FIGURE 9.

Expression of catalytically inactive AMSH or UBPY does not prolong the interaction between β-arrestin2 and PAR₂ in endosomes. HEK-PAR₂ cells were transiently transfected with β-arrestin2-HSV and pcDNA5 (control) (A), GFP-AMSH(D348A) (B), or GFP-UBPY(C786S) (C). Cell surface PAR₂ was labeled with FLAG antibody. Cells were challenged with AP (100 μm, 30 min) or with AP (100 μm, 30 min), followed by washing and recovery in AP-free medium for 1 h. In control cells, AP (30 min) induced trafficking of PAR₂ and β-arrestin2 to the same vesicles (presumably endosomes; arrows). After a 1-h recovery, PAR₂ was still present in vesicles, and β-arrestin2 had returned to the cytosol. Similar results were obtained in cells expressing GFP-AMSH(D348A) or GFP-UBPY(C786S). Scale bars, 10 μm. D, HEK cells transiently transfected with PAR₂ were stimulated with AP (100 μm) for 0 or 30 min. Endosomes were isolated and analyzed for β-arrestin2 and EEA1 by Western blotting. AP (30 min) increased the levels of β-arrestin2 in endosomes. E, HEK cells transiently transfected with PAR₂ and pcDNA5 (control), GFP-AMSH(D348A), or GFP-UBPY(C786S) were challenged with AP (100 μm, 30 min), followed by washing and recovery in AP-free medium for 1 h. Endosomes were isolated and analyzed for β-arrestin2 and EEA1 by Western blotting and quantified using densitometry. Expression of GFP-AMSH(D348A) or GFP-UBPY(C786S) did not cause retention of β-arrestin2 in endosomes. n = 3. IP, immunoprecipitation; WB, Western blot.