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.     

The G-protein coupling properties of the human sweet and amino acid taste receptors

The human T1R taste receptors are family C G-protein-coupled receptors (GPCRs) that act as heterodimers to mediate sweet (hT1R2 + hT1R3) and umami (hT1R1 + hT1R3) taste modalities. Each T1R has a large extracellular ligand-binding domain linked to a seven transmembrane-spanning core domain (7TMD). We demonstrate that the 7TMDs of hT1R1 and hT1R2 display robust ligand-independent constitutive activity, efficiently catalyzing the exchange of GDP for GTP on Galpha subunits. In contrast, relative to the 7TMDs of hT1R1 and hT1R2, the 7TMD of hT1R3 couples poorly to G-proteins, suggesting that in vivo signaling may proceed primarily through hT1R1 and hT1R2. In addition, we provide direct evidence that the hT1Rs selectively signal through Galpha(i/o) pathways, coupling to multiple Galpha(i/o) subunits as well as the taste cell specific Gbeta(1)gamma(13) dimer.     

Three sweet receptor genes are clustered in human Chromosome 1

A search of the human genome database led us to identify three human candidate taste receptors, hT1R1, hT1R2, and hT1R3, which contain seven transmembrane domains. All three genes map to a small region of Chromosome (Chr) 1. This region is syntenous to the distal end of Chr 4 in mouse, which contains the Sac (saccharin preference) locus that is involved in detecting sweet tastants. A genetic marker, DVL1, which is linked to the Sac locus, is within 1700 bp of human T1R3. Recently, the murine T1Rs and its human ortholog have been independently identified in combination as sweet and umami receptors near the Sac locus. All three hT1Rs genes are expressed selectively in human taste receptor cells in the fungiform papillae, consistent with their role in taste perception.     

Molecular Mechanisms for Sweet-suppressing Effect of Gymnemic Acids

Gymnemic acids are triterpene glycosides that selectively suppress taste responses to various sweet substances in humans but not in mice. This sweet-suppressing effect of gymnemic acids is diminished by rinsing the tongue with γ-cyclodextrin (γ-CD). However, little is known about the molecular mechanisms underlying the sweet-suppressing effect of gymnemic acids and the interaction between gymnemic acids versus sweet taste receptor and/or γ-CD. To investigate whether gymnemic acids directly interact with human (h) sweet receptor hT1R2 + hT1R3, we used the sweet receptor T1R2 + T1R3 assay in transiently transfected HEK293 cells. Similar to previous studies in humans and mice, gymnemic acids (100 μg/ml) inhibited the [Ca²⁺]_i responses to sweet compounds in HEK293 cells heterologously expressing hT1R2 + hT1R3 but not in those expressing the mouse (m) sweet receptor mT1R2 + mT1R3. The effect of gymnemic acids rapidly disappeared after rinsing the HEK293 cells with γ-CD. Using mixed species pairings of human and mouse sweet receptor subunits and chimeras, we determined that the transmembrane domain of hT1R3 was mainly required for the sweet-suppressing effect of gymnemic acids. Directed mutagenesis in the transmembrane domain of hT1R3 revealed that the interaction site for gymnemic acids shared the amino acid residues that determined the sensitivity to another sweet antagonist, lactisole. Glucuronic acid, which is the common structure of gymnemic acids, also reduced sensitivity to sweet compounds. In our models, gymnemic acids were predicted to dock to a binding pocket within the transmembrane domain of hT1R3.     

Identification of the cyclamate interaction site within the transmembrane domain of the human sweet taste receptor subunit T1R3

The artificial sweetener cyclamate tastes sweet to humans, but not to mice. When expressed in vitro, the human sweet receptor (a heterodimer of two taste receptor subunits: hT1R2 + hT1R3) responds to cyclamate, but the mouse receptor (mT1R2 + mT1R3) does not. Using mixed-species pairings of human and mouse sweet receptor subunits, we determined that responsiveness to cyclamate requires the human form of T1R3. Using chimeras, we determined that it is the transmembrane domain of hT1R3 that is required for the sweet receptor to respond to cyclamate. Using directed mutagenesis, we identified several amino acid residues within the transmembrane domain of T1R3 that determine differential responsiveness to cyclamate of the human versus mouse sweet receptors. Alanine-scanning mutagenesis of residues predicted to line a transmembrane domain binding pocket in hT1R3 identified six residues specifically involved in responsiveness to cyclamate. Using molecular modeling, we docked cyclamate within the transmembrane domain of T1R3. Our model predicts substantial overlap in the hT1R3 binding pockets for the agonist cyclamate and the inverse agonist lactisole. The transmembrane domain of T1R3 is likely to play a critical role in the interconversion of the sweet receptor from the ground state to the active state.     

Amiloride reduces the sweet taste intensity by inhibiting the human sweet taste receptor

In mammals, sweet taste perception is mediated by the heterodimeric G-protein-coupled receptor, T1R2/T1R3. An interesting characteristic of this sweet taste receptor is that it has multiple ligand binding sites. Although there have been several studies on agonists of sweet taste receptors, little is known about antagonists of these receptors. In this study, we constructed a cell line stably expressing the human sweet taste receptor (hT1R2/hT1R3) and a functional chimeric G-protein (hGα16gust44) using the Flp-In system for measuring the antagonistic activity against the receptor. This constructed cell line responded quite intensely and frequently to the compounds applied for activation of hT1R2/hT1R3. In the presence of 3 mM amiloride, the responses to sweet tastants such as sugar, artificial sweetener, and sweet protein were significantly reduced. The inhibitory activity of amiloride toward 1 mM aspartame was observed in a dose-dependent manner with an IC₅₀ value of 0.87 mM. Our analysis of a cell line expressing hT1R3 mutants (hT1R3-A733V or hT1R3-F778A) made us to conclude that the target site of amiloride is distinct from that of lactisole, a known sweet taste inhibitor. Our results strongly indicate that amiloride reduces the sweet taste intensity by inhibiting the human sweet taste receptor and also that this receptor has multiple inhibitor binding sites.     

Recombinant expression, in vitro refolding, and biophysical characterization of the N-terminal domain of T1R3 taste receptor

The sweet taste receptor is a heterodimeric receptor composed of the T1R2 and T1R3 subunits, while T1R1 and T1R3 assemble to form the umami taste receptor. T1R receptors belong to the family of class C G-protein coupled receptors (GPCRs). In addition to a transmembrane heptahelical domain, class C GPCRs have a large extracellular N-terminal domain (NTD), which is the primary ligand-binding site. The T1R2 and T1R1 subunits have been shown to be responsible for ligand binding, via their NTDs. However, little is known about the contribution of T1R3-NTD to receptor functions. To enable biophysical characterization, we overexpressed the human NTD of T1R3 (hT1R3-NTD) using Escherichia coli in the form of inclusion bodies. Using a fractional factorial screen coupled to a functional assay, conditions were determined for the refolding of hT1R3-NTD. Far-UV circular dichroism spectroscopic studies revealed that hT1R3-NTD was well refolded. Using size-exclusion chromatography, we found that the refolded protein behaves as a dimer. Ligand binding quantified by tryptophan fluorescence quenching and microcalorimetry showed that hT1R3-NTD is functional and capable of binding sucralose with an affinity in the millimolar range. This study also provides a strategy to produce functional hT1R3-NTD by heterologous expression in E. coli; this is a prerequisite for structural determination and functional analysis of ligand-binding regions of other class C GPCRs.     

The liaison of sweet and savory

The sense of taste provides humans with necessary information about the composition and quality of food. For humans, five basic tastes are readily distinguishable and include sweet, bitter, salty, sour, and savory (or umami). Although each of these qualities has individualized transduction pathways, sweet and umami tastes are believed to share a common receptor element, the T1R3 receptor subunit. The two G-protein-coupled heteromer receptors that comprise an umami stimulus receptor (T1R1-T1R3) and a sweetener receptor (T1R2-T1R3) constitute a potential link between these two qualities of perception. While the role of the individual monomers in each human heteromer has been examined in vitro, very little is known of the implication of this research for human perception, or specifically, how sweet and savory taste perceptions may be connected. Using a psychophysical approach, we demonstrate that lactisole, a potent sweetness inhibitor that binds in vitro to hT1R3, also inhibits a significant portion of the perception of umami taste from monosodium glutamate. Following the molecular logic put forward by Xu et al. (2004, Proc. Natl Acad. Sci. USA, 101, 14258-14263), our psychophysical data support the in vitro hypothesis that the shared T1R3 monomer moderates the activation of both T1R2 and T1R1 in humans and impairs suprathreshold perception, respectively, of sweetness and, to a lesser degree, umaminess in the presence of lactisole.     

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

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

Sucrose and monosodium glutamate taste thresholds and discrimination ability of T1R3 knockout mice

Molecular and behavioral studies have identified heterodimers of the T1R family as receptors for detecting the tastes of sweet (T1R2 + T1R3) and umami (T1R1 + T1R3). However, behavioral studies have reported conflicting findings with T1R3 knockout (KO) mice. One study showed a complete or nearly complete loss of preference for sweet and umami substances by KO mice, whereas KO mice in another study showed only a partial reduction in preferences for sucrose and monosodium glutamate (MSG), the prototypical umami substance. The present experiments used psychophysical methods to assess how sensitive T1R1-KO mice are to sucrose and MSG and discrimination methods to determine if these mice could distinguish between the tastes of sucrose and MSG. Detection thresholds of T1R3-KO mice and wild-type (WT) C57Bl mice were nearly identical for sucrose and MSG. Mice of both genotypes were easily able to discriminate between the tastes of sucrose and MSG. When amiloride (a sodium channel blocker) was added to all solutions to reduce the taste of Na+, discrimination accuracy of both genotypes of mice decreased but more so for the T1R3-KO mice than the WT mice. However, even when the sodium taste of MSG was neutralized, both genotypes could still discriminate between the two substances well above chance performance. These results suggest that sucrose and MSG can be detected by taste receptors other than T1R2 + T1R3 and T1R1 + T1R3 and that the conflicts between the previous studies may have been due to the methodological limitations.     

Taste-modifying sweet protein, neoculin, is received at human T1R3 amino terminal domain

This study examines taste reception of neoculin, a Curculigo latifolia sweet protein with taste-modifying activity which converts sourness to sweetness. Neoculin tastes sweet to humans, but not to mice, and is received by the human sweet taste receptor hT1R2-hT1R3. In the present study with calcium imaging analysis of HEK cells expressing human and mouse T1Rs, we demonstrated that hT1R3 is required for the reception of neoculin. Further experiments using human/mouse chimeric T1R3s revealed that the extracellular amino terminal domain (ATD) of hT1R3 is essential for the reception of neoculin. Although T1R2-T1R3 is known to have multiple potential ligand-binding sites to receive a wide variety of sweeteners, the present study is apparently the first to identify the ATD of hT1R3 as a new sweetener-binding region.     

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).     

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.     

Abnormal taste perception in mice lacking the type 3 inositol 1,4,5-trisphosphate receptor

Inositol 1,4,5-trisphosphate receptor (IP3R) is one of the important calcium channels expressed in the endoplasmic reticulum and has been shown to play crucial roles in various physiological phenomena. Type 3 IP3R is expressed in taste cells, but the physiological relevance of this receptor in taste perception in vivo is still unknown. Here, we show that mice lacking IP3R3 show abnormal behavioral and electrophysiological responses to sweet, umami, and bitter substances that trigger G-protein-coupled receptor activation. In contrast, responses to salty and acid tastes are largely normal in the mutant mice. We conclude that IP3R3 is a principal mediator of sweet, bitter, and umami taste perception and would be a missing molecule linking phospholipase C beta2 to TRPM5 activation.     

Genetic variation in taste and its influence on food selection

Abstract Taste perception plays a key role in determining individual food preferences and dietary habits. Individual differences in bitter, sweet, umami, sour, or salty taste perception may influence dietary habits, affecting nutritional status and nutrition-related chronic disease risk. In addition to these traditional taste modalities there is growing evidence that "fat taste" may represent a sixth modality. Several taste receptors have been identified within taste cell membranes on the surface of the tongue, and they include the T2R family of bitter taste receptors, the T1R receptors associated with sweet and umami taste perception, the ion channels PKD1L3 and PKD2L1 linked to sour taste, and the integral membrane protein CD36, which is a putative "fat taste" receptor. Additionally, epithelial sodium channels and a vanilloid receptor, TRPV1, may account for salty taste perception. Common polymorphisms in genes involved in taste perception may account for some of the interindividual differences in food preferences and dietary habits within and between populations. This variability could affect food choices and dietary habits, which may influence nutritional and health status and the risk of chronic disease. This review will summarize the present state of knowledge of the genetic variation in taste, and how such variation might influence food intake behaviors.     

Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25

Background  

Taste receptor cells are responsible for transducing chemical stimuli from the environment and relaying information to the nervous system. Bitter, sweet and umami stimuli utilize G-protein coupled receptors which activate the phospholipase C (PLC) signaling pathway in Type II taste cells. However, it is not known how these cells communicate with the nervous system. Previous studies have shown that the subset of taste cells that expresses the T2R bitter receptors lack voltage-gated Ca²⁺ channels, which are normally required for synaptic transmission at conventional synapses. Here we use two lines of transgenic mice expressing green fluorescent protein (GFP) from two taste-specific promoters to examine Ca²⁺ signaling in subsets of Type II cells: T1R3-GFP mice were used to identify sweet- and umami-sensitive taste cells, while TRPM5-GFP mice were used to identify all cells that utilize the PLC signaling pathway for transduction. Voltage-gated Ca²⁺ currents were assessed with Ca²⁺ imaging and whole cell recording, while immunocytochemistry was used to detect expression of SNAP-25, a presynaptic SNARE protein that is associated with conventional synapses in taste cells.     

Characterization of the modes of binding between human sweet taste receptor and low-molecular-weight sweet compounds

One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2-hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein-coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2-hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling-based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.     

Contrasting modes of evolution between vertebrate sweet/umami receptor genes and bitter receptor genes

Taste reception is fundamental to diet selection in many animals. The genetic basis underlying the evolution and diversity of taste reception, however, is not well understood. Recent discoveries of T1R sweet/umami receptor genes and T2R bitter receptor genes in humans and mice provided an opportunity to address this question. Here, we report the identification of 20 putatively functional T1R genes and 167 T2R genes from the genome sequences of nine vertebrates, including three fishes, one amphibian, one bird, and four mammals. Our comparative genomic analysis shows that orthologous T1R sequences are relatively conserved in evolution and that the T1R gene repertoire remains virtually constant in size across most vertebrates, except for the loss of the T1R2 sweet receptor gene in the sweet-insensitive chicken and the absence of all T1R genes in the tongueless western clawed frog. In contrast, orthologous T2R sequences are more variable, and the T2R repertoire diverges tremendously among species, from only three functional genes in the chicken to 49 in the frog. These evolutionary patterns suggest the relative constancy in the number and type of sweet and umami tastants encountered by various vertebrates or low binding specificities of T1Rs but a large variation in the number and type of bitter compounds detected by different species. Although the rate of gene duplication is much lower in T1Rs than in T2Rs, signals of positive selection are detected during the functional divergences of paralogous T1Rs, as was previously found among paralogous T2Rs. Thus, functional divergence and specialization of taste receptors generally occurred via adaptive evolution.     

Expression of gustducin overlaps with that of type III IP3 receptor in taste buds of the rat soft palate

Type III IP3 receptor (IP3R3) is one of the common critical calcium-signaling molecules for sweet, umami, and bitter signal transduction in taste cells, and the total IP3R3-expressing cell population represents all cells mediating these taste modalities in the taste buds. Although gustducin, a taste cell-specific G-protein, is also involved in sweet, umami, and bitter signal transduction, the expression of gustducin is restricted to different subsets of IP3R3-expressing cells by location in the tongue. Based on the expression patterns of gustducin and taste receptors in the tongue, the function of gustducin has been implicated primarily in bitter taste in the circumvallate (CV) papillae and in sweet taste in the fungiform (FF) papillae. However, in the soft palate (SP), the expression pattern of gustducin remains unclear and little is known about its function. In the present paper, the expression patterns of gustducin and IP3R3 in taste buds of the SP and tongue papillae in the rat were examined by double-color whole-mount immunohistochemistry. Gustducin was expressed in almost all (96.7%) IP3R3-expressing cells in taste buds of the SP, whereas gustducin-positive cells were 42.4% and 60.1% of IP3R3-expressing cells in FF and CV, respectively. Our data suggest that gustducin is involved in signal transduction of all the tastes of sweet, umami, and bitter in the SP, in contrast to its limited function in the tongue.