Figure 1.
Construction of tagged mouse T1r2 and T1r3, and human T1R2 and T1R3.
A. Schematic diagrams of tagged mouse T1r2 and T1r3, and human T1R2 and T1R3. The striped section corresponds to the signal peptide of mouse mGluR1, and the gray and black boxes represent c-Myc and FLAG tags, respectively. CH2, c-Myc-tagged human T1R2 (hT1R2); Cm2, c-Myc-tagged mouse T1r2 (mT1r2); CRD, cysteine-rich domain; FH3, FLAG-tagged human T1R3 (hT1R3); Fm3, FLAG-tagged mouse T1r3 (mT1r3); HD, heptahelical domain; VFTM, Venus flytrap modules. B. Surface expression of tagged mT1r2/mT1r3 and hT1R2/hT1R3. HEK293 cells stably expressing c-Myc-tagged T1r2 (hT1R2 and mT1r2) and FLAG-tagged T1r3 (hT1R3 and mT1r3) and labeled with rabbit anti-c-Myc antibody or rabbit anti-FLAG antibody under non-permeabilized conditions (scale bar = 50 µm). C. Tagged T1r2/T1r3s functions as a sweet taste receptor. The intensity of the response was represented as the ratio (ΔF) relative to the baseline (F) and was plotted versus ligand concentration. The cells expressing tagged T1r2/tagged T1r3 (filled circles) responded to sucralose. Error bars: SD (n = 3–6). D. Immunoblot analysis of cells expressing tagged T1r2s. The sample proteins were obtained through immunoprecipitation from 7.5×105 cells/well using an anti-c-Myc antibody. E. Immunoblot analysis of cells expressing tagged T1r3 with a rabbit anti-FLAG antibody using cell lysate from 2.5×104 cells/well.
Figure 2.
Surface expression of tagged T1r2 and T1r3.
A. Surface expression of tagged T1r2 and T1r3 solely in HEK293 cells. HEK293 cells stably expressing tagged T1r2 or tagged T1r3 were labeled with a rabbit anti-c-Myc antibody or rabbit anti-FLAG antibody under non-permeabilized conditions (scale bar = 50 µm). B. Surface expression of tagged T1r3 in COS-7 and CHO cells. Tagged T1r3 were transiently expressed in COS-7 or CHO cells and were labeled with a rabbit anti-FLAG antibody under non-permeabilized conditions (scale bars = 50 µm) C. Tagged T1rs expression in HEK293 cells. The cells were labeled with a rabbit anti-c-Myc antibody or rabbit anti-FLAG antibody under permeabilized conditions (scale bar = 50 µm). D. Surface expression of FH3 with transient CH2 expression. FH3 cells were labeled with a rabbit anti-FLAG antibody under non-permeabilized conditions after 24-h lipofection with CH2 (scale bar = 50 µm). E. Representative flow cytometry histogram data. HEK293 cells were used as a control. F. Flow cytometry quantification of cell surface expression of tagged T1r3 in intact cells. Data are expressed as the mean fluorescence intensity (MFI) ratio of FLAG labeling (MFI [FLAG]) in cells expressing tagged mouse T1r2, T1r3, T1r2/T1r3, human T1R2, T1R3, or T1R2/T1R3 to that in control HEK293 cells. Statistical significance was calculated by ANOVA followed by Tukey test (*: P<0.05). Error bars: SEM (n = 3).
Figure 3.
Surface expression of human-mouse T1r3 chimeras.
A. Construction of T1r3 chimeras. The mouse regions are shaded in grey. B. Surface expression of T1r3 chimeras. HEK293 cells expressing chimeras were labeled with rabbit anti-FLAG antibody under non-permeabilized conditions (scale bar = 50 µm). C. Immunoblot analysis of cells expressing chimeras with rabbit anti-FLAG (2.5×104 cells/well). D. Flow cytometry quantification of cell surface expression of chimeras in intact cells. Data are expressed as the MFI ratio of FLAG labeling (MFI [FLAG]) in chimera-expressing cells. Statistical significance was calculated by ANOVA followed by Tukey test (*: P<0.05). Error bars: SEM (n = 3). E. Representative flow cytometry histogram data.
Figure 4.
Surface expression of truncated human T1R3 mutants.
A. Construction of truncated human T1R3 mutants. All mutants were tagged with a FLAG epitope under the mGluR1 signal peptide. B. Truncated mutant surface expression. HEK293 cells expressing mutants were labeled with a rabbit anti-FLAG antibody under non-permeabilized conditions (scale bar = 50 µm). C. The responses of truncated mutants for the sweet taste substance cyclamate. FxxH3-expressing cells (open circles) or control cells (filled triangles) showed little response to cyclamate, whereas cells expressing hT1R2/hT1R3 (filled circles) did respond to cyclamate. Error bars: SD (n = 3–6). The intensity of the response was represented as the ratio (ΔF) relative to the baseline (F) and was plotted versus ligand concentration. D. Flow cytometry quantification of cell surface expression of truncated mutants in intact cells. Data are expressed as the MFI ratio of FLAG labeling (MFI [FLAG]) in chimera-expressing cells. Error bars: SEM (n = 3). E. Immunoblot analysis of truncated mutants treated with a rabbit anti-FLAG (7.5×104 cells/well). The filled arrowhead indicates a molecular weight of approximately 33 kDa for FxHH3, and the open arrowhead indicates approximately 27 kDa for FxxH3.
Figure 5.
Surface expression of VFTM-truncated human T1R2/T1R3 mutants.
A. Construction of the truncated mutants of human T1R2 and T1R3. All mutants were tagged with c-Myc or FLAG epitopes under the mGluR1 signal peptide. B. Surface expression of the truncated mutants. HEK293 cells expressing mutants were labeled with a rabbit anti-c-Myc antibody or rabbit anti-FLAG antibody under non-permeabilized conditions (scale bar = 50 µm). C. Immunoblot analysis of truncated mutant-expressing cells. (Left panel) The sample proteins for truncated hT1R2 (CxHH2) or c-Myc tagged hT1R2 (CH2) were obtained through immunoprecipitation from 7.5×105 cells/well using an anti-c-Myc antibody. (Right panel) The cell lysates from 2.5×104 cells/well were used for truncated hT1R3 (FxHH3) or FLAG tagged hT1R3 (FH3) proteins. Arrowheads indicate molecular weights of approximately 33 kDa for both CxHH2 and FxHH3. D. Flow cytometry quantification of cell surface expression of the truncated mutants. Data are expressed as the MFI ratio of FLAG (MFI [FLAG]) labeling in mutant-expressing cells. Statistical significance was calculated by ANOVA followed by Tukey test (*: P<0.05). Error bars: SEM (n = 3). E. Responses of truncated T1rs to cyclamate. Cells expressing CxHH2/FH3 (open triangles) or CH2/FxHH3 (filled squares) showed little response to cyclamate, whereas cells expressing CH2/FH3 (filled circles) did respond to cyclamate. The intensity of the response was represented as the ratio (ΔF) relative to the baseline (F) and was plotted versus ligand concentration. Error bars: SD (n = 3–6).