The Role of Palmitoylation in Signalling, Cellular Trafficking and Plasma Membrane Localization of Protease-Activated Receptor-2
Figure 5
PAR2 agonism stimulates palmitate incorporation which occurs during secretory trafficking in pre-medial Golgi vesicles.
A, CHO-PAR2-myc cells preincubated with cerulenin for 1 h were labelled with 17-ODYA for 1.5 h in the presence or absence of PAR2-AP (100 µM) for the indicated times. Membrane preparations were collected and biotin-azide reacted to 17-ODYA labelled proteins via click chemistry. Biotinylated proteins were isolated using streptavidin beads. PAR2 palmitoylation was assessed by anti-myc Western blot analysis of bead elutes. B, CHO-PAR2-myc cells preincubated with cerulenin were labelled with 17-ODYA for 2 h in the presence or absence of PAR2 AP (100 µM). Nocodazole (20 µg/mL; NOC) was added to medium 15 min prior to labelling with 17-ODYA and monensin (10 µM; MON) and brefeldin A (5 µg/mL; BFA) were added to medium 30 min prior to labelling with 17-ODYA. Labelled proteins were reacted to biotin-azide via click chemistry and biotinylated proteins isolated using streptavidin beads. Palmitoylated PAR2 was detected by anti-Myc Western blot analysis of bead elutes. Data shown in B is from cropped lanes from a single Western blot analysis. The data are representative of three independent experiments.