Fig. 7. Endocytosis and recycling do not require motifs in the C-terminal tail of CCR7 and are independent of G-protein activation. (A) Removing the entire C-terminus of CCR7 does not abolish chemokine-mediated receptor trafficking. 300-19 cells stably expressing wild-type CCR7 or C-terminal truncation mutants were stimulated with 2 µg/ml CCL19 or medium for 30 minutes at 37°C, followed by detection of CCR7 surface expression by flow cytometry using a CCR7 antibody to determine endocytosis (black bars). To analyze receptor recycling, chemokine-triggered cells were washed extensively to remove unbound CCL19 and subsequently incubated for 1 hour in the absence of chemokines, permitting recycling of CCR7 back to the plasma membrane (white bars). Relative CCR7 surface expression as a percentage of untreated cells is plotted. (B) CCR7 trafficking is PTx insensitive. 300-19 CCR7 cells were pretreated or not with PTx for 2 hours, followed by stimulation with CCL19. CCR7 internalization and recycling were determined by flow cytometry as described above. (C) The DRY motif is dispensable for CCR7 trafficking. 300-19 cells stably expressing wild-type CCR7 or the DNY mutant were incubated with 2 µg/ml CCL19 for 30 minutes for internalization assays. CCR7 endocytosis and recycling were assessed as outlined in A. Values represent the mean ± s.e.m. of three experiments.