Distinct Human and Mouse Membrane Trafficking Systems for Sweet Taste Receptors T1r2 and T1r3
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.