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 (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.
Hasdemir
Burcu
B
Department of Surgery and Physiology, University of California, San Francisco, California 94143-0660.
Murphy
Jane E
JE
Department of Surgery and Physiology, University of California, San Francisco, California 94143-0660.
Cottrell
Graeme S
GS
Department of Surgery and Physiology, University of California, San Francisco, California 94143-0660.
Bunnett
Nigel W
NW
Department of Surgery and Physiology, University of California, San Francisco, California 94143-0660. Electronic address: nigel.bunnett@ucsf.edu.
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
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
J Biol Chem. 2000 Dec 1;275(48):37481-7
10982817
Science. 1990 Jun 22;248(4962):1547-50
2163110
J Biol Chem. 1999 Jun 25;274(26):18524-35
10373461
J Cell Biol. 2007 Jun 4;177(5):905-16
17535965
J Biol Chem. 2006 May 5;281(18):12618-24
16520378
EMBO J. 2009 Jun 17;28(12):1684-96
19424180
J Cell Biol. 2004 Aug 16;166(4):487-92
15314065
Nat Rev Mol Cell Biol. 2007 May;8(5):355-68
17450176
Biochem J. 1994 Oct 1;303 ( Pt 1):177-86
7524481
Traffic. 2002 Feb;3(2):110-23
11929601
Mol Biol Cell. 2005 Nov;16(11):5163-74
16120644
Trends Cell Biol. 2006 Nov;16(11):551-9
16996268
Science. 1999 Jan 29;283(5402):655-61
9924018
Proc Natl Acad Sci U S A. 2009 Apr 21;106(16):6650-5
19363159
Biochem Soc Trans. 2006 Nov;34(Pt 5):754-6
17052190
Science. 1996 Jan 19;271(5247):363-6
8553074
J Biol Chem. 2007 Oct 5;282(40):29646-57
17675298
Genes Dev. 1998 Dec 1;12(23):3663-74
9851973
Dev Cell. 2003 Nov;5(5):709-22
14602072
Traffic. 2006 Aug;7(8):1017-31
16771824
Mol Cell Biol. 2007 Jul;27(13):5029-39
17452457
J Biol Chem. 2003 Apr 18;278(16):14498-506
12574160
J Biol Chem. 2007 Jan 19;282(3):1658-69
17121848
J Biol Chem. 1996 Sep 6;271(36):22003-16
8703006
J Biol Chem. 2005 Apr 22;280(16):16076-87
15708858
Nature. 1996 Oct 3;383(6599):447-50
8837779
Traffic. 2005 Apr;6(4):324-34
15752137
J Cell Biol. 2000 Mar 20;148(6):1267-81
10725339
Mol Biol Cell. 2000 Oct;11(10):3365-80
11029042
Biochem J. 2002 Dec 1;368(Pt 2):495-505
12171601
J Biol Chem. 1997 Dec 26;272(52):32785-91
9407053
J Biol Chem. 1996 Jun 21;271(25):14910-5
8663011
J Biol Chem. 2009 Aug 14;284(33):22411-22425
19531493
Am J Respir Cell Mol Biol. 2003 Mar;28(3):339-46
12594060
J Biol Chem. 2008 Aug 8;283(32):22166-76
18544533
Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):11086-91
10995467
Nature. 1996 Feb 8;379(6565):557-60
8596637
J Cell Biol. 2007 Dec 3;179(5):981-97
18039931
Proc Natl Acad Sci U S A. 2007 Jul 10;104(28):11838-43
17592116
EMBO J. 1998 Jun 15;17(12):3241-50
9628861
J Mol Endocrinol. 2003 Apr;30(2):117-26
12683936
J Cell Sci. 2003 Oct 15;116(Pt 20):4169-79
12953068
J Biol Chem. 1999 Jul 2;274(27):19129-35
10383417
Physiol Rev. 2004 Apr;84(2):579-621
15044683
Science. 2001 Nov 9;294(5545):1307-13
11588219
J Biol Chem. 2009 Jul 17;284(29):19361-70
19433584
Curr Biol. 2006 Jan 24;16(2):160-5
16431367
J Biol Chem. 2006 Feb 24;281(8):5094-105
16371348