Umami is one of the 5 basic taste qualities. The umami taste of L-glutamate can be drastically enhanced by 5 ribonucleotides and the synergy is a hallmark of this taste quality. The umami taste receptor is a heteromeric complex of 2 class C G-protein-coupled receptors, T1R1 and T1R3. Here we elucidate the molecular mechanism of the synergy using chimeric T1R receptors, site-directed mutagenesis, and molecular modeling. We propose a cooperative ligand-binding model involving the Venus flytrap domain of T1R1, where L-glutamate binds close to the hinge region, and 5 ribonucleotides bind to an adjacent site close to the opening of the flytrap to further stabilize the closed conformation. This unique mechanism may apply to other class C G-protein-coupled receptors.glutamate ͉ G protein-coupled receptors ͉ IMP ͉ T1R H umans can detect at least 5 basic taste qualities, including sweet, umami, bitter, salty, and sour. Umami, the savory taste of L-glutamate, was first discovered in 1908 by K. Ikeda, but only recently accepted as a basic taste quality by the general public. The most unique characteristic of umami taste is synergism. Purinic ribonucleotides, such as IMP and GMP, can strongly potentiate the umami taste intensity (1). In human taste tests, 200 M of IMP, which does not elicit any umami taste by itself, can increase one's umami taste sensitivity to glutamate by 15-fold (2).Among the 5 basic taste qualities, sweet, umami, and bitter taste are mediated by G protein-coupled receptors (GPCRs) (3). Receptors for umami taste and sweet taste are closely related to each other. The 3 subunits of the T1R family form 2 heteromeric receptors: umami (T1R1/T1R3) (2, 4) and sweet (T1R2/T1R3) (2, 5). T1R receptors belong to the class C GPCRs, along with metabotropic glutamate receptors (mGluRs), ␥-aminobutyric acid receptor B (GABA B R), calcium sensing receptors (CaSR), and so forth. The defining motif in these receptors is an outer membrane N-terminal Venus flytrap (VFT) domain that consists of 2 globular subdomains, the N-terminal upper lobe and the lower lobe, that are connected by a 3-stranded flexible hinge. The VFT domain of C-GPCRs contains the ligand-binding site (6), as demonstrated by studies on mGluRs, GABA B R, and the sweet taste receptor (7). The crystal structures of mGluR VFT domains revealed that the bi-lobed architecture can form an open or closed conformation (8, 9). Glutamate binding stabilizes both the active dimer and the closed conformation. This scheme in the initial receptor activation has been applied generally to other C-GPCRs.Studies on sweet taste-receptor functional domains revealed multiple binding sites for its structurally diverse ligands. Using human-rat chimeric receptors, we demonstrated the T1R2 VFT domain of the human sweet receptor interacts with 2 structurally related synthetic sweeteners aspartame and neotame, while the transmembrane domain (TMD) of human T1R3 interacts with another sweetener cyclamate and a human sweet-taste inhibitor lactisole (7). Works from several other laborator...

Positive allosteric modulators of the human sweet taste receptor have been developed as a new way of reducing dietary sugar intake. Besides their potential health benefit, the sweet taste enhancers are also valuable tool molecules to study the general mechanism of positive allosteric modulations of T1R taste receptors. Using chimeric receptors, mutagenesis, and molecular modeling, we reveal how these sweet enhancers work at the molecular level. Our data argue that the sweet enhancers follow a similar mechanism as the natural umami taste enhancer molecules. Whereas the sweeteners bind to the hinge region and induce the closure of the Venus flytrap domain of T1R2, the enhancers bind close to the opening and further stabilize the closed and active conformation of the receptor.positive allosteric modulators | sweet taste receptor | T1R H umans can detect at least five basic taste qualities, including sweet, umami, bitter, salty, and sour. The sweet and umami taste are mediated by closely related G protein-coupled receptors (GPCRs). The three members of the T1R family form two heteromeric taste receptors: umami (T1R1/T1R3) (1, 2) and sweet (T1R2/T1R3) (1, 3). T1R receptors belong to the class C GPCRs, along with metabotropic glutamate receptors (mGluRs), γ-aminobutyric acid receptor B (GABA B R), calcium sensing receptors (CaSR), and others. The defining motif in these receptors is an extracellular Venus flytrap (VFT) domain (4), which consists of two globular subdomains connected by a threestranded flexible hinge. The VFT domain contains the orthosteric ligand binding site. The crystal structures of mGluR VFT domains (5, 6) revealed that the bilobed architecture can form an "open" or "closed" conformation. Glutamate binding stabilizes both the "closed" and the "active" dimer conformation. This scheme in the initial receptor activation has been applied generally to other class C GPCRs.Over the years, researchers have been developing noncaloric sweeteners to reduce dietary sugar intake. Unfortunately, all existing noncaloric sweeteners are characterized by their off taste (7,8) and fail to mimic the real sugar taste. Since the identification of the sweet taste receptor, a new approach became available, which is to develop positive allosteric modulators (PAMs) of the receptor. These molecules work as sweet taste "enhancers," which possess no taste of their own but potentiate the sweet taste of sugars. Examples of taste enhancers can be found in umami taste, which is known for its unique characteristic of synergism (9). Purinic ribonucleotides such as inosine-5′-monophosphate (IMP) and guanosine-5′-monophosphate (GMP) can strongly potentiate the umami taste intensity of glutamate and are rare examples of naturally occurring GPCR PAMs. In taste tests, 200 μM IMP, which does not elicit any umami taste by itself, can increase human umami taste sensitivity to glutamate by 15-fold (1). We recently illustrated the molecular mechanism of IMP/GMP (10). Our data indicate that glutamate binds close to the hinge region of the VFT ...

To identify molecules that could enhance sweetness perception, we undertook the screening of a compound library using a cellbased assay for the human sweet taste receptor and a panel of selected sweeteners. In one of these screens we found a hit, SE-1, which significantly enhanced the activity of sucralose in the assay. At 50 μM, SE-1 increased the sucralose potency by >20-fold. On the other hand, SE-1 exhibited little or no agonist activity on its own. SE-1 effects were strikingly selective for sucralose. Other popular sweeteners such as aspartame, cyclamate, and saccharin were not enhanced by SE-1 whereas sucrose and neotame potency were increased only by 1.3-to 2.5-fold at 50 μM. Further assayguided chemical optimization of the initial hit SE-1 led to the discovery of SE-2 and SE-3, selective enhancers of sucralose and sucrose, respectively. SE-2 (50 μM) and SE-3 (200 μM) increased sucralose and sucrose potencies in the assay by 24-and 4.7-fold, respectively. In human taste tests, 100 μM of SE-1 and SE-2 allowed for a reduction of 50% to >80% in the concentration of sucralose, respectively, while maintaining the sweetness intensity, and 100 μM SE-3 allowed for a reduction of 33% in the concentration of sucrose while maintaining the sweetness intensity. These enhancers did not exhibit any sweetness when tasted on their own. Positive allosteric modulators of the human sweet taste receptor could help reduce the caloric content in food and beverages while maintaining the desired taste.enhancer | sweetness | perception | sucrose T he steady increase of the daily consumption of dietary sugar over the last decades may have contributed to the obesity crisis and the early onset of type-II diabetes observed in many developed countries (1, 2). As a result, food and beverage companies have launched a plethora of diet brands where sugar has been partly or fully replaced by noncaloric sweeteners to decrease caloric intake. Currently some of the commonly used noncaloric sweeteners include saccharin, aspartame, cyclamate, sucralose, and acesulfame K (3, 4). However, none of these substances can completely reproduce the taste of sugar. These sweeteners all suffer from one or more shortcomings including a bitter or metallic aftertaste at high concentrations, limiting their use to lower concentrations, or temporal issues such as a delayed sweet taste onset, a lingering sweet aftertaste, or a limited maximum sweetness intensity (4, 5). Another appealing approach to address the problem, in addition to looking for novel noncaloric sweeteners, would be to find molecules capable of enhancing sweetness perception. Ideally, such an enhancer molecule would not elicit sweetness on its own but it would boost the sweetness intensity of a lower amount of sweetener or sugar. Such enhancers could therefore allow for a reduction in the amount of sugar, and calories, in food and beverages while maintaining the desired taste. Similarly, a sweet taste enhancer could allow for a reduction in the amount of noncaloric sweeteners used in "0"-calorie...

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