FIGURE 3.

PAR₂ is ubiquitinated at the plasma membrane and early endosomes and deubiquitinated before transfer to lysosomes. HEK cells were transiently transfected with PAR₂ and pcDNA5 (control) or Hrs-Myc. A, cell surface PAR₂ was labeled with antibody to extracellular FLAG epitope. Cells were stimulated with AP (100 μm, 240 min), and PAR₂, Hrs, and LAMP1 were localized. In cells treated with hypertonic sucrose, PAR₂ remained at the plasma membrane (arrowheads). Expression of Hrs prevented trafficking of PAR₂ from early endosomes. In cells treated with lysosomal protease inhibitors, PAR₂ accumulated in LAMP1-positive lysosomes (arrows). Scale bar, 10 μm. B, cells were treated as in A. PAR₂ was immunoprecipitated (IP), and membranes analyzed by Western blotting (WB) for ubiquitin (ubi) and PAR₂. In unstimulated cells, there was no detectable PAR₂ ubiquitination. AP-induced PAR₂ ubiquitination was detected when trafficking of PAR₂ was blocked at the plasma membrane with sucrose or when PAR₂ was trapped in early endosomes by overexpression of Hrs. PAR₂ ubiquitination was not detected when PAR₂ had accumulated in lysosomes in the presence or absence of lysosomal inhibitors. Results shown are representative of n = 3 experiments.