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Vol. 17, Issue 3, 1228-1238, March 2006
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* Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365;
Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365; and
Department of Genetics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365
Submitted September 28, 2005;
Revised December 16, 2005;
Accepted January 3, 2006
Monitoring Editor: Sandra Schmid
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.
Abbreviations used: CI-MPR, cation-independent mannose 6-phosphate receptor; EGFR, epidermal growth factor receptor; GPCR, G protein-coupled receptor; Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; LAMP1, lysosomal-associated membrane protein-1; PAR1, protease-activated receptor-1; PX, phox homology domain; siRNA, small interfering RNA; SNX, sorting nexin; TGN, trans-Golgi network; Tsg101, tumor susceptibility gene 101; Vps, vacuolar protein sorting.
Address correspondence to: JoAnn Trejo (joann_trejo{at}med.unc.edu).
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