Part:BBa_K2368004
Introduction
T1R3
The protein encoded by the T1R3 gene is a G protein-coupled receptor with seven trans-membrane domains. And it is a component of the heterodimeric amino acid taste receptor T1R1+3 and sweet taste receptor T1R2+3. This receptor is formed as a protein dimer with either T1R1 or T1R2. Experiments have also shown that a homo-dimer of T1R3 is also sensitive to natural sweeteners. This has been hypothesized as the mechanism by which sugar substitutes do not have the same taste qualities as natural sugars.
Originally, T1R1+3 expressing cells are found in fungiform papillae at the tip and edges of the tongue and palate taste receptor cells in the roof of the mouth. These cells are shown to synapse upon the chorda tympani nerves to send their signals to the brain. T1R2+3 expressing cells are found in circumvallate papillae and foliate papillae near the back of the tongue and palate taste receptor cells in the roof of the mouth. These cells are shown to synapse upon the glossopharyngeal nerves to send their signals to the brain.
Fig. 1 The schematic diagram of T1R2/T1R3 in cytomembrane</i>
Part 1: Dry experiments of T1R3
1、Structure models
Purpose
In order to confirm whether this “radar” T1R2/T1R3 can “sense” the sweetness of different sweetener, we simulate the receptor structure model. It helps us to understand how the sweeteners binding to T1R2/T1R3 receptor visually. Otherwise we hope to find some unknown sweeteners binding sites based on this model, even some ideal but unknown sweeteners.
Method
Initially, to make the signal input more accurate and reliable, we simulate the T1R2/T1R3 receptor structure model using SWISS-MODEL. Additionally, we prepare some sweeteners’ PDB file based on Chemdraw 2D and Chemdraw 3D. And the docking process is performed by using Autodock Vina.
Result
We used homology modeling method to get the structure of human sweet receptorT1R2/T1R3. The quality of the simulate structure within normal range and it only contain the ligand-binding-domain based on the crystal protein structure of Mice.
Fig. 2 The simulate structure of human sweetness receptors ligand-binding domain (LBD)
Then we use software Chemdraw 2D and Chemdraw 3D to build PDB files of some classic sweeteners.
Fig. 3 The 3D structure of some sweeteners
The docking process is carried out under Autodock Vina (Fig. 4-6).
Fig. 4 The docking result of different sweeteners
Fig. 5 The docking result of aspartame
Fig. 6 The docking result of stevioside
2、T1R2/T1R3 receptor activation part
The part is about the sweeteners binding to the receptor and the GPCR produce the initial signal through changing structure. For T1R2/T1R3, we divide the process of inducing signal into four conditions of T1R2/T1R3 as show in Fig. 7. And the reaction between different conditions of protein is used ODEs to describe it. The all ODEs are shown follow.
Fig. 7 T1R2/T1R3 receptor activation reaction diagram
(T1R2/3: the T1R2/T1R3 heterodimer)
The parameters of this part are listed in the Table 1.
The output of the T1R2/T1R3 receptor activation part is showed. (Fig.8)
Fig. 8 The output signal, activated state of T1R2/T1R3, induced by different concentration ligand </i>
As we can see, the activated state of T1R2/T1R3 establishes clear distant under different concentration of ligand for binding. It can prove that this pathway can sense different strength of signal and the signal will decrease soon, which means our system will have less signal interfere.
3、The result of T1R2 expression
As the result of combination model established, we can see the curve of RFP intensity does not increase at first 5 hours, and then the RFP intensity increases rapidly among 5-15 hours. At the same time, the differences of each ligand concentration are displayed in the middle of culture time (around at 15 hours), which will is similar to our experimental data. Finally, the RFP of all groups reduce slowly.
Fig. 9 The simulation result of RFP intensity in population level.
Part 2: Wet experiments of T1R3
Design:
According to data mining, we synthesized the T1R3 gene by OE PCR. we fused myc, his tag at the N-terminus of sweetness receptor to detect whether it localize the cell membrane. And they are ligated to the plasmid by Gibson assembly.
Fig. 10 The schematic diagram of expressing T1R3.
Result
In our experiment, the T1R3 gene is used to accept the signal inputted in our whole pathway. We synthesized this part by OE PCR.
Fig.11 The electrophoresis of the positive result of T1R3. <p>Then the expression and location of the receptor are detected by immunofluorescence. The principle of immunofluorescence is that the antibody A binds to the tag, and then the fluorescent antibody B binds to the A. This is the positive result of our immunofluorescence.
Fig.12 Immunofluorescence of T1R2/T1R3 in yeast. The scale bars represent 10µm
As the result of combination model established, we can see the curve of RFP intensity does not increase at first 5 hours, and then the RFP intensity increases rapidly among 5-15 hours. At the same time, the differences of each ligand concentration are displayed in the middle of culture time (around at 15 hours), which will is similar to our experimental data. Finally, the RFP of all groups reduce slowly after 15 hours.
Fig. 13 The fluorescence intensity of different Sweeteners
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[3] Bohlmann L, Tredwell G D, Yu X, et al. Nature Chemical Biology, 2015, 11(12):955.
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[6] Richardson, Kathryn. Mechanisms of GPCR signal regulation in fission yeast[J]. University of Warwick, 2014.
[7] Nie Y, Vigues S, Hobbs J R, et al. Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli.[J]. Current Biology Cb, 2005, 15(21):1948-52.
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Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1369
Illegal BglII site found at 1789
Illegal BglII site found at 1951
Illegal XhoI site found at 396 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 880
Illegal AgeI site found at 2098 - 1000COMPATIBLE WITH RFC[1000]
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