The three members of the T1R class of taste-specific G proteincoupled receptors have been hypothesized to function in combination as heterodimeric sweet taste receptors. Here we show that human T1R2͞T1R3 recognizes diverse natural and synthetic sweeteners. In contrast, human T1R1͞T1R3 responds to the umami taste stimulus L-glutamate, and this response is enhanced by 5-ribonucleotides, a hallmark of umami taste. The ligand specificities of rat T1R2͞T1R3 and T1R1͞T1R3 correspond to those of their human counterparts. These findings implicate the T1Rs in umami taste and suggest that sweet and umami taste receptors share a common subunit. Large-scale sequencing of a subtracted cDNA library derived from rat taste tissue identified a new C-family G proteincoupled receptor, T1R1, that is expressed selectively in taste receptor cells; T1R1-based degenerate PCR led to the identification of a related taste-specific receptor, T1R2 (1). Recently, a third and possibly final member of the T1R family, T1R3, was identified in the human DNA databank (2-7). Tellingly, mouse T1R3 maps to a genomic interval containing Sac, a locus that influences sweet taste in the mouse (8, 9). Recent highresolution genetic mapping and complementation studies have strengthened the connection between mouse T1R3 and Sac (2-7). Although T1R1 and T1R2 appear to be expressed in predominantly nonoverlapping regions of the tongue, they each are coexpressed with T1R3 (1, 3, 4, 6). These overlapping expression patterns and precedent from the structurally related heterodimeric ␥-aminobutyric acid type B receptor (10-13) suggested that T1R1 and T1R2 may combine with T1R3 to form heterodimeric sweet taste receptors. Indeed, rat T1R2 has been shown recently to function in combination with T1R3 to recognize a subset of sweet taste stimuli, a finding that has been proposed to reflect the involvement of additional combinations of T1Rs in sweet taste (6). In this study we cloned and functionally expressed human and rat T1Rs. Human and rat T1R2͞ T1R3 recognized all sweet taste stimuli tested, and human and rat T1R1͞T1R3 recognized umami taste stimuli. These findings suggest that different combinations of T1Rs function as heterodimeric sweet and umami taste receptors.Material and Methods T1R Cloning. Intronless human T1R expression constructs were generated in a pEAK10-derived vector (Edge Biosystems, Gaithersburg, MD) by a combination of cDNA-based and genomic DNA-based methods. To generate the full-length T1R1 expression construct, two 5Ј coding exons identified in a cloned T1R1 interval (GenBank accession no. AL159177) were combined by PCR overlap and then joined to a 5Ј-truncated testis cDNA clone. The T1R2 expression construct was generated from a partially sequenced T1R2 genomic interval. Two missing T1R2 5Ј introns were identified by screening shotgun libraries of the cloned genomic interval using probes derived from the corresponding rat coding sequence. Coding exons then were combined by PCR overlap to produce the full-length expression construct. The T1R3 e...

The T1R receptors, a family of taste-specific class C G proteincoupled receptors, mediate mammalian sweet and umami tastes. The structure-function relationships of T1R receptors remain largely unknown. In this study, we demonstrate the different functional roles of T1R extracellular and transmembrane domains in ligand recognition and G protein coupling. Similar to other family C G protein-coupled receptors, the N-terminal Venus flytrap domain of T1R2 is required for recognizing sweeteners, such as aspartame and neotame. The G protein coupling requires the transmembrane domain of T1R2. Surprisingly, the C-terminal transmembrane domain of T1R3 is required for recognizing sweetener cyclamate and sweet taste inhibitor lactisole. Because T1R3 is the common subunit in the sweet taste receptor and the umami taste receptor, we tested the interaction of lactisole and cyclamate with the umami taste receptor. A family of class C G protein-coupled receptors (GPCRs), T1Rs, is selectively expressed in the taste buds (1-6). Functional expression of T1Rs in human embryonic kidney (HEK)-293 cells revealed that different combinations of T1Rs respond to sweet and umami taste stimuli (6, 7). T1R2 and T1R3, when coexpressed in HEK-293 cells, recognize diverse natural and synthetic sweeteners. Similarly, T1R1 and T1R3, when coexpressed in HEK-293 cells, respond to the umami taste stimulus L-glutamate. This response is enhanced by 5Ј ribonucleotides, a hallmark of umami taste. Recent experiments with knockout mice confirmed that T1Rs indeed mediate mouse sweet and umami tastes (8, 9).The class C GPCRs possess a large N-terminal extracellular domain, often referred to as the Venus flytrap domain (10), and are known to function as either homodimers, in the cases of metabotropic glutamate receptors (mGluRs) and calciumsensing receptor, or heterodimers, in the case of ␥-aminobutyric acid type B receptor (GABA B R) (10). The functional expression data suggest a heterodimer mechanism for T1Rs: both T1R1 and T1R2 need to be coexpressed with T1R3 to be functional, which is supported by the overlapping expression patterns of T1Rs in rodent tongue. Nonetheless, there has been no direct evidence that T1Rs function as heteromeric complexes. It is possible that T1R3 is not a functional component of sweet and umami taste receptors, but merely a chaperone protein, which facilitates the proper folding or intracellular translocation of T1R1 and T1R2. The distinct ligand specificities of T1R1͞T1R3 and T1R2͞T1R3 receptors suggest that T1R1 and T1R2 play more important roles in ligand binding in sweet and umami taste receptors than T1R3. Support for this hypothesis was provided recently by results from mouse genetics where human T1R2 transgenic mice, generated on the T1R2 knockout background, displayed sweetener taste preferences similar to those of humans (9). However, the functional role of T1R3 and the overall structure͞function relationship of T1R taste receptors remain largely unknown.Another intriguing observation about the T1R2͞T1R3 receptor is th...

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...

TAS1R- and TAS2R-type taste receptors are expressed in the gustatory system, where they detect sweet- and bitter-tasting stimuli, respectively. These receptors are also expressed in subsets of cells within the mammalian gastrointestinal tract, where they mediate nutrient assimilation and endocrine responses. For example, sweeteners stimulate taste receptors on the surface of gut enteroendocrine L cells to elicit an increase in intracellular Ca2+ and secretion of the incretin hormone glucagon-like peptide-1 (GLP-1), an important modulator of insulin biosynthesis and secretion. Because of the importance of taste receptors in the regulation of food intake and the alimentary responses to chemostimuli, we hypothesized that differences in taste receptor efficacy may impact glucose homeostasis. To address this issue, we initiated a candidate gene study within the Amish Family Diabetes Study and assessed the association of taste receptor variants with indicators of glucose dysregulation, including a diagnosis of type 2 diabetes mellitus and high levels of blood glucose and insulin during an oral glucose tolerance test. We report that a TAS2R haplotype is associated with altered glucose and insulin homeostasis. We also found that one SNP within this haplotype disrupts normal responses of a single receptor, TAS2R9, to its cognate ligands ofloxacin, procainamide and pirenzapine. Together, these findings suggest that a functionally compromised TAS2R receptor negatively impacts glucose homeostasis, providing an important link between alimentary chemosensation and metabolic disease.

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 ...