Lactisole Interacts with the Transmembrane Domains of Human T1R3 to Inhibit Sweet Taste

Peihua Jiang,Meng Cui,Baohua Zhao,Zhan Liu,Lenore A Snyder,Lumie M.J Benard,Roman Osman,Robert F Margolskee,Marianna Max

doi:10.1074/jbc.m414287200

Peihua Jiang, Meng Cui + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m414287200

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Abstract

The detection of sweet-tasting compounds is mediated in large part by a heterodimeric receptor comprised of T1R2+T1R3. Lactisole, a broad-acting sweet antagonist, suppresses the sweet taste of sugars, protein sweeteners, and artificial sweeteners. Lactisole's inhibitory effect is specific to humans and other primates; lactisole does not affect responses to sweet compounds in rodents. By heterologously expressing interspecies combinations of T1R2+T1R3, we have determined that the target for lactisole's action is human T1R3. From studies with mouse/human chimeras of T1R3, we determined that the molecular basis for sensitivity to lactisole depends on only a few residues within the transmembrane region of human T1R3. Alanine substitution of residues in the transmembrane region of human T1R3 revealed 4 key residues required for sensitivity to lactisole. In our model of T1R3's seven transmembrane helices, lactisole is predicted to dock to a binding pocket within the transmembrane region that includes these 4 key residues.

Highlights

The detection of sweet-tasting compounds is mediated in large part by a heterodimeric receptor comprised of T1R2؉T1R3
To determine whether one or both components of the heterodimeric human sweet receptor is required for sensitivity to lactisole, we tested the responses of human, mouse and human ϩ mouse mismatched heterodimers to D-tryptophan with and without lactisole (Fig. 1B)
As reported previously [17], the other mismatched pair does not produce a functional receptor, precluding us from analyzing its sensitivity to lactisole. These results demonstrate that human T1R3 (hT1R3) is required for lactisole sensitivity

Summary

EXPERIMENTAL PROCEDURES

Materials—Acesulfame-K, cyclamate, D-tryptophan, neohesperidin dihydrochalcone (NHDC), saccharin, sucrose, thaumatin, and lactisole were obtained from Sigma. Based on the sequence alignment, the hT1R3 TM homology model was constructed by residue replacement using the InsightII (Accelrys, San Diego, CA) biopolymer module. The other intracellular and extracellular loop regions were generated by the modeler program [26, 27] based on the bovine rhodopsin template. The final docked conformation was selected by comparing the available mutagenesis results followed by some manual adjustments of the positions of lactisole and the side chains of hT1R3 before employing model refinement by MD simulations. A similar MD protocol was used for the docked lactisole-hT1R3 complex structure refinement that except the ␣-carbon atoms of the helixes were restricted by 1.0 kcal/mol/Å2 harmonic restraint force instead of fixing the ␣-carbon atoms during the simulations

RESULTS

Lactisole Inhibits the Sweet Taste Receptor

DISCUSSION