Receptors for bitter and sweet taste

The identification of two families of receptors, T1Rs and T2Rs, for sweet and bitter taste stimuli has opened the door to understanding some of the basic mechanisms underlying taste transduction in mammals. Studies of the functions of these receptors and their patterns of expression provide important information regarding the detection of structurally diverse taste compounds and the manner in which different taste qualities are encoded in the mouth.     

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.     

Arachidonic acid can function as a signaling modulator by activating the TRPM5 cation channel in taste receptor cells

Vertebrate sensory cells such as vomeronasal neurons and Drosophila photoreceptor cells use TRP channels to respond to exogenous stimuli. In mammalian taste cells, bitter and sweet substances as well as some amino acids are received by G protein-coupled receptors (T2Rs or T1Rs). As a result of activation of G protein and phospholipase Cbeta2, the TRPM5 channel is activated. Intracellular Ca(2+) is known to be a TRPM5 activator, but the participation of lipid activators remains unreported. To clarify the effect of arachidonic acid on TRPM5 in taste cells, we investigated the expression profile of a series of enzymes involved in controlling the intracellular free arachidonic acid level, with the result that in a subset of taste bud cells, monoglyceride lipase (MGL) and cyclooxygenase-2 (COX-2) are expressed as well as the previously reported group IIA phospholipase A(2) (PLA(2)-IIA). Double-labeling analysis revealed that MGL, COX-2 and PLA(2)-IIA are co-expressed in some cells that express TRPM5. We then investigated whether arachidonic acid activates TRPM5 via a heterologous expression system in HEK293 cells, and found that its activation occurred at 10 microM arachidonic acid. These results strongly suggest the possibility that arachidonic acid acts as a modulator of TRPM5 in taste signaling pathways.     

Two families of candidate taste receptors in fishes

Vertebrates receive tastants, such as sugars, amino acids, and nucleotides, via taste bud cells in epithelial tissues. In mammals, two families of G protein-coupled receptors for tastants are expressed in taste bud cells-T1Rs for sweet tastants and umami tastants (l-amino acids) and T2Rs for bitter tastants. Here, we report two families of candidate taste receptors in fish species, fish T1Rs and T2Rs, which show significant identity to mammalian T1Rs and T2Rs, respectively. Fish T1Rs consist of three types: fish T1R1 and T1R3 that show the highest degrees of identity to mammalian T1R1 and T1R3, respectively, and fish T1R2 that shows almost equivalent identity to both mammalian T1R1 and T1R2. Unlike mammalian T1R2, fish T1R2 consists of two or three members in each species. We also identified two fish T2Rs that show low degrees of identity to mammalian T2Rs. In situ hybridization experiments revealed that fish T1R and T2R genes were expressed specifically in taste bud cells, but not in olfactory receptor cells. Fish T1R1 and T1R2 genes were expressed in different subsets of taste bud cells, and fish T1R3 gene was co-expressed with either fish T1R1 or T1R2 gene as in the case of mammals. There were also a significant number of cells expressing fish T1R2 genes only. Fish T2R genes were expressed in different cells from those expressing fish T1R genes. These results suggest that vertebrates commonly have two kinds of taste signaling pathways that are defined by the types of taste receptors expressed in taste receptor cells.     

Mammalian sweet taste receptors

The sense of taste provides animals with valuable information about the quality and nutritional value of food. Previously, we identified a large family of mammalian taste receptors involved in bitter taste perception (the T2Rs). We now report the characterization of mammalian sweet taste receptors. First, transgenic rescue experiments prove that the Sac locus encodes T1R3, a member of the T1R family of candidate taste receptors. Second, using a heterologous expression system, we demonstrate that T1R2 and T1R3 combine to function as a sweet receptor, recognizing sweet-tasting molecules as diverse as sucrose, saccharin, dulcin, and acesulfame-K. Finally, we present a detailed analysis of the patterns of expression of T1Rs and T2Rs, thus providing a view of the representation of sweet and bitter taste at the periphery.     

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.     

Capsaicin receptors are colocalized with sweet/bitter receptors in the taste sensing cells of circumvallate papillae

We examined co-localization of vanilloid receptor (VR1) with sweet receptors T1R2, T1R3, or bitter receptor T2R6 in taste receptor cells of rat circumvallate papillae. Tissue sections of rat circumvallate papillae were doubly reacted with anti-VR1 antibodies and anti-T1R2, anti-T1R3 or anti-T2R6 antibodies, using double-immunofluorescence histochemistry technique. Localizations of VR1, T1Rs and T2R6 in the vallate taste cells containing α-gustducin were also examined. VR1 immunoreactivities (-ir) were observed in subsets of taste cells in the circumvallate papillae, and 96–99% of the vallate taste cells exhibiting T1R2-, T1R3- or T2R6-ir co-exhibited VR1-ir. Approximately half of T2R6-ir cells (~49%), and 50–58% of T1Rs-ir cells, co-exhibited α-gustducin-ir in the vallate taste buds. About 58% of VR1-ir cells in the vallate exhibited α-gustducin-ir as well. Results support the idea that capsaicin may interact with the transduction pathways of sweet and bitter taste stimuli, possibly in mediation of its receptor VR1 localized in taste receptor cells. Additionally, the partial co-localization of α-gustducin with VR1 suggests that a tentative modulatory function of capsaicin in sweet and bitter transductions in the rat circumvallate comprises of both α-gustducin-mediated and non-mediated transduction pathways.     

Apparent specific volumes and tastes of amino acids

The apparent specific volumes of 17 amino acids are determinedand compared with their tastes and predicted ranges of tastereported in earlier work. Most amino acids elicit many basictastes although one taste usually predominates. All D-aminoacids tasted (except proline and hydroxy prohne) are sweet andseven of the L-amino acids are sweet. Ten of the 16 L-aminoacids are bitter but two of these are both bitter and sweetin accordance with prediction from their apparent specific volumes.‘Spatial barriers’ account for the exclusion ofthe larger L-amino acids from sweet receptors. However, D-proline,the only reportedly purely bitter D-amino acid, is cyclic andthis explains its borderline position between the sweet andbitter regions, and the sweet-bitter taste of L-proline. Lowmol. wt amino acids, irrespective of whether they are D- orL-enantiomers, are always sweet, providing that their apparentspecific volumes are below 0.66 and that they do not generatesuprathreshold concentrations of protons. The two dicarboxylicamino acids, glutamic and aspartic are sour. Apparent specificvolumes represent the effective size of solutes in water dueto their intrinsic molecular architecture. The values reportedhere are used to interpret steric exclusion of certain enantiomersfrom the taste receptors. These results support the idea thatapparent specific volume is a determinant of taste quality.     

The receptors for mammalian sweet and umami taste

Sweet and umami (the taste of monosodium glutamate) are the main attractive taste modalities in humans. T1Rs are candidate mammalian taste receptors that combine to assemble two heteromeric G-protein-coupled receptor complexes: T1R1+3, an umami sensor, and T1R2+3, a sweet receptor. We now report the behavioral and physiological characterization of T1R1, T1R2, and T1R3 knockout mice. We demonstrate that sweet and umami taste are strictly dependent on T1R-receptors, and show that selective elimination of T1R-subunits differentially abolishes detection and perception of these two taste modalities. To examine the basis of sweet tastant recognition and coding, we engineered animals expressing either the human T1R2-receptor (hT1R2), or a modified opioid-receptor (RASSL) in sweet cells. Expression of hT1R2 in mice generates animals with humanized sweet taste preferences, while expression of RASSL drives strong attraction to a synthetic opiate, demonstrating that sweet cells trigger dedicated behavioral outputs, but their tastant selectivity is determined by the nature of the receptors.     

Bitter taste receptors: Novel insights into the biochemistry and pharmacology

Bitter taste receptors (T2Rs) belong to the super family of G protein-coupled receptors (GPCRs). There are 25 T2Rs expressed in humans, and these interact with a large and diverse group of bitter ligands. T2Rs are expressed in many extra-oral tissues and can perform diverse physiological roles. Structure-function studies led to the identification of similarities and dissimilarities between T2Rs and Class A GPCRs including amino acid conservation and novel motifs. However, the efficacy of most of the T2R ligands is not yet elucidated and the biochemical pharmacology of T2Rs is poorly understood. Recent studies on T2Rs characterized novel ligands including blockers for these receptors that include inverse agonist and antagonists. In this review we discuss the techniques used for elucidating bitter blockers, concept of ligand bias, generic amino acid numbering, the role of cholesterol, and conserved water molecules in the biochemistry and pharmacology of T2Rs.     

Faithful expression of GFP from the PLCbeta2 promoter in a functional class of taste receptor cells

Phospholipase C-type beta2 (PLCbeta2) is expressed in a subset of cells within mammalian taste buds. This enzyme is involved in the transduction of sweet, bitter, and umami stimuli and thus is believed to be a marker for gustatory sensory receptor cells. We have developed transgenic mice expressing green fluorescent protein (GFP) under the control of the PLCbeta2 promoter to enable one to identify these cells and record their physiological activity in living preparations. Expression of GFP (especially in lines with more than one copy integrated) is strong enough to be detected in intact tissue preparations using epifluorescence microscopy. By immunohistochemistry, we confirmed that the overwhelming majority of cells expressing GFP are those that endogenously express PLCbeta2. Expression of the GFP transgene in circumvallate papillae occurs at about the same time during development as endogenous PLCbeta2 expression. When loaded with a calcium-sensitive dye in situ, GFP-positive taste cells produce typical Ca2+ responses to a taste stimulus, the bitter compound cycloheximide. These PLCbeta2 promoter-GFP transgenic lines promise to be useful for studying taste transduction, sensory signal processing, and taste bud development.     

Identification and characterization of human taste receptor genes belonging to the TAS2R family

The sense of taste is a chemosensory system responsible for basic food appraisal. Humans distinguish between five primary tastes: bitter, sweet, sour, salty and umami. The molecular events in the perception of bitter taste are believed to start with the binding of specific water-soluble molecules to G-protein-coupled receptors encoded by the TAS2R/T2R family of taste receptor genes. TAS2R receptors are expressed at the surface of taste receptor cells and are coupled to G proteins and second messenger pathways. We have identified, cloned and characterized 11 new bitter taste receptor genes and four new pseudogenes that belong to the human TAS2R family. Their encoded proteins have between 298 and 333 amino acids and share between 23 and 86% identity with other human TAS2R proteins. Screening of a mono-chromosomal somatic cell hybrid panel to assign the identified bitter taste receptor genes to human chromosomes demonstrated that they are located in chromosomes 7 and 12. Including the 15 sequences identified, the human TAS2R family is composed of 28 full-length genes and 16 pseudogenes. Phylogenetic analyses suggest a classification of the TAS2R genes in five groups that may reflect a specialization in the detection of specific types of bitter chemicals.     

The sweet and the bitter of mammalian taste

The discovery of two families of mammalian taste receptors has provided important insights into taste recognition and taste perception. Recent studies have examined the receptors and signaling pathways that mediate sweet, bitter, and amino acid taste detection in mammals. These studies demonstrate that taste cells are selectively tuned to different taste modalities and clarify the logic of taste coding in the periphery.     

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 .     

Molecular mechanisms underlying the reception and transmission of sour taste information

Taste enables organisms to determine the properties of ingested substances by conveying information regarding the five basic taste modalities: sweet, salty, sour, bitter, and umami. The sweet, salty, and umami taste modalities convey the carbohydrate, electrolyte, and glutamate content of food, indicating its desirability and stimulating appetitive responses. The sour and bitter modalities convey the acidity of food and the presence of potential toxins, respectively, stimulating aversive responses to such tastes. In recent years, the receptors mediating sweet, bitter, and umami tastes have been identified as members of the T1R and T2R G-protein-coupled receptor families; however, the molecular mechanisms underlying sour taste detection have yet to be clearly elucidated. This review covers the molecular mechanisms proposed to mediate the detection and transmission of sour stimuli, focusing on polycystic kidney disease 1-like 3 (Pkd1l3), Pkd2l1, and carbonic anhydrase 4 (Car4).     

Modulation of Sweet Taste by Umami Compounds via Sweet Taste Receptor Subunit hT1R2

Although the five basic taste qualities—sweet, sour, bitter, salty and umami—can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5’-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.     

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.     

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 sweet taste quality is linked to a cluster of taste fibers in primates: lactisole diminishes preference and responses to sweet in S fibers (sweet best) chorda tympani fibers of M. fascicularis monkey

Background

Psychophysically, sweet and bitter have long been considered separate taste qualities, evident already to the newborn human. The identification of different receptors for sweet and bitter located on separate cells of the taste buds substantiated this separation. However, this finding leads to the next question: is bitter and sweet also kept separated in the next link from the taste buds, the fibers of the taste nerves? Previous studies in non-human primates, P. troglodytes, C. aethiops, M. mulatta, M. fascicularis and C. jacchus, suggest that the sweet and bitter taste qualities are linked to specific groups of fibers called S and Q fibers. In this study we apply a new sweet taste modifier, lactisole, commercially available as a suppressor of the sweetness of sugars on the human tongue, to test our hypothesis that sweet taste is conveyed in S fibers.

Results

We first ascertained that lactisole exerted similar suppression of sweetness in M. fascicularis, as reported in humans, by recording their preference of sweeteners and non- sweeteners with and without lactisole in two-bottle tests. The addition of lactisole significantly diminished the preference for all sweeteners but had no effect on the intake of non-sweet compounds or the intake of water. We then recorded the response to the same taste stimuli in 40 single chorda tympani nerve fibers. Comparison between single fiber nerve responses to stimuli with and without lactisole showed that lactisole only suppressed the responses to sweeteners in S fibers. It had no effect on the responses to any other stimuli in all other taste fibers.

Conclusion

In M. fascicularis, lactisole diminishes the attractiveness of compounds, which taste sweet to humans. This behavior is linked to activity of fibers in the S-cluster. Assuming that lactisole blocks the T1R3 monomer of the sweet taste receptor T1R2/R3, these results present further support for the hypothesis that S fibers convey taste from T1R2/R3 receptors, while the impulse activity in non-S fibers originates from other kinds of receptors. The absence of the effect of lactisole on the faint responses in some S fibers to other stimuli as well as the responses to sweet and non-sweet stimuli in non-S fibers suggest that these responses originate from other taste receptors.