Endosomal Deubiquitinating Enzymes Control Ubiquitination and Down-regulation of Protease-activated Receptor 2

The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled 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. 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 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. The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled 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. 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 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. Ubiquitination of certain G protein-coupled receptors (GPCRs) 3The abbreviations used are: GPCRG protein-coupled receptorβ2ARβ2-adrenergic receptorDORδ-opioid receptorDUBdeubiquitinating enzymeEGFepidermal growth factorEGFRepidermal growth factor receptorERKextracellular signal-regulated kinaseMAPKmitogen activated protein kinaseMVBmultivesicular bodyPAR2protease-activated receptor 2pERKphosphorylated extracellular signal-regulated kinaseTfRtransferrin receptorUSPubiquitin-specific proteaseE3ubiquitin-protein isopeptide ligasesiRNAsmall interfering RNACFPcyan fluorescent proteinHEKhuman embryonic kidney 293PBSphosphate-buffered salineAPactivating peptideRIPAradioimmune precipitationHAhemagglutinin. 3The abbreviations used are: GPCRG protein-coupled receptorβ2ARβ2-adrenergic receptorDORδ-opioid receptorDUBdeubiquitinating enzymeEGFepidermal growth factorEGFRepidermal growth factor receptorERKextracellular signal-regulated kinaseMAPKmitogen activated protein kinaseMVBmultivesicular bodyPAR2protease-activated receptor 2pERKphosphorylated extracellular signal-regulated kinaseTfRtransferrin receptorUSPubiquitin-specific proteaseE3ubiquitin-protein isopeptide ligasesiRNAsmall interfering RNACFPcyan fluorescent proteinHEKhuman embryonic kidney 293PBSphosphate-buffered salineAPactivating peptideRIPAradioimmune precipitationHAhemagglutinin. is an essential signal for their postendocytic trafficking to lysosomes, which prevents uncontrolled signaling during chronic stimulation. Agonists stimulate ubiquitination of the β2-adrenergic receptor (β2AR), chemokine (CXC motif) receptor 4, and protease-activated receptor 2 (PAR2), and the E3 ubiquitin ligases that mediate ubiquitination of these GPCRs and associated proteins, such as β-arrestins, have been identified (1Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (706) PubMed Scopus PubMed Scopus ubiquitination of these receptors is not required for receptors postendocytic sorting to lysosomes and impaired of the of is the of deubiquitinating proteases in the postendocytic trafficking and signaling of of the of in postendocytic receptor trafficking from of receptor such as growth factor receptor endosomal AMSH with the of and UBPY as deubiquitination and postendocytic trafficking of PubMed Scopus AMSH to the of and for ubiquitin PubMed Scopus PubMed Scopus UBPY is of the and not between and ubiquitin PubMed Scopus PubMed Scopus the E3 ligase which ubiquitinates the receptor PubMed Scopus with the growth in early endosomes PubMed Scopus of the sorting required for that receptors in to that with lysosomes, where degradation occurs PubMed Scopus receptors are the are which to of ubiquitin PubMed Scopus AMSH and UBPY with through its PubMed Scopus PubMed Scopus PubMed Scopus The of ubiquitination c-Cbl and deubiquitination AMSH and UBPY the postendocytic trafficking and down-regulation of the c-Cbl lysosomal degradation of the PubMed Scopus AMSH c-Cbl and PubMed Scopus and UBPY is required for lysosomal sorting and degradation of PubMed Scopus PubMed Scopus PubMed Scopus PubMed Scopus The of AMSH and UBPY in regulating deubiquitination, trafficking, and signaling of GPCRs in endosomes is unknown. that AMSH and UBPY the down-regulation of the receptor that is and PubMed Scopus Expression of catalytically inactive of AMSH or UBPY or knockdown of AMSH or UBPY down-regulation of the of AMSH and UBPY in down-regulation to of and of did not have PubMed Scopus and have been to ubiquitination of the S.K. PubMed Scopus the of AMSH and UBPY in the postendocytic trafficking, and lysosomal degradation of PAR2. We also AMSH and UBPY PAR2 association with in endosomes and extracellular signal-regulated activation. PAR2 is receptor for proteases that are during and PubMed Scopus PAR2 and and PAR2 to of the and PAR2 are during to expression or receptor which the of proteases PubMed Scopus the of and the receptor the of protein to endosomes PubMed Scopus of PAR2 signaling receptor degradation in lysosomes, which in is PubMed Scopus S.K. PubMed Scopus is to mechanisms of PAR2 ubiquitination and lysosomal and also these We have that activated PAR2 is the E3 ligase c-Cbl and to lysosomes an PubMed Scopus PubMed Scopus is the and of PAR2 the of AMSH and UBPY in regulating the deubiquitination, lysosomal trafficking, and degradation of the of PAR2 with and ERK2 activation. We that endosomal are of of certain GPCRs is signal for postendocytic sorting to lysosomes ubiquitination is of trafficking of the of which in ubiquitination and have the to the of identified endosomal AMSH and as of PAR2 ubiquitination and of the the of PAR2 ubiquitination and deubiquitination PAR2 ubiquitination of with or PAR2 in early endosomes of PubMed Scopus Thus, PAR2 is the and in early endosomes, which is with of PAR2 and c-Cbl PubMed Scopus PAR2 is between early endosomes and lysosomes, the receptor lysosomes in the of lysosomal are that ubiquitin to from receptors to their to lysosomes for degradation in to of ubiquitin PubMed Scopus Thus, PAR2 is in lysosomes, ubiquitination the of the for lysosomal sorting PubMed Scopus Ubiquitination of the where ubiquitination the receptor the and is not required for lysosomal sorting and degradation PubMed Scopus and UBPY are for deubiquitinating PAR2. PAR2 with AMSH in endosomes, and expression of catalytically inactive AMSH or UBPY of PAR2 and in early trafficking of to endosomes AMSH or UBPY in PubMed Scopus PubMed Scopus AMSH and UBPY have the of which with through PubMed Scopus PubMed Scopus PubMed Scopus to the association of PAR2 with AMSH and UBPY in endosomes is of or an the PAR2 with in early endosomes PubMed Scopus The is also the PubMed Scopus of AMSH or UBPY did not PAR2 trafficking, or that of AMSH and UBPY are in is with that the of AMSH and UBPY not ubiquitination PubMed Scopus of UBPY also been to ubiquitination PubMed Scopus of catalytically inactive AMSH or UBPY of PAR2 ubiquitination in caused of PAR2 in early endosomes, with which AMSH(D348A) and UBPY(C786S) are that their is for dissociation from endosomal AMSH(D348A) and UBPY(C786S) endosomal of proteins, such as or and of the endosomal sorting such as PubMed Scopus PubMed Scopus PubMed Scopus the of is AMSH(D348A) but not AMSH(D348A) or UBPY(C786S) also lysosomal trafficking and degradation of which deubiquitination of PAR2 in endosomes and receptor of that the of AMSH and UBPY is required for PAR2 deubiquitination and lysosomal trafficking and catalytically inactive are associated with endosomal AMSH or UBPY. the of AMSH or UBPY in PAR2 used of AMSH or UBPY PAR2 deubiquitination and which in PAR2 deubiquitination, AMSH or UBPY with the of the receptor of thereby lysosomal and as for the UBPY knockdown PubMed Scopus deubiquitination endosomal of PAR2 or which the sorting and receptor knockdown PAR2 trafficking from endosomes to lysosomes to in endosomal UBPY knockdown caused PAR2 in early UBPY knockdown in early endosomes the and of endosomal PubMed Scopus PubMed Scopus PubMed Scopus PubMed Scopus UBPY knockdown lysosomal trafficking and degradation of the which is to of of UBPY PubMed Scopus PubMed Scopus The of AMSH knockdown PAR2 degradation is to to in endosomal endosomes AMSH or UBPY knockdown the ubiquitination of endosomal sorting proteins, such as and Thus, the PAR2 to in the of sorting in UBPY knockdown that UBPY is to which for degradation PubMed Scopus PubMed Scopus AMSH in PubMed Scopus of in UBPY knockdown the in degradation PubMed Scopus UBPY AMSH knockdown not the of of the endosomal sorting such as PubMed Scopus PubMed Scopus Thus, the of AMSH knockdown PAR2 degradation is to to in the of the endosomal sorting AMSH is UBPY knockdown PubMed Scopus PubMed Scopus which the of UBPY PAR2. AMSH or UBPY knockdown of ubiquitin and the ubiquitination and lysosomal sorting of PAR2. of the protein which is to ubiquitin and and degradation of the PubMed Scopus the is required to ubiquitin from receptors to the PubMed Scopus UBPY knockdown in not of ubiquitin PubMed Scopus PubMed Scopus that in the of ubiquitin are to for lysosomal degradation of PAR2 UBPY We that AMSH or UBPY knockdown PAR2 deubiquitination and thereby of the receptor of and to lysosomes for the that AMSH and UBPY PAR2 and thereby lysosomal and degradation of the receptor of AMSH or UBPY these to have in PAR2 trafficking to PAR2 deubiquitination occurs of and the lysosomal sorting been been for the where AMSH and UBPY in PubMed Scopus UBPY knockdown degradation PubMed Scopus PubMed Scopus AMSH knockdown degradation PubMed Scopus that UBPY and AMSH degradation. AMSH an early of the sorting to ubiquitination to to the lysosomal and thereby the receptor to PubMed Scopus PubMed Scopus AMSH PAR2 an with lysosomal sorting and thereby PAR2 which not to the PAR2 that been AMSH an early c-Cbl and UBPY to Thus, the of ubiquitination and deubiquitination the which degradation AMSH or UBPY also that PAR2 trafficking to mediate of GPCRs to and to mediate and as for of PubMed Scopus Lefkowitz R.J. Science. PubMed Scopus PubMed Scopus Science. PubMed Scopus PubMed Scopus Lefkowitz R.J. Science. PubMed Scopus is in to and expression of (1Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (706) S.K. Lefkowitz R.J. PubMed Scopus is which is to the dissociation of from activated GPCRs and the of S.K. Lefkowitz R.J. PubMed Scopus Thus, that the trafficking and of activated PAR2 to ubiquitination and AMSH and UBPY also have in the down-regulation of that is and PubMed Scopus are to the mechanisms which AMSH and UBPY PAR2 and down-regulation to the of these also the ubiquitination of with the of the the E3 ligase to the to mediate receptor which is for lysosomal degradation and down-regulation chronic S.K. PubMed Scopus and ubiquitination and thereby down-regulation and and of S.K. PubMed Scopus Thus, AMSH and UBPY PAR2 and to receptor and to receptors to degradation and of from the to the and of PAR2 and to the endosomes PubMed Scopus PAR2 and mechanisms PubMed Scopus PAR2 the of signaling and activated PubMed Scopus of PAR2 is for of is unknown. PAR2 and in endosomes PAR2 and of an signaling that PAR2 in early endosomes expression of catalytically inactive AMSH and UBPY prolong PAR2 association with and PAR2-mediated activation. expression of catalytically inactive AMSH or UBPY did not of PAR2 and β-arrestin2 in endosomes, association of β-arrestin2 with endosomes, or PAR2-induced signaling. Thus, AMSH and UBPY not to these proteases trafficking and signaling of GPCRs and in and in endosomes to the which to the and receptor to the PubMed Scopus PubMed Scopus also PubMed Scopus the for PAR2 is to the endosomal proteases the to the and thereby of and PAR2 degradation to that PAR2 degradation and thereby the uncontrolled that to from PAR2 PubMed Scopus also to the of AMSH and UBPY in receptor down-regulation to GPCRs PAR2 and PubMed Scopus Ubiquitination of certain G protein-coupled receptors (GPCRs) 3The abbreviations used are: GPCRG protein-coupled receptorβ2ARβ2-adrenergic receptorDORδ-opioid receptorDUBdeubiquitinating enzymeEGFepidermal growth factorEGFRepidermal growth factor receptorERKextracellular signal-regulated kinaseMAPKmitogen activated protein kinaseMVBmultivesicular bodyPAR2protease-activated receptor 2pERKphosphorylated extracellular signal-regulated kinaseTfRtransferrin receptorUSPubiquitin-specific proteaseE3ubiquitin-protein isopeptide ligasesiRNAsmall interfering RNACFPcyan fluorescent proteinHEKhuman embryonic kidney 293PBSphosphate-buffered salineAPactivating peptideRIPAradioimmune precipitationHAhemagglutinin. 3The abbreviations used are: GPCRG protein-coupled receptorβ2ARβ2-adrenergic receptorDORδ-opioid receptorDUBdeubiquitinating enzymeEGFepidermal growth factorEGFRepidermal growth factor receptorERKextracellular signal-regulated kinaseMAPKmitogen activated protein kinaseMVBmultivesicular bodyPAR2protease-activated receptor 2pERKphosphorylated extracellular signal-regulated kinaseTfRtransferrin receptorUSPubiquitin-specific proteaseE3ubiquitin-protein isopeptide ligasesiRNAsmall interfering RNACFPcyan fluorescent proteinHEKhuman embryonic kidney 293PBSphosphate-buffered salineAPactivating peptideRIPAradioimmune precipitationHAhemagglutinin. is an essential signal for their postendocytic trafficking to lysosomes, which prevents uncontrolled signaling during chronic stimulation. Agonists stimulate ubiquitination of the β2-adrenergic receptor (β2AR), chemokine (CXC motif) receptor 4, and protease-activated receptor 2 (PAR2), and the E3 ubiquitin ligases that mediate ubiquitination of these GPCRs and associated proteins, such as β-arrestins, have been identified (1Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (706) PubMed Scopus PubMed Scopus ubiquitination of these receptors is not required for receptors postendocytic sorting to lysosomes and impaired of the of is the of deubiquitinating proteases in the postendocytic trafficking and signaling of G protein-coupled receptor β2-adrenergic receptor receptor deubiquitinating growth factor growth factor receptor extracellular signal-regulated activated protein protease-activated receptor 2 extracellular signal-regulated receptor isopeptide ligase interfering RNA fluorescent protein embryonic kidney G protein-coupled receptor β2-adrenergic receptor receptor deubiquitinating growth factor growth factor receptor extracellular signal-regulated activated protein protease-activated receptor 2 extracellular signal-regulated receptor isopeptide ligase interfering RNA fluorescent protein embryonic kidney of the of in postendocytic receptor trafficking from of receptor such as growth factor receptor endosomal AMSH with the of and UBPY as deubiquitination and postendocytic trafficking of PubMed Scopus AMSH to the of and for ubiquitin PubMed Scopus PubMed Scopus UBPY is of the and not between and ubiquitin PubMed Scopus PubMed Scopus the E3 ligase which ubiquitinates the receptor PubMed Scopus with the growth in early endosomes PubMed Scopus of the sorting required for that receptors in to that with lysosomes, where degradation occurs PubMed Scopus receptors are the are which to of ubiquitin PubMed Scopus AMSH and UBPY with through its PubMed Scopus PubMed Scopus PubMed Scopus The of ubiquitination c-Cbl and deubiquitination AMSH and UBPY the postendocytic trafficking and down-regulation of the c-Cbl lysosomal degradation of the PubMed Scopus AMSH c-Cbl and PubMed Scopus and UBPY is required for lysosomal sorting and degradation of PubMed Scopus PubMed Scopus PubMed Scopus PubMed Scopus The of AMSH and UBPY in regulating deubiquitination, trafficking, and signaling of GPCRs in endosomes is unknown. that AMSH and UBPY the down-regulation of the receptor that is and PubMed Scopus Expression of catalytically inactive of AMSH or UBPY or knockdown of AMSH or UBPY down-regulation of the of AMSH and UBPY in down-regulation to of and of did not have PubMed Scopus and have been to ubiquitination of the S.K. PubMed Scopus We the of AMSH and UBPY in the postendocytic trafficking, and lysosomal degradation of PAR2. We also AMSH and UBPY PAR2 association with in endosomes and extracellular signal-regulated activation. PAR2 is receptor for proteases that are during and PubMed Scopus PAR2 and and PAR2 to of the and PAR2 are during to expression or receptor which the of proteases PubMed Scopus the of and the receptor the of protein to endosomes PubMed Scopus of PAR2 signaling receptor degradation in lysosomes, which in is PubMed Scopus S.K. PubMed Scopus is to mechanisms of PAR2 ubiquitination and lysosomal and also these We have that activated PAR2 is the E3 ligase c-Cbl and to lysosomes an PubMed Scopus PubMed Scopus is the and of PAR2 the of AMSH and UBPY in regulating the deubiquitination, lysosomal trafficking, and degradation of the of PAR2 with and ERK2 activation. We that endosomal are of of certain GPCRs is signal for postendocytic sorting to lysosomes ubiquitination is of trafficking of the of which in ubiquitination and have the to the of identified endosomal AMSH and as of PAR2 ubiquitination and of the the of PAR2 ubiquitination and deubiquitination PAR2 ubiquitination of with or PAR2 in early endosomes of PubMed Scopus Thus, PAR2 is the and in early endosomes, which is with of PAR2 and c-Cbl PubMed Scopus PAR2 is between early endosomes and lysosomes, the receptor lysosomes in the of lysosomal are that ubiquitin to from receptors to their to lysosomes for degradation in to of ubiquitin PubMed Scopus Thus, PAR2 is in lysosomes, ubiquitination the of the for lysosomal sorting PubMed Scopus Ubiquitination of the where ubiquitination the receptor the and is not required for lysosomal sorting and degradation PubMed Scopus and UBPY are for deubiquitinating PAR2. PAR2 with AMSH in endosomes, and expression of catalytically inactive AMSH or UBPY of PAR2 and in early trafficking of to endosomes AMSH or UBPY in PubMed Scopus PubMed Scopus AMSH and UBPY have the of which with through PubMed Scopus PubMed Scopus PubMed Scopus to the association of PAR2 with AMSH and UBPY in endosomes is of or an the PAR2 with in early endosomes PubMed Scopus The is also the PubMed Scopus of AMSH or UBPY did not PAR2 trafficking, or that of AMSH and UBPY are in is with that the of AMSH and UBPY not ubiquitination PubMed Scopus of UBPY also been to ubiquitination PubMed Scopus of catalytically inactive AMSH or UBPY of PAR2 ubiquitination in caused of PAR2 in early endosomes, with which AMSH(D348A) and UBPY(C786S) are that their is for dissociation from endosomal AMSH(D348A) and UBPY(C786S) endosomal of proteins, such as or and of the endosomal sorting such as PubMed Scopus PubMed Scopus PubMed Scopus the of is AMSH(D348A) but not AMSH(D348A) or UBPY(C786S) also lysosomal trafficking and degradation of which deubiquitination of PAR2 in endosomes and receptor of that the of AMSH and UBPY is required for PAR2 deubiquitination and lysosomal trafficking and catalytically inactive are associated with endosomal AMSH or UBPY. the of AMSH or UBPY in PAR2 used of AMSH or UBPY PAR2 deubiquitination and which in PAR2 deubiquitination, AMSH or UBPY with the of the receptor of thereby lysosomal and as for the UBPY knockdown PubMed Scopus deubiquitination endosomal of PAR2 or which the sorting and receptor knockdown PAR2 trafficking from endosomes to lysosomes to in endosomal UBPY knockdown caused PAR2 in early UBPY knockdown in early endosomes the and of endosomal PubMed Scopus PubMed Scopus PubMed Scopus PubMed Scopus UBPY knockdown lysosomal trafficking and degradation of the which is to of of UBPY PubMed Scopus PubMed Scopus The of AMSH knockdown PAR2 degradation is to to in endosomal endosomes AMSH or UBPY knockdown the ubiquitination of endosomal sorting proteins, such as and Thus, the PAR2 to in the of sorting in UBPY knockdown that UBPY is to which for degradation PubMed Scopus PubMed Scopus AMSH in PubMed Scopus of in UBPY knockdown the in degradation PubMed Scopus UBPY AMSH knockdown not the of of the endosomal sorting such as PubMed Scopus PubMed Scopus Thus, the of AMSH knockdown PAR2 degradation is to to in the of the endosomal sorting AMSH is UBPY knockdown PubMed Scopus PubMed Scopus which the of UBPY PAR2. AMSH or UBPY knockdown of ubiquitin and the ubiquitination and lysosomal sorting of PAR2. of the protein which is to ubiquitin and and degradation of the PubMed Scopus the is required to ubiquitin from receptors to the PubMed Scopus UBPY knockdown in not of ubiquitin PubMed Scopus PubMed Scopus that in the of ubiquitin are to for lysosomal degradation of PAR2 UBPY We that AMSH or UBPY knockdown PAR2 deubiquitination and thereby of the receptor of and to lysosomes for the that AMSH and UBPY PAR2 and thereby lysosomal and degradation of the receptor of AMSH or UBPY these to have in PAR2 trafficking to PAR2 deubiquitination occurs of and the lysosomal sorting been been for the where AMSH and UBPY in PubMed Scopus UBPY knockdown degradation PubMed Scopus PubMed Scopus AMSH knockdown degradation PubMed Scopus that UBPY and AMSH degradation. AMSH an early of the sorting to ubiquitination to to the lysosomal and thereby the receptor to PubMed Scopus PubMed Scopus AMSH PAR2 an with lysosomal sorting and thereby PAR2 which not to the PAR2 that been AMSH an early c-Cbl and UBPY to Thus, the of ubiquitination and deubiquitination the which degradation AMSH or UBPY also that PAR2 trafficking to mediate of GPCRs to and to mediate and as for of PubMed Scopus Lefkowitz R.J. Science. PubMed Scopus PubMed Scopus Science. PubMed Scopus PubMed Scopus Lefkowitz R.J. Science. PubMed Scopus is in to and expression of (1Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (706) S.K. Lefkowitz R.J. PubMed Scopus is which is to the dissociation of from activated GPCRs and the of S.K. Lefkowitz R.J. PubMed Scopus Thus, that the trafficking and of activated PAR2 to ubiquitination and AMSH and UBPY also have in the down-regulation of that is and PubMed Scopus are to the mechanisms which AMSH and UBPY PAR2 and down-regulation to the of these also the ubiquitination of with the of the the E3 ligase to the to mediate receptor which is for lysosomal degradation and down-regulation chronic S.K. PubMed Scopus and ubiquitination and thereby down-regulation and and of S.K. PubMed Scopus Thus, AMSH and UBPY PAR2 and to receptor and to receptors to degradation and of from the to the and of PAR2 and to the endosomes PubMed Scopus PAR2 and mechanisms PubMed Scopus PAR2 the of signaling and activated PubMed Scopus of PAR2 is for of is unknown. PAR2 and in endosomes PAR2 and of an signaling that PAR2 in early endosomes expression of catalytically inactive AMSH and UBPY prolong PAR2 association with and PAR2-mediated activation. expression of catalytically inactive AMSH or UBPY did not of PAR2 and β-arrestin2 in endosomes, association of β-arrestin2 with endosomes, or PAR2-induced signaling. Thus, AMSH and UBPY not to these proteases trafficking and signaling of GPCRs and in and in endosomes to the which to the and receptor to the PubMed Scopus PubMed Scopus also PubMed Scopus the for PAR2 is to the endosomal proteases the to the and thereby of and PAR2 degradation to that PAR2 degradation and thereby the uncontrolled that to from PAR2 PubMed Scopus also to the of AMSH and UBPY in receptor down-regulation to GPCRs PAR2 and PubMed Scopus Ubiquitination of certain GPCRs is signal for postendocytic sorting to lysosomes ubiquitination is of trafficking of the of which in ubiquitination and have the to the of identified endosomal AMSH and as of PAR2 ubiquitination and of the the of PAR2 ubiquitination and deubiquitination PAR2 ubiquitination of with or PAR2 in early endosomes of PubMed Scopus Thus, PAR2 is the and in early endosomes, which is with of PAR2 and c-Cbl PubMed Scopus PAR2 is between early endosomes and lysosomes, the receptor lysosomes in the of lysosomal are that ubiquitin to from receptors to their to lysosomes for degradation in to of ubiquitin PubMed Scopus Thus, PAR2 is in lysosomes, ubiquitination the of the for lysosomal sorting PubMed Scopus Ubiquitination of the where ubiquitination the receptor the and is not required for lysosomal sorting and degradation PubMed Scopus AMSH and UBPY are for deubiquitinating PAR2. PAR2 with AMSH in endosomes, and expression of catalytically inactive AMSH or UBPY of PAR2 and in early trafficking of to endosomes AMSH or UBPY in PubMed Scopus PubMed Scopus AMSH and UBPY have the of which with through PubMed Scopus PubMed Scopus PubMed Scopus to the association of PAR2 with AMSH and UBPY in endosomes is of or an the PAR2 with in early endosomes PubMed Scopus The is also the PubMed Scopus of AMSH or UBPY did not PAR2 trafficking, or that of AMSH and UBPY are in is with that the of AMSH and UBPY not ubiquitination PubMed Scopus of UBPY also been to ubiquitination PubMed Scopus of catalytically inactive AMSH or UBPY of PAR2 ubiquitination in caused of PAR2 in early endosomes, with which AMSH(D348A) and UBPY(C786S) are that their is for dissociation from endosomal AMSH(D348A) and UBPY(C786S) endosomal of proteins, such as or and of the endosomal sorting such as PubMed Scopus PubMed Scopus PubMed Scopus the of is AMSH(D348A) but not AMSH(D348A) or UBPY(C786S) also lysosomal trafficking and degradation of which deubiquitination of PAR2 in endosomes and receptor of that the of AMSH and UBPY is required for PAR2 deubiquitination and lysosomal trafficking and degradation. catalytically inactive are associated with endosomal AMSH or UBPY. the of AMSH or UBPY in PAR2 used of AMSH or UBPY PAR2 deubiquitination and which in PAR2 deubiquitination, AMSH or UBPY with the of the receptor of thereby lysosomal and as for the UBPY knockdown PubMed Scopus deubiquitination endosomal of PAR2 or which the sorting and receptor knockdown PAR2 trafficking from endosomes to lysosomes to in endosomal UBPY knockdown caused PAR2 in early UBPY knockdown in early endosomes the and of endosomal PubMed Scopus PubMed Scopus PubMed Scopus PubMed Scopus UBPY knockdown lysosomal trafficking and degradation of the which is to of of UBPY PubMed Scopus PubMed Scopus The of AMSH knockdown PAR2 degradation is to to in endosomal endosomes AMSH or UBPY knockdown the ubiquitination of endosomal sorting proteins, such as and Thus, the PAR2 to in the of sorting in UBPY knockdown that UBPY is to which for degradation PubMed Scopus PubMed Scopus AMSH in PubMed Scopus of in UBPY knockdown the in degradation PubMed Scopus UBPY AMSH knockdown not the of of the endosomal sorting such as PubMed Scopus PubMed Scopus Thus, the of AMSH knockdown PAR2 degradation is to to in the of the endosomal sorting AMSH is UBPY knockdown PubMed Scopus PubMed Scopus which the of UBPY PAR2. AMSH or UBPY knockdown of ubiquitin and the ubiquitination and lysosomal sorting of PAR2. of the protein which is to ubiquitin and and degradation of the PubMed Scopus the is required to ubiquitin from receptors to the PubMed Scopus UBPY knockdown in not of ubiquitin PubMed Scopus PubMed Scopus that in the of ubiquitin are to for lysosomal degradation of PAR2 UBPY We that AMSH or UBPY knockdown PAR2 deubiquitination and thereby of the receptor of and to lysosomes for degradation. the that AMSH and UBPY PAR2 and thereby lysosomal and degradation of the receptor of AMSH or UBPY these to have in PAR2 trafficking to PAR2 deubiquitination occurs of and the lysosomal sorting been been for the where AMSH and UBPY in PubMed Scopus UBPY knockdown degradation PubMed Scopus PubMed Scopus AMSH knockdown degradation PubMed Scopus that UBPY and AMSH degradation. AMSH an early of the sorting to ubiquitination to to the lysosomal and thereby the receptor to PubMed Scopus PubMed Scopus AMSH PAR2 an with lysosomal sorting and thereby PAR2 which not to the PAR2 that been AMSH an early c-Cbl and UBPY to Thus, the of ubiquitination and deubiquitination the which degradation AMSH or UBPY also that PAR2 trafficking to mediate of GPCRs to and to mediate and as for of PubMed Scopus Lefkowitz R.J. Science. PubMed Scopus PubMed Scopus Science. PubMed Scopus PubMed Scopus Lefkowitz R.J. Science. PubMed Scopus is in to and expression of (1Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (706) S.K. Lefkowitz R.J. PubMed Scopus is which is to the dissociation of from activated GPCRs and the of S.K. Lefkowitz R.J. PubMed Scopus Thus, that the trafficking and of activated PAR2 to ubiquitination and AMSH and UBPY also have in the down-regulation of that is and PubMed Scopus are to the mechanisms which AMSH and UBPY PAR2 and down-regulation to the of these also the ubiquitination of with the of the the E3 ligase to the to mediate receptor which is for lysosomal degradation and down-regulation chronic S.K. PubMed Scopus and ubiquitination and thereby down-regulation and and of S.K. PubMed Scopus Thus, AMSH and UBPY PAR2 and to receptor and to receptors to degradation and PAR2 of from the to the and of PAR2 and to the endosomes PubMed Scopus PAR2 and mechanisms PubMed Scopus PAR2 the of signaling and activated PubMed Scopus of PAR2 is for of is unknown. PAR2 and in endosomes PAR2 and of an signaling that PAR2 in early endosomes expression of catalytically inactive AMSH and UBPY prolong PAR2 association with and PAR2-mediated activation. expression of catalytically inactive AMSH or UBPY did not of PAR2 and β-arrestin2 in endosomes, association of β-arrestin2 with endosomes, or PAR2-induced signaling. Thus, AMSH and UBPY not to these proteases trafficking and signaling of GPCRs and in and in endosomes to the which to the and receptor to the PubMed Scopus PubMed Scopus also PubMed Scopus the for PAR2 is to the endosomal proteases the to the and thereby signaling. The of and PAR2 degradation to that PAR2 degradation and thereby the uncontrolled that to from PAR2 PubMed Scopus also to the of AMSH and UBPY in receptor down-regulation to GPCRs PAR2 and PubMed Scopus with with