Human receptors for sweet and umami taste

Abstract

The three members of the T1R class of taste-specific G protein-coupled receptors have been hypothesized to function in combination as heterodimeric sweet taste receptors. Here we show that human T1R2/T1R3 recognizes diverse natural and synthetic sweeteners. In contrast, human T1R1/T1R3 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 T1R2/T1R3 and T1R1/T1R3 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.

Figure 1

Sequence alignment of human and rat T1Rs. T1R sequences determined in this study, human T1R1 (GenBank accession no. BK000153), T1R2 (accession no. BK000151), T1R3 (accession no. BK000152), and rat T1R3 (accession no. AF456324), are aligned here with previously described rat T1Rs (accession nos. AAD18069 and AAD18070) and the rat mGluR1 metabotropic glutamate receptor (accession no. P23385). The mGluR1 C terminus is not shown. Potential transmembrane segments are boxed in blue. mGluR1 ligand-binding residues are highlighted following the color scheme used in Fig. 3A.

Figure 2

Human and rat T1R2/T1R3 recognize sweet taste stimuli. (A) G_α15 cells transiently transfected with human T1R2 and T1R3 and HEK-293T cells transiently transfected with rat T1R2, T1R3, and G_α15/i1 were assayed for intracellular calcium increases in response to 300 mM sucrose in the presence (+) and absence (−) of 1.25 mM lactisole and to 10 nM isoproterenol in the presence and absence of 1.25 mM lactisole. Each imaged field shown contains ≈1,000 confluent cells. (B) The responses of human T1R2/T1R3 and rat T1R2/T1R3 were determined for sweet taste stimuli at the concentrations listed in Materials and Methods. (C) Human T1R2/T1R3 dose responses were determined for sucrose, d-tryptophan, aspartame, and saccharin. Dose responses were normalized to the maximal percentage of responding cells, which ranged from 10 to 40% for different sweeteners. Values represent the mean ± SE of four independent responses. The x axis circles represent average psychophysical detection threshold values for these four sweeteners. (D) The dose responses of G_α15 cells stably transfected with human T1R2 and T1R3 were determined for sucrose, d-tryptophan, aspartame, and saccharin. Responses are shown as the percentage of fluorescence values relative to fluorescence increases elicited by 1 μM ionomycin. Values represent the mean ± SE of four independent responses. (E) HEK-293T cells were transiently transfected with rat T1R2, rat T1R3, and each G_α15 chimera, and assayed for intracellular calcium increases in response to 75 mM sucrose. The five C-terminal residues of each G_α15 chimera are shown. The activities in B and E represent the mean ± SE number of responding cells for four imaged fields of ≈1,000 confluent cells.

Figure 3

Human and rat T1R1/T1R3 recognize umami taste stimuli. (A) mGluR1 residues (PDB entry no. 1EWK) that contact the l-glutamate side chain carboxylate are shown in red, and residues that contact the l-glutamate α-amino acid moiety are shown in green. (B) G_α15 cells transiently transfected with human T1R1 and T1R3 were assayed for intracellular calcium increases in response to increasing concentrations of l-glutamate in the presence or absence of 1 mM IMP. Each imaged field shown contains ≈1,000 confluent cells. (C) Human T1R1/T1R3 dose responses were determined for l-glutamate in the presence and absence of 0.2 mM IMP. Dose responses were normalized to the maximal percentage of responding cells, which was ≈5% for l-glutamate and ≈10% for l-glutamate plus IMP. Values represent the mean ± SE of four independent responses. The x axis circles represent average psychophysical detection threshold values for l-glutamate in the presence and absence of 0.2 mM IMP. (D) Human T1R1/T1R3 responses to 25 mM d-glutamate, 25 mM l-glutamate, 25 mM l-aspartate, 25 mM l-AP4, and binary mixtures of these compounds with 2.5 mM IMP, 2.5 mM GMP, or 2.5 mM CMP were determined. (E) Human T1R1/T1R3 dose responses were determined for IMP in the presence of 0.2 and 2 mM l-glutamate and normalized to the maximal percentages of responding cells, which were ≈10%. Values represent the mean ± SE of four independent responses. (F) Human T1R2/T1R3 responses to 25 mM l-glutamate, 100 mM sucrose (SUC), 2.5 mM d-tryptophan, 1.5 mM aspartame (ASP), and 0.4 mM saccharin (SAC) were determined in the presence and absence of 2.5 mM IMP. (G) Human T1R1/T1R3 dose responses were determined for l-aspartate and l-AP4 in the presence of 0.2 mM IMP. Dose responses were normalized to the maximal percentage of responding cells, which was ≈5% for l-aspartate plus IMP and ≈10% for l-AP4 plus IMP. Values represent the mean ± SE of four independent responses. The x axis circles represent average psychophysical detection threshold values for l-aspartate plus 0.2 mM IMP and l-AP4 plus 0.2 mM IMP. (H) Human T1R1/T1R3 responses to sucrose (100 mM), d-tryptophan (20 mM), aspartame (2 mM), saccharin (1 mM), l-tyrosine (5 mM), and d-glutamate, l-glutamine, d-aspartate, glycine, l-histidine, l-leucine, l-lysine, l-proline, and l-serine (each at 10 mM) were determined in the presence and absence of 1 mM IMP. (I) HEK-293T cells were transiently transfected with rat T1R1, rat T1R3, and G_α15/i1 and assayed for increases in intracellular calcium in response to 25 mM d-glutamate, 25 mM l-glutamate, 25 mM l-aspartate, 25 mM l-AP4, and binary mixtures with 2.5 mM IMP, 2.5 mM GMP, or 2.5 mM CMP. (J) Rat T1R1/T1R3 dose responses were determined for l-aspartate and l-glutamate in the presence of 0.2 mM IMP. Dose responses were normalized to the maximal percentage of responding cells, which was ≈10% for l-aspartate plus IMP and ≈15% for l-glutamate plus IMP. Values represent the mean ± SE of four independent responses. The activities in D, F, H, and I represent the mean ± SE number of responding cells for four imaged fields of ≈1,000 confluent cells.