Sweeteners map to different domains/subunits of the human sweet taste receptor. (A) Responses of human and rat sweet taste receptors to sucrose (200 mM), aspartame (10 mM), neotame (0.1 mM), cyclamate (10 mM), and sucrose (200 mM) in the presence of lactisole (1 mM) (Suc/Lac). HEK-293T cells were transiently transfected with human or rat T1R2, T1R3, and a Gα₁₅ chimera, Gα_15/i1 (7), and assayed for intracellular calcium increases in response to sweeteners. (B) Aspartame and neotame were mapped to the N-terminal extracellular domain of human T1R2. Combinations of T1R chimeras were transiently transfected into HEK-293T cells with Gα_15/i1 and assayed for responses to sweeteners at the concentrations listed in A. The T1R2H-R/rat T1R3 combination generated a significantly weaker response to the control sweetener sucrose than did the WT receptors, possibly because of less than perfect folding of the artificial receptor subunit. Nonetheless, the same receptor responds well to aspartame and neotame. Because of the potential differences in folding, surface targeting, and coupling efficiency, we avoided comparing the relative activities of different combinations. Instead, we looked for the presence or absence of response to different sweeteners within each combination. (C) Cyclamate was mapped to the C-terminal transmembrane domain of human T1R3. (D) Lactisole was mapped to the transmembrane domain of human T1R3. Different combinations of T1R chimeras were transiently transfected into HEK-293T cells with Gα_15/i1 and assayed for responses to sucrose (200 mM) and AceK (10 mM) in the absence or presence of lactisole (1 mM). The activities in B–D represent the mean ± SE of the number of responding cells for four imaged fields of ≈1,000 confluent cells. H, Human; R, rat.

Mutations in T1R2 or T1R3 selectively affect the activity of different sweeteners. (A) Sequence alignment of the N-terminal ligand binding domain of rat mGluR5 with human and rodent T1R2s. The eight critical amino acids involved in ligand binding in mGluR5 are labeled with *; three of the eight amino acids are conserved in T1R2 and labeled with green *. (B) Two point mutations in the human T1R2 N-terminal extracellular domain abolish response to aspartame and neotame without affecting cyclamate. Stable cell lines of human T1R2/human T1R3 (WT), human T1R2 S144A/human T1R3 (S144A), and E302A/human T1R3 (E302A) were generated as described (7). The dose–responses of these stable lines were determined by FLIPR for sucrose, aspartame, neotame, and cyclamate. The activities represent the mean ± SE of fold increases in fluorescence intensities for four recorded wells. (C) Sequence alignment of human and rodent T1R3 transmembrane domains. The three extracellular loops are underlined and labeled EL1, EL2, or EL3, according to their order in the protein sequences. (D) Mutations in the extracellular loop of human T1R3 abolish response to cyclamate without affecting aspartame. Each of the three extracellular loops of human T1R3 was replaced with rat protein sequence separately, and the resulting human T1R3 mutants were transiently transfected into HEK-293T cells together with Gα_15/i1 and assayed for responses to sucrose (200 mM), aspartame (10 mM), and cyclamate (10 mM), and sucrose (200 mM) in the presence of lactisole (1 mM). The activities represent the mean ± SE of the number of responding cells for four imaged fields of ≈1,000 confluent cells.

Human T1R2 is required for Gα₁₅ coupling. (A) Responses of human (H), rat (R), and chimeric sweet taste receptors to sucrose (200 mM) and AceK (10 mM). Stable Gα₁₅ cells were transiently transfected with human, rat, or chimeric T1Rs and assayed for intracellular calcium increases in response to sweeteners. (B) Gα₁₅ coupling is mediated by human T1R2. The activities represent the mean ± SE of the number of responding cells for four imaged fields of ≈1,000 confluent cells.

The effect of lactisole and cyclamate on the human T1R1/T1R3 umami taste receptor. (A) Response of the human T1R1/T1R3 stable cell line tol-glutamate (5 mM) and l-glutamate/IMP (1/0.2 mM) in the absence and presence of lactisole (5 mM). (B) The lactisole dose-dependent inhibition curves were determined for l-glutamate (Glu) and l-glutamate with 0.2 mM IMP (Glu/IMP), each at two different concentrations. The IC₅₀s are 0.19 ± 0.02 and 0.21 ± 0.01 mM for l-glutamate at 8 and 80 mM, respectively, and 0.35 ± 0.03 and 0.82 ± 0.06 mM for l-glutamate with IMP at 0.8 and 8 mM, respectively. (B) The dose–responses for l-glutamate, with or without 0.2 mM IMP, were determined in the presence of different concentrations of lactisole. In the presence of 0, 25, or 50 μM lactisole, the EC₅₀s are 9.9 ± 1.5, 7.9 ± 0.5, and 7.0 ± 0.3 mM, respectively, for l-glutamate. In the presence of 0, 100, or 200 μM lactisole, the EC₅₀s are 0.53 ± 0.04, 0.71 ± 0.10, and 0.84 ± 0.10 mM, respectively, for l-glutamate with IMP. Values represent the mean ± SE for four independent responses. (D) The detection thresholds for sweet, umami, and salty taste stimuli were determined in the presence or absence of lactisole. The inhibition effect of lactisole is shown as fold increases in detection thresholds. The detection threshold values were averaged over four trials for three subjects. (E) The responses of the human T1R1/T1R3 stable cell line to threshold level of l-glutamate (4 mM) and endogenous M2 receptor agonist carbachol were assayed on FLIPR in the absence and presence of various concentrations of cyclamate. (F) Dose–responses of the human T1R1/T1R3 stable cell line were determined on FLIPR for l-glutamate with or without 0.2 mM IMP in the absence and presence of cyclamate (8 mM). The activities in B, C, E, and F represent the mean ± SE of fold increases in fluorescence intensities for four recorded wells. The dose–responses in B, C, E, and F were reproduced at least six times independently.

A working model for the sweet and umami taste receptor structure–function relationships. Filled arrows indicate direct activation, open arrows indicate enhancement, and bar heads indicate inhibition. Solid lines indicate proposed mechanisms based on experimental evidence; broken lines indicate mechanisms based on our speculations.