Figure 3.

Regulation of D₂R trafficking by NCAM. A, Myc-D₂R-expressing HEK293 cells were mock-transfected or transfected with NCAM180. Two days later, cells were stimulated with 10 μm DA for 30 min, followed by cell surface biotinylation with Sulfo-NHS-LC-biotin. Biotin-labeled surface proteins (surf) and total proteins (total) were detected by Western blot analysis using antibodies against D₂R, APP, or NCAM, respectively. The levels of selected proteins at the cell surface relative to their total levels were determined, with the ratio obtained from the mock-transfected cells in the absence of dopamine being set to 100%. *p < 0.05 by unpaired t test (n = 3). B, Myc-D₂R-expressing HEK293 cells transfected with NCAM180 were subjected to live staining with myc antibody to label cell surface D₂Rs. After treatment with 10 μm dopamine, cells were stained with antibodies against D₂R before permeabilization (surface D₂Rs) and after permeabilization (internalized D₂Rs). Percentage of internalized D₂Rs, defined as the ratio of internalized to total fluorescence intensities, was presented as mean + SEM for 50 cells. Results are representative of two independent experiments. *p < 0.05 by unpaired t test. Scale bar, 25 μm. C, Myc-D₂R-expressing HEK293 cells transiently transfected with NCAM180 were stimulated with 10 μm DA for 0, 30, 90, or 180 min in the presence of 10 μg/ml cycloheximide to block de novo protein synthesis. The levels of D₂R and NCAM were evaluated by Western blot analysis with D₂R and NCAM antibodies, respectively. Determination of GAPDH levels served as control for loading. The proteins levels obtained by dopamine stimulation for 0 min were set to 100%. The dashed lines indicate 50% degradation of D₂Rs. Means + SEM are shown here (n = 3).