the mouth, they monitor the contents of foods, drinks and other substances as they are ingested. When bitter compounds are encountered, TAS2Rs respond by triggering neural pathways leading to sensation. The importance of this role placed TAS2Rs under selective pressures in the course of their evolution, leaving signatures in patterns of gene gain and loss, sequence polymorphism, and population structure consistent with vertebrates' diverse feeding ecologies. The protective value of bitter taste is reduced in modern humans because contemporary food supplies are safe and abundant. However, this is not always the case. Some crops, particularly in the developing world, retain surprisingly high toxicity and bitterness remains an important measure of safety. Bitter perception also shapes health through its influence on preference driven behaviors such as diet choice, alcohol intake and tobacco use. Further, allelic variation in TAS2R s is extensive, leading to individual differences in taste sensitivity that drive these behaviors, shaping susceptibility to disease. Thus, bitter taste perception occupies a critical intersection between ancient evolutionary processes and modern human health. [ABSTRACT FROM AUTHOR]

The careful evaluation of food is important for survival throughout the animal kingdom, and specialized chemoreceptors have evolved to recognize nutrients, minerals, acids, and many toxins. Vertebrate bitter taste, mediated by the taste receptor type 2 (T2R) family, warns against potentially toxic compounds. During evolution T2R receptors appear first in bony fish, but the functional properties of bony fish T2R receptors are mostly unknown. We performed a phylogenetic analysis showing the "living fossil" coelacanth (Latimeria chalumnae) and zebrafish (Danio rerio) to possess T2R repertoires typical for early-diverged species in the lobe-finned and the ray-finned clade, respectively. Receptors from these two species were selected for heterologous expression assays using a diverse panel of bitter substances. Remarkably, the ligand profile of the most basal coelacanth receptor, T2R01, is identical to that of its ortholog in zebrafish, consistent with functional conservation across >400 Myr of separate evolution. The second coelacanth receptor deorphaned, T2R02, is activated by steroid hormones and bile acids, evolutionary old molecules that are potentially endogenously synthesized agonists for extraoral T2Rs. For zebrafish, we report the presence of both specialized and promiscuous T2R receptors. Moreover, we identified an antagonist for one of the zebrafish receptors suggesting that bitter antagonism contributed to shape this receptor family throughout evolution. [ABSTRACT FROM AUTHOR]

Salt taste is one of the 5 basic taste qualities. Depending on the concentration, table salt is perceived either as appetitive or aversive, suggesting the contribution of several mechanisms to salt taste, distinguishable by their sensitivity to the epithelial sodium channel (ENaC) blocker amiloride. A taste-specific knockout of the α-subunit of the ENaC revealed the relevance of this polypeptide for low-salt transduction, whereas the response to other taste qualities remained normal. The fully functional ENaC is composed of α-, β-, and γ-subunits. In taste tissue, however, the precise constitution of the channel and the cell population responsible for detecting table salt remain uncertain. In order to examine the cells and subunits building the ENaC, we generated mice carrying modified alleles allowing the synthesis of green and red fluorescent proteins in cells expressing the α- and β-subunit, respectively. Fluorescence signals were detected in all types of taste papillae and in taste buds of the soft palate and naso-incisor duct. However, the lingual expression patterns of the reporters differed depending on tongue topography. Additionally, immunohistochemistry for the γ-subunit of the ENaC revealed a lack of overlap between all potential subunits. The data suggest that amiloride-sensitive recognition of table salt is unlikely to depend on the classical ENaCs formed by α-, β-, and γ-subunits and ask for a careful investigation of the channel composition. [ABSTRACT FROM AUTHOR]

Human bitter taste receptors (TAS2Rs) are a subfamily of 25 G protein-coupled receptors that mediate bitter taste perception. TAS2R14 is the most broadly tuned bitter taste receptor, recognizing a range of chemically diverse agonists with micromolar-range potency. The receptor is expressed in several extra-oral tissues and is suggested to have physiological roles related to innate immune responses, male fertility, and cancer. Higher potency ligands are needed to investigate TAS2R14 function and to modulate it for future clinical applications. Here, a structure-based modeling approach is described for the design of TAS2R14 agonists beginning from flufenamic acid, an approved non-steroidal anti-inflammatory analgesic that activates TAS2R14 at sub-micromolar concentrations. Structure-based molecular modeling was integrated with experimental data to design new TAS2R14 agonists. Subsequent chemical synthesis and in vitro profiling resulted in new TAS2R14 agonists with improved potency compared to the lead. The integrated approach provides a validated and refined structural model of ligand–TAS2R14 interactions and a general framework for structure-based discovery in the absence of closely related experimental structures. [ABSTRACT FROM AUTHOR]

A respected number of drugs suffer from bitter taste which results in patient incompliance. With the aim of solving the bitterness of guaifenesin, dimethyl maleate, maleate, glutarate, succinate, and dimethyl succinate prodrugs were designed and synthesized. Molecular orbital methods were utilized for the design of the ester prodrugs. The density functional theory (DFT) calculations revealed that the hydrolysis efficiency of the synthesized prodrugs is significantly sensitive to the pattern of substitution on C=C bond and distance between the nucleophile and the electrophile. The hydrolysis of the prodrugs was largely affected by the pH of the medium. The experimental t1/2 for the hydrolysis of guaifenesin dimaleate ester prodrugs in 1N HCl was the least and for guaifenesin dimethyl succinate was the highest. Functional heterologous expression of TAS2R14, a broadly tuned bitter taste receptor responding to guaifenesin, and experiments using these prodrugs revealed that, while some of the prodrugs still activated the receptor similarly or even stronger than the parent substance, succinate derivatization resulted in the complete loss of receptor responses. The predicted binding modes of guaifenesin and its prodrugs to the TAS2R14 homology model suggest that the decreased activity of the succinate derivatives may be caused by a clash with Phe247. A number of novel bitterless ester guaifenesin prodrugs were designed, synthesized, and characterized. The prodrugs have shown sufficient stability at neutral pH values and rapid release under low pH conditions (stomach). Using bitter taste receptor assays, it was confirmed that the most promiscuous human bitter taste receptor responsible for the bitter perception of many drugs, the TAS2R14, is not activated by some of the prodrugs. We conclude that our approach could be used to mask the bitterness of other similar drugs. [ABSTRACT FROM AUTHOR]

The field of chemical senses has made major progress in understanding the cellular mechanisms of olfaction and taste in the past 2 decades. However, the molecular understanding of odor and taste recognition is still lagging far behind and will require solving multiple structures of the relevant full-length receptors in complex with native ligands to achieve this goal. However, the development of multiple complimentary strategies for the structure determination of G protein-coupled receptors (GPCRs) makes this goal realistic. The common conundrum of how multi-specific receptors that recognize a large number of different ligands results in a sensory perception in the brain will only be fully understood by a combination of high-resolution receptor structures and functional studies. This review discusses the first steps on this pathway, including biochemical and physiological assays, forward genetics approaches, molecular modeling, and the first steps towards the structural biology of olfactory and taste receptors. [ABSTRACT FROM AUTHOR]

The number and variety of bitter compounds originating from plants are vast. Whereas some bitter chemicals are toxic and should not be ingested, other compounds exhibit health beneficial effects, which is manifest in the cross‐cultural believe that the bitterness of medicine is correlated with the desired medicinal activity. The bitter taste receptors in the oral cavity serve as sensors for bitter compounds and, as they are expressed in numerous extraoral tissues throughout the body, may also be responsible for some physiological effects exerted by bitter compounds. Chinese herbal medicine uses bitter herbs since ancient times for the treatment of various diseases; however, the routes by which these herbs modify physiology are frequently not well understood. We therefore screened 26 bitter substances extracted from medical herbs for the activation of the 25 human bitter taste receptors. We identified six receptors activated by in total 17 different bitter compounds. Interestingly, we observed a bias in bitter taste receptor activation with 10 newly identified agonists for the broadly tuned receptor TAS2R46, seven agonists activating the TAS2R14 and two compounds activating narrowly tuned receptors, suggesting that these receptors play dominant roles in the evaluation and perhaps physiological activities of Chinese herbal medicines. [ABSTRACT FROM AUTHOR]

The chemical variability of the intestinal lumen requires the presence of molecular receptors detecting the various substances naturally occurring in the diet and as a result of the activity of the microbiota. Despite their early discovery, intestinal bitter taste receptors (Tas2r) have not yet been assigned an unambiguous physiological function. Recently, using a CRE-recombinant approach we showed that the Tas2r131 gene is expressed in a subset of mucin-producing goblet cells in the colon of mice. Moreover, we also demonstrated that the expression of the Tas2r131 locus is not restricted to this region. In the present study we aimed at characterizing the presence of positive cells also in other gastrointestinal regions. Our results show that Tas2r131 cells appear in the jejunum and the ileum, and are absent from the stomach and the duodenum. We identified the positive cells as a subpopulation of deep-crypt Paneth cells in the ileum, strengthening the notion of a defensive role for Tas2rs in the gut. To get a broader perspective on the expression of bitter taste receptors in the alimentary canal, we quantified the expression of all 35 Tas2r genes along the gastrointestinal tract by qRT-PCR. We discovered that the number and expression level of Tas2r genes profoundly vary along the alimentary canal, with the stomach and the colon expressing the largest subsets. [ABSTRACT FROM AUTHOR]

Carbonic anhydrases form an enzyme family of 16 members, which reversibly catalyze the hydration of carbon dioxide to bicarbonate and protons. In lung, kidney, and brain, presence of carbonic anhydrases is associated with protons and bicarbonate transport in capillary endothelium of lung, reabsorption of bicarbonate in proximal renal tubule and extracellular buffering. In contrast, their role in taste is less clear. Recently, carbonic anhydrase IV expression was detected in sour-sensing presynaptic taste cells and was associated with the taste of carbonation, yet the precise role and cell population remained uncertain. To examine the role of carbonic anhydrase 4-expressing cells in taste reception, we generated a mouse strain carrying a modified allele of the carbonic anhydrase 4 gene in which the coding region of the red fluorescent protein monomeric Cherry is attached to that of carbonic anhydrase 4 via an internal ribosome entry site. Monomeric Cherry fluorescence was detected in lingual papillae as well as taste buds of soft palate and naso-incisor duct. However, expression patterns on the tongue differ between posterior and fungiform papillae. Whereas monomeric Cherry auto-fluorescence was almost always co-localized with presynaptic cell markers aromatic L-amino-acid decarboxylase, synaptosomal-associated protein 25 or glutamic acid decarboxylase 67 in fungiform papillae and taste buds of palate and naso-incisor duct, monomeric Cherry-positive cells in posterior tongue papillae represent only a subpopulation of presynaptic cells. We conclude that this model is well suited for detailed investigation into the role of carbonic anhydrase in gustation and other processes. [ABSTRACT FROM AUTHOR]

Caffeine, generally known as a stimulant of gastric acid secretion (GAS), is a bitter-tasting compound that activates several taste type 2 bitter receptors (TAS2Rs). TAS2Rs are expressed in the mouth and in several extraoral sites, e.g., in the gastrointestinal tract, in which their functional role still needs to be clarified. We hypothesized that caffeine evokes effects on GAS by activation of oral and gastric TAS2Rs and demonstrate that caffeine, when administered encapsulated, stimulates GAS, whereas oral administration of a caffeine solution delays GAS in healthy human subjects. Correlation analysis of data obtained from ingestion of the caffeine solution revealed an association between the magnitude of the GAS response and the perceived bitterness, suggesting a functional role of oral TAS2Rs in GAS. Expression of TAS2Rs, including cognate TAS2Rs for caffeine, was shown in human gastric epithelial cells of the corpus/fundus and in HGT-1 cells, a model for the study of GAS. In HGT-1 cells, various bitter compounds as well as caffeine stimulated proton secretion, whereby the caffeine-evoked effect was (i) shown to depend on one of its cognate receptor, TAS2R43, and adenylyl cyclase; and (ii) reduced by homoeriodictyol (HED), a known inhibitor of caffeine's bitter taste. This inhibitory effect of HED on caffeine-induced GAS was verified in healthy human subjects. These findings (i) demonstrate that bitter taste receptors in the stomach and the oral cavity are involved in the regulation of GAS and (ii) suggest that bitter tastants and bitter-masking compounds could be potentially useful therapeutics to regulate gastric pH. [ABSTRACT FROM AUTHOR]

Lineage-specific gene losses can be driven by selection or environmental adaptations. However, a lack of studies on the original function of species-specific pseudogenes leaves a gap in our understanding of their role in evolutionary histories. Pseudogenes are of particular relevance for taste perception genes, which encode for receptors that confer the ability to both identify nutritionally valuable substances and avoid potentially harmful substances. To explore the role of bitter taste pseudogenization events in human origins, we restored the open reading frames of the three human-specific pseudogenes and synthesized the reconstructed functional hTAS2R2, hTAS2R62 and hTAS2R64 receptors. We have identified ligands that differentially activate the human and chimpanzee forms of these receptors and several other human functional TAS2Rs. We show that these receptors are narrowly tuned, suggesting that bitter-taste sensitivities evolved independently in different species, and that these pseudogenization events occurred because of functional redundancy. The restoration of function of lineage-specific pseudogenes can aid in the reconstruction of their evolutionary history, and in understanding the forces that led to their pseudogenization. [ABSTRACT FROM AUTHOR]

Bitter taste elicits an aversive reaction, and is believed to protect against consuming poisons. Bitter molecules are detected by the Tas2r family of G-protein-coupled receptors, with a species-dependent number of subtypes. Chickens demonstrate bitter taste sensitivity despite having only three bitter taste receptors—ggTas2r1, ggTas2r2 and ggTas2r7. This minimalistic bitter taste system in chickens was used to determine relationships between in-vitro (measured in heterologous systems) and in-vivo (behavioral) detection thresholds. ggTas2r-selective ligands, nicotine (ggTas2r1), caffeine (ggTas2r2), erythromycin and (+)-catechin (ggTas2r7), and the Tas2r-promiscuous ligand quinine (all three ggTas2rs) were studied. Ligands of the same receptor had different in-vivo:in-vitro ratios, and the ggTas2r-promiscuous ligand did not exhibit lower in-vivo:in-vitro ratios than ggTas2r-selective ligands. In-vivo thresholds were similar or up to two orders of magnitude higher than the in-vitro ones. [ABSTRACT FROM AUTHOR]

Bitter taste receptor genes (TAS2Rs) harbor extensive diversity, which is broadly distributed across human populations and strongly associated with taste response phenotypes. The majority of TAS2R variation is composed of single-nucleotide polymorphisms. However, 2 closely positioned loci at 12p13, TAS2R43 and -45, harbor high-frequency deletion (Δ) alleles in which genomic segments are absent, resulting in copy number variation (CNV). To resolve their chromosomal structure and organization, we generated maps using long-range contig alignments and local sequencing across the TAS2R43–45 region. These revealed that the deletion alleles (43Δ and 45Δ) are 37.8 and 32.2 kb in length, respectively and span the complete coding region of each gene (~1 kb) along with extensive up- and downstream flanking sequence, producing separate CNVs at the 2 loci. Comparisons with a chimpanzee genome, which contained intact homologs of TAS2R43, -45, and nearby TAS2Rs, indicated that the deletions evolved recently, through unequal recombination in a cluster of closely related loci. Population genetic analyses in 946 subjects from 52 worldwide populations revealed that copy number ranged from 0 to 2 at both TAS2R43 and TAS2R45, with 43Δ and 45Δ occurring at high global frequencies (0.33 and 0.18). Estimated recombination rates between the loci were low (ρ = 2.7 × 10-4; r = 6.6 × 10-9) and linkage disequilibrium was high (D′ = 1.0), consistent with their adjacent genomic positioning and recent origin. Geographic variation pointed to an African origin for the deletions. However, no signatures of natural selection were found in population structure or integrated haplotype scores spanning the region, suggesting that patterns of diversity at TAS2R43 and -45 are primarily due to genetic drift. [ABSTRACT FROM AUTHOR]

Sensing potentially harmful bitter substances in the oral cavity is achieved by a group of ˜25 receptors, named TAS2Rs, which are expressed in specialized sensory cells and recognize individual but overlapping sets of bitter compounds. The receptors differ in their tuning breadths ranging from narrowly to broadly tuned receptors. One of the most broadly tuned human bitter taste receptors is the TAS2R14 recognizing an enormous variety of chemically diverse synthetic and natural bitter compounds, including numerous medicinal drugs. This suggests that this receptor possesses a large readily accessible ligand binding pocket. To allow probing the accessibility and size of the ligand binding pocket, we chemically modified cognate agonists and tested receptor responses in functional assays. The addition of large functional groups to agonists was usually possible without abolishing agonistic activity. The newly synthesized agonist derivatives were modeled in the binding site of the receptor, providing comparison to the mother substances and rationalization of the in vitro activities of this series of compounds. [ABSTRACT FROM AUTHOR]

The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects’ genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen, e.g. for absinthin, the bitter principle of the beverage absinth. These findings illustrate the extent to which genetic influences on taste are complex, yet arise from both receptor activation patterns and linkage structure among receptor genes. [ABSTRACT FROM AUTHOR]

Bitter taste perception in vertebrates relies on a variable number of bitter taste receptor (Tas2r) genes, ranging from only three functional genes in chicken to as many as approximately 50 in frogs. Humans possess a medium-sized Tas2r repertoire encoding three broadly and several narrowly tuned receptors plus receptors with intermediate tuning properties. Such tuning information is not available for bitter taste receptors of other vertebrate species. In particular it is not known, whether a small Tas2r repertoire may be compensated for by broad tuning of these receptors, and on the other side, whether a large repertoire might entail a preponderance of narrowly tuned receptors. To elucidate this question, we cloned all three chicken Tas2rs, the two turkey Tas2rs, three zebra finch Tas2rs, and six Tas2rs of the Western clawed frog representative of major branches of the phylogenetic tree, and screened them with 46 different bitter compounds. All chicken and turkey Tas2rs were broadly tuned, the zebra finch Tas2rs were narrowly tuned, and frog Tas2rs ranged from broadly to narrowly tuned receptors. We conclude that a low number of functional Tas2r genes does not imply a reduced importance of bitter taste per se, as it can be compensated by large tuning width. A high number of functional Tas2r genes appears to allow the evolution of specialized receptors, possibly for toxins with species-specific relevance. In sum, we show that variability in tuning breadth, overlapping agonist profiles, and staggered effective agonist concentration ranges are shared features of human and other vertebrate Tas2rs. [ABSTRACT FROM PUBLISHER]

G-protein-coupled receptors (GPCRs) are key mediators in cardiovascular physiology, yet frontline therapies for heart disease target only a small fraction of the cardiac GPCR repertoire. Moreover, there is emerging evidence that GPCRs implicated in taste (Tas1r and Tas2rs) have specific functions beyond the oral cavity. Our recent description of these receptors in heart tissue foreshadows a potential novel role in cardiac cells. In this study, we identified novel agonist ligands for cardiac-Tas2rs to enable the functional investigation of these receptors in heart tissue. Five Tas2rs cloned from heart tissue were screened against a panel of 102 natural or synthetic bitter compounds in a heterologous expression system. We identified agonists for Tas2r108, Tas2r137, and Tas2r143 that were then tested in Langendorff-perfused mouse hearts (from 8-wk-old male C57BL/6 mice). All Tas2r agonists tested exhibited concentration-dependent effects, with agonists for Tas2rl08 and Tas2rl37, leading to a ~40% decrease in left ventricular developed pressure and an increase in aortic pressure, respectively. These responses were abrogated in the presence of Gαi and Gβγ inhibitors (pertussis toxin and gallein). This study represents the first demonstration of profound Tas2r agonist-induced, G protein-dependent effects on mouse heart function. [ABSTRACT FROM AUTHOR]

Bitter taste receptors (TAS2Rs) mediate aversive response to toxic food, which is often bitter. These G-protein-coupled receptors are also expressed in extraoral tissues, and emerge as novel targets for therapeutic indications such as asthma and infection. Our goal was to identify ligands of the broadly timed TAS2R14 among clinical drugs. Molecular properties of known human bitter taste receptor TAS2R14 agonists were incorporated into pharmacophore- and shape-based models and used to computationally predict additional ligands. Predictions were tested by calcium imaging of TAS2R14-transfected HEK293 cells. In vitro testing of the virtual screening predictions resulted in 30-80% success rates, and 15 clinical drugs were found to activate the TAS2R14. hERG potassium channel, which is predominantly expressed in the heart, emerged as a common off-target of bitter drugs. Despite immense chemical diversity of known TAS2R14 ligands, novel ligands and previously unknown polypharmacology of drugs were unraveled by in vitro screening of computational predictions. This enables rational repurposing of traditional and standard drugs for bitter taste signaling modulation for therapeutic indications. [ABSTRACT FROM AUTHOR]

The concept that gut nutrient sensing involves taste receptors has been fueled by recent reports associating the expression of taste receptors and taste-associated signaling molecules in the gut and in gut-derived cell lines with physiological responses induced by known taste stimuli. However, for bitter taste receptors (Tas2rs), direct evidence for their functional role in gut physiology is scarce and their cellular expression pattern remained unknown. We therefore investigated Tas2r expression in mice. RT-PCR experiments assessed the presence of mRNA for Tas2rs and taste signaling molecules in the gut. A gene-targeted mouse strain was established to visualize and identify cell types expressing the bitter receptor Tas2r131. Messenger RNA for various Tas2rs and taste signaling molecules were detected by RT-PCR in the gut. Using our knock-in mouse strain we demonstrate that a subset of colonic goblet cells express Tas2r131. Cells that express this receptor are absent in the upper gut and do not correspond to enteroendocrine and brush cells. Expression in colonic goblet cells is consistent with a role of Tas2rs in defense mechanisms against potentially harmful xenobiotics. [ABSTRACT FROM AUTHOR]

Mutational polymorphism in the TAS2R38 bitter taste receptor is a key determinant of threshold taste detection of isolated compounds, such as phenylthiocarbamide (PTC) and propylthiouracil (PROP), as well as complex orosensation-mediated traits such as diet choice and smoking habits. These relationships are accounted for, in part, by 2 common alleles differing in functionality, TAS2R38-PAV and TAS2R38-AVI. However, TAS2R38 harbors extensive additional polymorphism whose functional significance remains unknown. To examine this variation, we ascertained genetic diversity in 56 Caucasian subjects via whole-gene sequencing, analyzed allele-specific responses to 5 TAS2R38 agonists (PTC, PROP, goitrin, methimazole, and sinigrin) using in vitro assays, and assessed genotypic associations with threshold detection phenotypes. Sequencing identified 3 single-nucleotide substitutions encoding 3 amino acid changes (C145G/P49A, C785T/A262V, and A886G/I296V), which combined to form 6 haplotypes in our sample. In vitro assays revealed a continuous range of response across alleles, and associations with threshold were significant for all single nucleotide polymorphisms (P < 0.002) and PAV/AVI haplotypes (P < 0.001). Haplotypes other than PAV and AVI did not exhibit phenotypic associations in our sample, possibly as a result of their low frequencies. However, prior studies have indicated that these alleles are common in some global regions, suggesting that alleles rare in our sample may be phenotypically relevant in other populations. [ABSTRACT FROM AUTHOR]

Bitter molecules in humans are detected by ∼25 G protein-coupled receptors (GPCRs). The lack of atomic resolution structure for any of them is complicating an in depth understanding of the molecular mechanisms underlying bitter taste perception. Here, we investigate the molecular determinants of the interaction of the TAS2R38 bitter taste receptor with its agonists phenylthiocarbamide (PTC) and propylthiouracil (PROP). We use the recently developed hybrid Molecular Mechanics/Coarse Grained (MM/CG) method tailored specifically for GPCRs. The method, through an extensive exploration of the conformational space in the binding pocket, allows the identification of several residues important for agonist binding that would have been very difficult to capture from the standard bioinformatics/docking approach. Our calculations suggest that both agonists bind to Asn103, Phe197, Phe264 and Trp201, whilst they do not interact with the so-called extra cellular loop 2, involved in cis-retinal binding in the GPCR rhodopsin. These predictions are consistent with data sets based on more than 20 site-directed mutagenesis and functional calcium imaging experiments of TAS2R38. The method could be readily used for other GPCRs for which experimental information is currently lacking. [ABSTRACT FROM AUTHOR]

Bitter taste is a basic taste modality, required to safeguard animals against consuming toxic substances. Bitter compounds are recognized by G-protein-coupled bitter taste receptors (TAS2Rs). The human TAS2R10 responds to the toxic strychnine and numerous other compounds. The mechanism underlying the development of the broad tuning of some TAS2Rs is not understood. Using comparative modeling, site-directed mutagenesis, and functional assays, we identified residues involved in agonist-induced activation of TAS2R10, and investigated the effects of different substitutions on the receptor's response profile. Most interestingly, mutations in S853.29 and Q1755.40 have differential impact on stimulation with different agonists. The fact that single point mutations lead to improved responses for some agonists and to decreased activation by others indicates that the binding site has evolved to optimally accommodate multiple agonists at the expense of reduced potency. TAS2R10 shares the agonist strychnine with TAS2R46, another broadly tuned receptor. Engineering the key determinants for TAS2R46 activation by strychnine in TAS2R10 caused a loss of response to strychnine, indicating that these paralog receptors display different strychnine-binding modes, which suggests independent acquisition of agonist specificities. This implies that the gene duplication event preceding primate speciation was accompanied by independent evolution of the strychnine-binding sites. [ABSTRACT FROM AUTHOR]

Taste buds are chemosensory structures widely distributed on the surface of the oral cavity and larynx. Taste cells, exposed to the oral environment, face great challenges in defense against potential pathogens. While immune cells, such as T-cells and macrophages, are rarely found in taste buds, high levels of expression of some immune-response-associated molecules are observed in taste buds. Yet, the cellular origins of these immune molecules such as cytokines in taste buds remain to be determined. Here, we show that a specific subset of taste cells selectively expresses high levels of the inflammatory cytokine tumor necrosis factor-α (TNF-α). Based on immuno- colocalization experiments using taste-cell-type markers, the TNF-α producing cells are predominantly type II taste cells expressing the taste receptor T1R3. These cells can rapidly increase TNF-α production and secretion upon inflammatory challenges, both in vivo and in vitro. The lipopolysaccharide (LPS)-induced TNF-α expression in taste cells was completely eliminated in TLR2-/-/TLR4-/- double-gene- knockout mice, which confirms that the induction of TNF-α in taste buds by LPS is mediated through TLR signaling pathways. The taste-cell-produced TNF- α may contribute to local immune surveillance, as well as regulate taste sensation under normal and pathological conditions. [ABSTRACT FROM AUTHOR]

The sense of taste plays an important role in the evaluation of the nutrient composition of consumed food. Bitter taste in particular is believed to serve a warning function against the ingestion of poisonous substances. In the past years enormous progress was made in the characterization of bitter taste receptors, including their gene expression patterns, pharmacological features and presumed physiological roles in gustatory as well as in non-gustatory tissues. However, due to a lack in TAS2R-specifc antibodies the localization of receptor proteins within gustatory tissues has never been analyzed. In the present study we have screened a panel of commercially available antisera raised against human bitter taste receptors by immunocytochemical experiments. One of these antisera was found to be highly specific for the human bitter taste receptor TAS2R38. We further demonstrate that this antibody is able to detect heterologously expressed TAS2R38 protein on Western blots. The antiserum is, however, not able to interfere significantly with TAS2R38 function in cell based calcium imaging analyses. Most importantly, we were able to demonstrate the presence of TAS2R38 protein in human gustatory papillae. Using double immunofluorescence we show that TAS2R38-positive cells form a subpopulation of PLCbeta2 expressing cells. On a subcellular level the localization of this bitter taste receptor is neither restricted to the cell surface nor particularly enriched at the level of the microvilli protruding into the pore region of the taste buds, but rather evenly distributed over the entire cell body. [ABSTRACT FROM AUTHOR]

In contrast to carbohydrates and proteins, which are detected by specialized taste receptors in the forms of their respective building blocks, sugars, and L-amino acids, the third macronutrient, lipids, has until now not been associated with gustatory receptors. Instead, the recognition of fat stimuli was believed to rely mostly on textural, olfactory, and postingestive cues. During the recent years, however, research done mainly in rodent models revealed an additional gustatory component for the detection of long-chain fatty acids (LCFAs), the main taste-activating component of lipids. Concomitantly, a number of candidate fat taste receptors were proposed to be involved in rodent's gustatory fatty acid perception. Compared with rodent models, much less is known about human fat taste. In order to investigate the ability of the human gustatory system to respond to fat components, we performed sensory experiments with fatty acids of different chain lengths and derivatives thereof. We found that our panelists discriminated a "fatty" and an irritant "scratchy" taste component, with the "fatty" percept restricted to LCFAs. Using functional calcium–imaging experiments with the human orthologs of mouse candidate fat receptors belonging to the G protein-coupled receptor family, we correlated human sensory data with receptor properties characterized in vitro. We demonstrated that the pharmacological activation profile of human GPR40 and GPR120, 2 LCFAspecific receptors associated with gustatory fat perception in rodents, is inconsistent with the "scratchy" sensation of human subjects and more consistent with the percept described as "fatty." Expression analysis of GPR40 and GPR120 in human gustatory tissues revealed that, while the GPR40 gene is not expressed, GPR120 is detected in gustatory and nongustatory epithelia. On a cellular level, we found GPR120 mRNA and protein in taste buds as well as in the surrounding epithelial cells. We conclude that GPR120 may indeed participate in human gustatory fatty acid perception. [ABSTRACT FROM AUTHOR]

Food contains complex blends of structurally diverse bitter compounds that trigger bitterness through activation of one or more of the β25 human TAS2 bitter taste receptors. It remains unsolved, however, whether the perceived bitterness of binary bitter-compound mixtures can be considered an additive function of all bitter-inducing chemicals in the mouth, suggesting that little mutual interaction takes place among bitter substances or if mixture suppression and synergism occurs. Here we report on two natural sesquiterpene lactones from edible plants, which stimulate distinct sets of hTAS2Rs in transfected cells. Both chemicals also robustly inhibit different but overlapping subsets of agonist-activated hTAS2Rs. These findings demonstrate that mixtures of bitter compounds, because they normally occur in human foodstuff, likely elicit bitter perception in a complex and not in a merely additive manner. An unexpected implication of this discovery is that, during evolution, the naturally occurring bitter taste receptor antagonists have shaped some of the pharmacological properties of the receptors, such as overlapping recognition profiles and breadth of tuning. [ABSTRACT FROM AUTHOR]

BSTRACT Bitter taste prevents mammals from ingesting food potentially contaminated with bitter-tasting toxins, which are frequent and structurally diverse. It is mediated by a family of heptahelical G protein-coupled receptors, called taste 2 receptors or TAS2Rs or T2Rs. The number of TAS2R genes differs greatly across mammalian species, suggesting that some species depend less than others on the protective effect of TAS2Rs. TAS2Rs are glycosylated to become substrates for chaperones required for them to complete biosynthesis and cell surface expression. TAS2Rs recognize several to many bitter compounds, a property that accounts for our ability to detect the numerous bitter substances. Although TAS2Rs oligomerize, heteromers apparently do not increase the number of detectable bitter substances. A small molecule antagonist that inhibits bitter taste in vivo blocks several TAS2Rs in vitro, thereby sharing the property with bitter agonists to interact with several receptors. TAS2Rs possess only one binding pocket, in which they accommodate their ligands by contacting different but overlapping sets of amino acids in the upper parts of transmembrane segments 2-7. Pronounced genetic variability has been observed for human TAS2R genes, including single nucleotide polymorphisms, copy number variations and indels, which determine functionally different receptor variants and influence subjects' sensitivity for the bitterness of specific compounds. However, whether differences in taste sensitivity influence preferences in nutrition remains elusive and is only indirectly supported. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

An important question in taste research is how 25 receptors of the human TAS2R family detect thousands of structurally diverse compounds. An answer to this question may arise from the observation that TAS2Rs in general are broadly tuned to interact with numerous substances. Ultimately, interaction with chemically diverse agonists requires architectures of binding pockets tailored to combine flexibility with selectivity. The present study determines the structure of hTAS2R binding pockets. We focused on a subfamily of closely related hTAS2Rs exhibiting pronounced amino acid sequence identities but unique agonist activation spectra. The generation of chimeric and mutant receptors followed by calcium imaging analyses identified receptor regions and amino acid residues critical for activation of hTAS2R46. -R43, and -R31. We found that the carboxyl-terminal regions of the investigated receptors are crucial for agonist selectivity. Intriguingly, exchanging two residues located in transmembrane domain seven between hTAS2R46, activated by strychnine, and hTAS2R31, activated by aristolochic acid, was sufficient to invert agonist selectivity. Further mutagenesis revealed additional positions involved in agonist interaction. The transfer of functionally relevant amino acids identified in hTAS2R46 to the corresponding positions of hTAS2R43 and -R31 resulted in pharmacological properties indistinguishable from the parental hTAS2R46. In silico modeling of hTAS2R46 allowed us to visualize the putative mode of interaction between agonists and hTAS2Rs. Detailed structure-function analyses of hTAS2Rs may ultimately pave the way for the development of specific antagonists urgently needed for more sophisticated analyses of human bitter taste perception. [ABSTRACT FROM AUTHOR]