FIGURE 8.

Mutation of the GASP-1-binding motif alters receptor fate. A–C, HEK293 cells stably expressing either D2NR (A), D3NR (B), or D4KL (C) were surface-biotinylated and incubated in the absence or presence of either DA (10 μm, D2NR and D4KL) or PMA (100 nm, D3NR) for 30, 90, or 180 min. The fate of the surface-labeled receptors was assessed after anti-FLAG immunoprecipitation followed by SDS-PAGE and streptavidin overlay. 100% lane shows total surface receptor labeled. Strip lane shows efficiency of thio cleavage. A representative immunoblot is shown. D–F, densitometric quantification (Scion Image) of D2NR (D), D3NR (E), and D4NR (F) receptor stability from several experiments performed in A–C compared with their wild type counterparts (Fig. 3, D–F). Bars represent the mean recovery of surface biotinylated receptors (relative to 30 min of stimulation). Data are represented as the mean of at least four independent experiments analyzed using two-way ANOVA with Bonferroni t test. ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05 compared with wild type receptors.