title: Endosomal deubiquitinating enzymes control ubiquitination and down-regulation of protease-activated receptor 2.
abstract: The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled receptor protease-activated receptor 2 (PAR(2)), which is required for postendocytic sorting of activated receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR(2) deubiquitination and its importance in trafficking and signaling of endocytosed PAR(2) are unknown. We report that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR(2) 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 PAR(2). Trapping PAR(2) in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR(2) with beta-arrestin2 or the duration of PAR(2)-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR(2) but not for regulating PAR(2) dissociation from beta-arrestin2 or PAR(2)-mediated ERK2 activation.
authors.affiliation: Department of Surgery and Physiology, University of California, San Francisco, California 94143-0660.
authors.forename: Burcu
authors.initials: B
authors.lastname: Hasdemir
authors.affiliation: Department of Surgery and Physiology, University of California, San Francisco, California 94143-0660.
authors.forename: Jane E
authors.initials: JE
authors.lastname: Murphy
authors.affiliation: Department of Surgery and Physiology, University of California, San Francisco, California 94143-0660.
authors.forename: Graeme S
authors.initials: GS
authors.lastname: Cottrell
authors.affiliation: Department of Surgery and Physiology, University of California, San Francisco, California 94143-0660. Electronic address: nigel.bunnett@ucsf.edu.
authors.forename: Nigel W
authors.initials: NW
authors.lastname: Bunnett
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism; D002460:Cell Line; D015536:Down-Regulation; D004705:Endocytosis / Q000502:physiology; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D056827:Endosomal Sorting Complexes Required for Transport; D034741:RNA, Small Interfering; D044464:Receptor, PAR-2; D011993:Recombinant Fusion Proteins; C486696:STAMBP protein, human; D000071557:beta-Arrestins; D019950:Mitogen-Activated Protein Kinase 1; D010450:Endopeptidases; C541534:USP8 protein, human; D043222:Ubiquitin Thiolesterase; D010196:Pancreatic Elastase; D014357:Trypsin
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