FIGURE 10.

GPR17 is internalized via clathrin-dependent endocytosis. In A, differentiated Oli-neu cells were surface-labeled with anti-Nt-GPR17 antibodies and then incubated for 12 min at 37 °C without (control (CTR)) or with agonists (LTD₄ or UDP-glucose (UDPglc)) in Sato medium minus (−) or supplemented (+) with 450 mm sucrose. After glycine washing, cells were fixed and labeled with monoclonal antibodies against Lamp1 followed by incubation with anti-rat IgG conjugated to fluorescein (LAMP) and anti-rabbit IgG conjugated to Cy3 (GPR17). Confocal microscopy images show no accumulation of GPR17 immunostaining in the cells incubated in hypertonic sucrose. Images are representative of three independent experiments. The merged images are shown. Bar = 10 μm. B, to quantify the effects of clathrin lattice inhibition on the endocytosis of GPR17, confocal images were collected from Oli-neu cells incubated with or without hypertonic sucrose (+), and the pixel intensity was determined by ImageJ software. The graph represents the increase of GPR17 immunoreactivity in the cells over background (background = the immunofluorescence detected in the cells after labeling at 4 °C followed by acid stripping). The values are the mean (± S.E.) of three independent experiments. *, p > 0.05; **, p < 0.01; ***, p < 0.0001 (two-tailed, non-paired Student's t test).