FIGURE 7.

Mutation of the cytoplasmic tail does not alter D₂-like dopamine receptor endocytosis. A, HEK293 cells expressing N-terminally FLAG-tagged D2NR, D3NR, or D4KL were incubated with anti-FLAG M1 antibody and then treated with 10 μm DA, 10 μm quinpirole, or 100 nm PMA for 45 min. Cells were then fixed, permeabilized, and incubated with Alexa Fluor® 488-conjugated anti-mouse secondary antibody. Shown are representative confocal images. B–D, endocytosis of the D₂-like receptor mutants was quantified using flow cytometry at 10, 20, 45, and 90 min (see “Materials and Methods”). D2NR receptors and D4KL receptors endocytosed in response to dopamine (B) and quinpirole (C), whereas D3NR did so only in response to PMA (D). Data are represented as the mean of at least four independent experiments performed in duplicate and analyzed using one-way ANOVA with Bonferroni t test. ***, p ≤ 0.001; *, p ≤ 0.05 compared with untreated controls.