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...
This paper presents a simple and efficient method for oligonucleotide-directed mutagenesis using vectors derived from single-stranded phage. This modification of our previously published procedure (Zoller and Smith, 1982) features the use of two primers, one of which is a standard M13 sequencing primer and the other is the mutagenic oligonucleotide. Both primers are simultaneously annealed to single-stranded template DNA, extended by DNA polymerase I (large fragment), and ligated together to form a mutant wild-type gapped heteroduplex. Escherichia coli is transformed directly with this DNA; the isolation of covalently closed circular DNA as in our previous report is not necessary. Mutants are identified by plaque lift hybridization using the mutagenic oligonucleotide as a probe. As an example of the method, a heptadecanucleotide was used to create a T----G transversion in the MATa gene of Saccharomyces cerevisiae cloned into the vector M13mp5. The efficiency of mutagenesis was approximately 50%. Production of the desired mutation was verified by DNA sequencing. The same procedure has been used without modification to create insertions of restriction sites as well as specific deletions of 500 bases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.