An essential role for SNX1 in lysosomal sorting of protease-activated receptor-1: evidence for retromer-, Hrs-, and Tsg101-independent functions of sorting nexins.
Sorting nexin 1 (SNX1) and SNX2 are the mammalian homologues of the yeast Vps5p retromer component that functions in endosome-to-Golgi trafficking. SNX1 is also implicated in endosome-to-lysosome sorting of cell surface receptors, although its requirement in this process remains to be determined. To assess SNX1 function in endocytic sorting of protease-activated receptor-1 (PAR1), we used siRNA to deplete HeLa cells of endogenous SNX1 protein. PAR1, a G-protein-coupled receptor, is proteolytically activated by thrombin, internalized, sorted predominantly to lysosomes, and efficiently degraded. Strikingly, depletion of endogenous SNX1 by siRNA markedly inhibited agonist-induced PAR1 degradation, whereas expression of a SNX1 siRNA-resistant mutant protein restored agonist-promoted PAR1 degradation in cells lacking endogenous SNX1, indicating that SNX1 is necessary for lysosomal degradation of PAR1. SNX1 is known to interact with components of the mammalian retromer complex and Hrs, an early endosomal membrane-associated protein. However, activated PAR1 degradation was not affected in cells depleted of retromer Vps26/Vps35 subunits, Hrs or Tsg101, an Hrs-interacting protein. We further show that SNX2, which dimerizes with SNX1, is not essential for lysosomal sorting of PAR1, but rather can regulate PAR1 degradation by disrupting endosomal localization of endogenous SNX1 when ectopically expressed. Together, our findings establish an essential role for endogenous SNX1 in sorting activated PAR1 to a distinct lysosomal degradative pathway that is independent of retromer, Hrs, and Tsg101.
Gullapalli
Anuradha
A
Department of Pharmacology, University of North Carolina at Chapel Hill, NC 27599-7365, USA.
Wolfe
Breann L
BL
Griffin
Courtney T
CT
Magnuson
Terry
T
Trejo
JoAnn
J
D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010750:Phosphoproteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D044463:Receptor, PAR-1 / Q000819:agonists / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D058305:Sorting Nexins; D014157:Transcription Factors / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D000099177:Tsg101 Protein
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D044463:Receptor, PAR-1; D011993:Recombinant Fusion Proteins; D058305:Sorting Nexins; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; D000099177:Tsg101 Protein; C513978:SNX1 protein, human; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate