FIGURE 4.

Action of specific ligands as pharmacological chaperones. A, dose-dependent effects of Y-24180 on cell surface expression of the mutant HA-hPAFRs. These experiments were carried out using cell lines HeLa-WT, HeLa-D63A, HeLa-P247A, and HeLa-mock cells, which are induced to produce the receptors when Dox is added. Twenty-four hours after addition of Dox, the cells were treated with the indicated concentrations of Y-24180 for 24 h. The expression levels were evaluated by the mean fluorescence intensity (MFI), represented as a percentage of WT HA-hPAFR at 0 nm compound. Data are represented as mean ± S.E. (n = 3). Bottom, the protein levels of each receptor under the conditions of Y-24180 (1 μm) treatment were examined by Western blotting using anti-HA antibody. Approximate molecular sizes are shown in kDa at the right. B, effects of Y-24180 (1 μm) on subcellular localization of WT and mutant HA-hPAFRs were observed with immunofluorescence confocal microscopy. Twenty-four hours after transfection of HeLa cells with WT HA-hPAFR, D63A, or P247A, the cells were treated with vehicle (ethanol; upper) or Y-24180 (lower) for 24 h, then subjected to immunocytochemical analysis. Calreticulin and HA-tagged hPAFRs were visualized using anti-calreticulin (red) and anti-HA (green) antibodies, respectively. White bars indicate 10 μm. The data are representative of three independent experiments with identical results. C, dose-dependent effects of mc-PAF on cell surface expression of the mutant HA-hPAFRs. These experiments were carried out using the same cells and methods described in A. Bottom, the protein levels of each receptor under mc-PAF (1 μm) treatment conditions were examined by Western blotting using anti-HA antibody. Approximate molecular sizes are shown in kDa at the right.