FIGURE 2.

AP and trypsin, but not elastase, induce PAR₂ ubiquitination, trafficking to lysosomes, and degradation. A, HEK-PAR₂ cells were stimulated for 0 or 30 min with AP (100 μm), trypsin (10 nm), or elastase (0.5 μm). PAR₂ was immunoprecipitated (IP), and membranes were analyzed by Western blotting (WB) for ubiquitin (ubi) and PAR₂. Only AP and trypsin induced ubiquitination of PAR₂. B, HEK-PAR₂ cells were challenged for 0 or 120 min with AP (100 μm), trypsin (10 nm), or elastase (0.5 μm). Cells were fixed, and proteins were localized by indirect immunofluorescence with antibodies to HA11 (PAR₂) and LAMP1. In unstimulated cells, PAR₂ was located at the plasma membrane (arrowheads) and in a perinuclear pool. Stimulation with AP or trypsin induced PAR₂ translocation to LAMP1-containing vesicles (arrows). In contrast, PAR₂ remained at the plasma membrane following treatment with elastase (arrowheads). Scale bar, 10 μm. C, cell surface proteins on HEK-PAR₂ cells were biotinylated prior to stimulation with AP (100 μm) or trypsin (10 nm) for 0 or 6 h. Biotinylated proteins were precipitated using avidin beads, and PAR₂ and TfR were detected by Western blotting. Stimulation with AP or trypsin both induced degradation of PAR₂. Results shown representative of n = 3 experiments.