Endosomal deubiquitinating enzymes control ubiquitination and down-regulation of protease-activated receptor 2

The E3 ubiquitin ligase c-Cbl ubiquitinates the G proteincoupled receptor protease-activated receptor 2 (PAR₂), which is required for postendocytic sorting of activated receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR₂ deubiquitination and its importance in trafficking and signaling of endocytosed PAR₂ are unknown.Wereport that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteasesAMSHand UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR₂ ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR₂. Trapping PAR₂ in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR₂ with β-arrestin2 or the duration of PAR₂-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR₂ but not for regulating PAR2 dissociation from β-arrestin2 or PAR₂-mediated ERK2 activation.