Sensitivity of human sweet taste receptor subunits T1R2 and T1R3 to activation by glucose enantiomers.

We have previously shown that l-glucose, the non-caloric enantiomer of d-glucose, activates the human sweet taste receptor T1R2/T1R3 transiently expressed in HEK293T cells. Here, we show that d- and l-glucose can also activate T1R2 and T1R3 expressed without the counterpart monomer. Serine mutation to alanine in residue 147 in the binding site of T1R3 VFT domain, completely abolishes T1R3S147A activation by either l- or d-glucose, while T1R2/T1R3S147A responds in the same way as T1R2 expressed without its counterpart. We further show that the original T1R2 reference sequence (NM_152232.1) is less sensitive by almost an order of magnitude than the reference sequence at the time this study was performed (NM_152232.4). We find that out of the four differing positions, it is the R317G in the VFT domain of T1R2, that is responsible for this effect in vitro. It is significant for both practical assay sensitivity and because glycine is found in this position in ~20% of the world population. While the effects of the mutations and the partial transfections were similar for d and l enantiomers, their dose-response curves remained distinct, with l-glucose reaching an early plateau.

Dubovski N ，Ben-Shoshan Galeczki Y ，Malach E ，Niv MY ... -  《-》

The T1R3 subunit of the sweet taste receptor is activated by D2O in transmembrane domain-dependent manner.

Ben Abu N ，Ben Shoshan-Galeczki Y ，Malach E ，Y Niv M ... -  《-》

The Heptahelical Domain of the Sweet Taste Receptor T1R2 Is a New Allosteric Binding Site for the Sweet Taste Modulator Amiloride That Modulates Sweet Taste in a Species-Dependent Manner.

Zhao M ，Xu XQ ，Meng XY ，Liu B ... -  《-》

Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor.

The sweet protein brazzein [recombinant protein with sequence identical with the native protein lacking the N-terminal pyroglutamate (the numbering system used has Asp2 as the N-terminal residue)] activates the human sweet receptor, a heterodimeric G-protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (T1R3). In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: site 1 (Loop43), site 2 (N- and C-termini and adjacent Glu36, Loop33), and site 3 (Loop9-19). Basic residues in site 1 and acidic residues in site 2 were essential for positive responses from each assay. Mutation of Y39A (site 1) greatly reduced positive responses. A bulky side chain at position 54 (site 2), rather than a side chain with hydrogen-bonding potential, was required for positive responses, as was the presence of the native disulfide bond in Loop9-19 (site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus flytrap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in brazzein response. The exception, hT1R2 (human T1R2 subunit of the sweet receptor):R217A/hT1R3 (human T1R3 subunit of the sweet receptor), which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site is involved in subunit-subunit interaction rather than in direct brazzein binding. Results from this study support a multi-point interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.

Assadi-Porter FM ，Maillet EL ，Radek JT ，Quijada J ，Markley JL ，Max M ... -  《-》

Distinct human and mouse membrane trafficking systems for sweet taste receptors T1r2 and T1r3.

Shimizu M ，Goto M ，Kawai T ，Yamashita A ，Kusakabe Y ... -  《PLoS One》