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.     

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 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.     

Artificial Sweeteners Stimulate Adipogenesis and Suppress Lipolysis Independently of Sweet Taste Receptors

G protein-coupled receptors mediate responses to a myriad of ligands, some of which regulate adipocyte differentiation and metabolism. The sweet taste receptors T1R2 and T1R3 are G protein-coupled receptors that function as carbohydrate sensors in taste buds, gut, and pancreas. Here we report that sweet taste receptors T1R2 and T1R3 are expressed throughout adipogenesis and in adipose tissues. Treatment of mouse and human precursor cells with artificial sweeteners, saccharin and acesulfame potassium, enhanced adipogenesis. Saccharin treatment of 3T3-L1 cells and primary mesenchymal stem cells rapidly stimulated phosphorylation of Akt and downstream targets with functions in adipogenesis such as cAMP-response element-binding protein and FOXO1; however, increased expression of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α was not observed until relatively late in differentiation. Saccharin-stimulated Akt phosphorylation at Thr-308 occurred within 5 min, was phosphatidylinositol 3-kinase-dependent, and occurred in the presence of high concentrations of insulin and dexamethasone; phosphorylation of Ser-473 occurred more gradually. Surprisingly, neither saccharin-stimulated adipogenesis nor Thr-308 phosphorylation was dependent on expression of T1R2 and/or T1R3, although Ser-473 phosphorylation was impaired in T1R2/T1R3 double knock-out precursors. In mature adipocytes, artificial sweetener treatment suppressed lipolysis even in the presence of forskolin, and lipolytic responses were correlated with phosphorylation of hormone-sensitive lipase. Suppression of lipolysis by saccharin in adipocytes was also independent of T1R2 and T1R3. These results suggest that some artificial sweeteners have previously uncharacterized metabolic effects on adipocyte differentiation and metabolism and that effects of artificial sweeteners on adipose tissue biology may be largely independent of the classical sweet taste receptors, T1R2 and T1R3.     

Allelic variation of the Tas1r3 taste receptor gene selectively affects taste responses to sweeteners: evidence from 129.B6-Tas1r3 congenic mice.

The Tas1r3 gene encodes the T1R3 receptor protein, which is involved in sweet taste transduction. To characterize ligand specificity of the T1R3 receptor and the genetic architecture of sweet taste responsiveness, we analyzed taste responses of 129.B6-Tas1r3 congenic mice to a variety of chemically diverse sweeteners and glucose polymers with three different measures: consumption in 48-h two-bottle preference tests, initial licking responses, and responses of the chorda tympani nerve. The results were generally consistent across the three measures. Allelic variation of the Tas1r3 gene influenced taste responsiveness to nonnutritive sweeteners (saccharin, acesulfame-K, sucralose, SC-45647), sugars (sucrose, maltose, glucose, fructose), sugar alcohols (erythritol, sorbitol), and some amino acids (D-tryptophan, D-phenylalanine, L-proline). Tas1r3 genotype did not affect taste responses to several sweet-tasting amino acids (L-glutamine, L-threonine, L-alanine, glycine), glucose polymers (Polycose, maltooligosaccharide), and nonsweet NaCl, HCl, quinine, monosodium glutamate, and inosine 5'-monophosphate. Thus Tas1r3 polymorphisms affect taste responses to many nutritive and nonnutritive sweeteners (all of which must interact with a taste receptor involving T1R3), but not to all carbohydrates and amino acids. In addition, we found that the genetic architecture of sweet taste responsiveness changes depending on the measure of taste response and the intensity of the sweet taste stimulus. Variation in the T1R3 receptor influenced peripheral taste responsiveness over a wide range of sweetener concentrations, but behavioral responses to higher concentrations of some sweeteners increasingly depended on mechanisms that could override input from the peripheral taste system.     

Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli

Animals utilize hundreds of distinct G protein-coupled receptor (GPCR)-type chemosensory receptors to detect a diverse array of chemical signals in their environment, including odors, pheromones, and tastants. However, the molecular mechanisms by which these receptors selectively interact with their cognate ligands remain poorly understood. There is growing evidence that many chemosensory receptors exist in multimeric complexes, though little is known about the relative contributions of individual subunits to receptor functions. Here, we report that each of the two subunits in the heteromeric T1R2:T1R3 sweet taste receptor binds sweet stimuli though with distinct affinities and conformational changes. Furthermore, ligand affinities for T1R3 are drastically reduced by the introduction of a single amino acid change associated with decreased sweet taste sensitivity in behaving mice. Thus, individual T1R subunits increase the receptive range of the sweet taste receptor, offering a functional mechanism for phenotypic variations in sweet taste.     

The history of sweet taste: not exactly a piece of cake

Understanding the molecular bases of sweet taste is of crucial importance not only in biotechnology but also for its medical implications, since an increasing number of people is affected by food-related diseases like, diabetes, hyperlipemia, caries, that are more or less directly linked to the secondary effects of sugar intake. Despite the interest paid to the field, it is only through the recent identification and functional expression of the receptor for sweet taste that new perspectives have been opened, drastically changing our approach to the development of new sweeteners. We shall give an overview of the field starting from the early days up to discussing the newest developments. After a review of early models of the active site, the mechanisms of interaction of small and macromolecular sweet molecules will be examined in the light of accurate modeling of the sweet taste receptor. The analysis of the homology models of all possible dimers allowed by combinations of the human T1R2 and T1R3 sequences of the sweet receptor and the closed (A) and open (B) conformations of the mGluR1 glutamate receptor shows that only 'type B' sites, either T1R2(B) and T1R3(B), can host the majority of small molecular weight sweeteners. Simultaneous binding to the A and B sites is not possible with two large sweeteners but is possible with a small molecule in site A and a large one in site B. This observation accounted for the first time for the peculiar phenomenon of synergy between some sweeteners. In addition to these two sites, the models showed an external binding site that can host sweet proteins.     

Identification of a novel member of the T1R family of putative taste receptors

In the gustatory system, the recognition of sugars, amino acids and bitter-tasting compounds is the function of specialized G protein-coupled receptors. Recently, two members of novel subfamily of G protein-coupled receptors were proposed to function as taste receptors based on their specific expression in taste receptor cells. Here, we report the identification of a third member, T1R3, of this family of receptors. T1R3 maps near the telomere of mouse chromosome 4 rendering it a candidate for the Sac locus, a primary determinant of sweet preference in mice. Consistent with its candidacy for the Sac locus, T1R3 displays taste receptor cell-specific expression. In addition, taster and non-taster strains of mouse harbor different alleles of T1R3.     

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.     

Lactisole interacts with the transmembrane domains of human T1R3 to inhibit sweet taste

The detection of sweet-tasting compounds is mediated in large part by a heterodimeric receptor comprised of T1R2+T1R3. Lactisole, a broad-acting sweet antagonist, suppresses the sweet taste of sugars, protein sweeteners, and artificial sweeteners. Lactisole's inhibitory effect is specific to humans and other primates; lactisole does not affect responses to sweet compounds in rodents. By heterologously expressing interspecies combinations of T1R2+T1R3, we have determined that the target for lactisole's action is human T1R3. From studies with mouse/human chimeras of T1R3, we determined that the molecular basis for sensitivity to lactisole depends on only a few residues within the transmembrane region of human T1R3. Alanine substitution of residues in the transmembrane region of human T1R3 revealed 4 key residues required for sensitivity to lactisole. In our model of T1R3's seven transmembrane helices, lactisole is predicted to dock to a binding pocket within the transmembrane region that includes these 4 key residues.     

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.     

Identification of novel sweet protein for nutritional applications

The prevalence of obesity and diabetes has increased exponentially in recent years around the globe, especially in India. Sweet proteins have the potential to substitute the sugars, by acting as natural, good and low calorie sweeteners. They also do not trigger a demand for insulin in diabetic patients unlike sucrose. In humans, the sweet taste perception is mainly due to taste-specific G protein-coupled heterodimeric receptors T1R2-T1R3. These receptors recognize diverse natural and synthetic sweeteners such as monelin, brazzein, thaumatin, curculin, mabinlin, miraculin and pentadin. Structural modeling of new sweetener proteins will be a great leap in further advancement of knowledge and their utility as sweeteners. We have explored the fingerprints of sweetness by studying the aminoacid composition and structure properties of the above proteins. The structural analysis of monellin revealed that the individual A or B chains of monellin are not contributing to its sweetness. However, the native conformation and ionic interaction between AspB7 of monellin with active site of T1R2-T1R3 receptor, along with hydrogen bonding stability of IleB6 and IleB8 are responsible for the sweet taste. Based on structural similarity search, we found a new hypothetical protein from Shewanella loihica, which has the presence of Asp(32) with adjacent isoleucine residues. Further, we examined the lead protein by two-step docking for the study of interaction of functionally conserved residues with receptors. The identified protein showed similar ionic and hydrophobic interactions with monelin. This gives a promising opportunity to explore this protein for potential health application in the low calorie sweetener industry viz., soft drinks, snacks, food, chocolate industries etc.     

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.     

Interaction of sweet proteins with their receptor. A conformational study of peptides corresponding to loops of brazzein, monellin and thaumatin.

The mechanism of interaction of sweet proteins with the T1R2-T1R3 sweet taste receptor has not yet been elucidated. Low molecular mass sweeteners and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor. The presence on the surface of the proteins of "sweet fingers", i.e. protruding features with chemical groups similar to those of low molecular mass sweeteners that can probe the active site of the receptor, would be consistent with a single mechanism for the two classes of compounds. We have synthesized three cyclic peptides corresponding to the best potential "sweet fingers" of brazzein, monellin and thaumatin, the sweet proteins whose structures are well characterized. NMR data show that all three peptides have a clear tendency, in aqueous solution, to assume hairpin conformations consistent with the conformation of the same sequences in the parent proteins. The peptide corresponding to the only possible loop of brazzein, c[CFYDEKRNLQC(37-47)], exists in solution in a well ordered hairpin conformation very similar to that of the same sequence in the parent protein. However, none of the peptides has a sweet taste. This finding strongly suggests that sweet proteins recognize a binding site different from the one that binds small molecular mass sweeteners. The data of the present work support an alternative mechanism of interaction, the "wedge model", recently proposed for sweet proteins [Temussi, P. A. (2002) FEBS Lett.526, 1-3.].     

Positional cloning of the mouse saccharin preference (Sac) locus

Differences in sweetener intake among inbred strains of mice are partially determined by allelic variation of the saccharin preference (Sac) locus. Genetic and physical mapping limited a critical genomic interval containing Sac to a 194 kb DNA fragment. Sequencing and annotation of this region identified a gene (Tas1r3) encoding the third member of the T1R family of putative taste receptors, T1R3. Introgression by serial backcrossing of the 194 kb chromosomal fragment containing the Tas1r3 allele from the high-sweetener-preferring C57BL/6ByJ strain onto the genetic background of the low-sweetener-preferring 129P3/J strain rescued its low-sweetener-preference phenotype. Polymorphisms of Tas1r3 that are likely to have functional significance were identified using analysis of genomic sequences and sweetener-preference phenotypes of genealogically distant mouse strains. Tas1r3 has two common haplotypes, consisting of six single nucleotide polymorphisms: one haplotype was found in mouse strains with elevated sweetener preference and the other in strains relatively indifferent to sweeteners. This study provides compelling evidence that Tas1r3 is equivalent to the Sac locus and that the T1R3 receptor responds to sweeteners.     

The mechanism of interaction of sweet proteins with the T1R2-T1R3 receptor: evidence from the solution structure of G16A-MNEI

The mechanism by which sweet proteins elicit a response on the T1R2-T1R3 sweet taste receptor is still mostly unknown but has been so far related to the presence of "sweet fingers" on the protein surface able to interact with the same mechanism as that of low molecular mass sweeteners. In the search for the identification of sweet fingers, we have solved the solution structure of G16A MNEI, a structural mutant that shows a reduction of one order of magnitude in sweetness with respect to its parent protein, MNEI, a single-chain monellin. Comparison of the structures of wild-type monellin and its G16A mutant shows that the mutation does not affect the structure of potential glucophores but produces a distortion of the surface owing to the partial relative displacement of elements of secondary structure. These results show conclusively that sweet proteins do not possess a sweet finger and strongly support the hypothesis that the mechanism of interaction of sweet-tasting proteins with the recently identified T1R2-T1R3 GPC receptor is different from that of low molecular mass sweeteners.     

The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, thaumatin

Thaumatin is an intensely sweet-tasting protein perceived by humans but not rodents. Its threshold value of sweetness in humans is 50 nM, the lowest of any sweet-tasting protein. In the present study, the sites where sweet receptors interact with thaumatin were investigated using human embryonic kidney 293 (HEK293) cells expressing the sweet receptors T1R2–T1R3. Chimeric human– mouse sweet receptors were constructed and their responses to sweeteners were investigated. The human (h) T1R2– mouse (m) T1R3 combination responded to sucralose but not to thaumatin, clearly indicating that a T1R3 subunit from humans is necessary for the interaction with thaumatin. Furthermore, results obtained from using chimeric T1R3s showed that the cysteine-rich domain (CRD) of human T1R3 is important for the interaction with thaumatin. The CRD of T1R3 would be a prominent target for designing new sweeteners.     

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.     

Loss of the nutrient receptor Tas1R3 reduces atherosclerotic plaque accumulation and hepatic steatosis in ApoE−/− mice

Journal of Physiology and Biochemistry - The taste receptor type I (Tas1R) family consists of three G protein-coupled receptors (T1R1, T1R2, and T1R3) that form heterodimers recognizing sweet...     

Sweet-taste-suppressing compounds: current knowledge and perspectives of application

Sweet-tasting compounds are recognized by a heterodimeric receptor composed of the taste receptor, type 1, members 2 (T1R2) and 3 (T1R3) located in the mouth. This receptor is also expressed in the gut where it is involved in intestinal absorption, metabolic regulation, and glucose homeostasis. These metabolic functions make the sweet taste receptor a potential novel therapeutic target for the treatment of obesity and related metabolic dysfunctions such as diabetes. Existing sweet taste inhibitors or blockers that are still in development would constitute promising therapeutic agents. In this review, we will summarize the current knowledge of sweet taste inhibitors, including a sweet-taste-suppressing protein named gurmarin, which is only active on rodent sweet taste receptors but not on that of humans. In addition, their potential applications as therapeutic tools are discussed.