FIGURE 4.

PAR₂ colocalizes with AMSH and UBPY in endosomes. HEK-PAR₂ cells were transiently transfected with GFP-AMSH (A), GFP-AMSH(D348A) (B), GFP-UBPY (C), GFP-UBPY(C786S) (D), GFP-AMSH and Rab5aQ79L-CFP (E), or GFP-UBPY and Rab5aQ79L-CFP (F). Cell surface PAR₂ was labeled with FLAG antibody. Cells were challenged with AP (100 μm; 0, 30, 120 min), and PAR₂ was localized. In unstimulated cells, PAR₂ was at the plasma membrane (arrowheads), GFP-AMSH was cytosolic and present in endosomes (arrows), GFP-UBPY was cytosolic, and GFP-AMSH(D348A) and GFP-UBPY(C786S) were in enlarged cytosolic vesicles (arrows). AP (30 min) induced trafficking of PAR₂ from the plasma membrane to colocalize with GFP-AMSH, GFP-AMSH(D348A), and GFP-UBPY(C786S) in vesicles (presumably endosomes; arrows, yellow in merged images). At 120 min, PAR₂ was still present in vesicles (presumably lysosomes) and did not colocalize with GFP-AMSH or GFP-UBPY. In GFP-AMSH(D348A)- and GFP-UBPY(C786S)-expressing cells, GFP-AMSH(D348A) and GFP-UBPY(C786S) remained colocalized with PAR₂. E and F, AP (30 min) induced trafficking of PAR₂ to enlarged endosomes containing Rab5aQ79L-CFP (arrows). GFP-AMSH and GFP-UBPY were also present in these enlarged endosomes (arrows). Scale bars, 10 μm.