Gullapalli Anuradha, Wolfe Breann L, Griffin Courtney T, Magnuson Terry, Trejo JoAnn
Department of Pharmacology, University of North Carolina at Chapel Hill, NC 27599-7365, USA.
Mol Biol Cell. 2006 Mar;17(3):1228-38. doi: 10.1091/mbc.e05-09-0899. Epub 2006 Jan 11.
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.
分选连接蛋白1（SNX1）和SNX2是酵母Vps5p逆向转运复合物成分的哺乳动物同源物，该复合物在从内体到高尔基体的运输过程中发挥作用。SNX1也参与细胞表面受体从内体到溶酶体的分选，尽管其在这一过程中的必要性仍有待确定。为了评估SNX1在蛋白酶激活受体-1（PAR1）内吞分选过程中的功能，我们使用小干扰RNA（siRNA）耗尽HeLa细胞中的内源性SNX1蛋白。PAR1是一种G蛋白偶联受体，可被凝血酶蛋白水解激活，内化后主要分选至溶酶体并有效降解。引人注目的是，通过siRNA耗尽内源性SNX1可显著抑制激动剂诱导的PAR1降解，而在缺乏内源性SNX1的细胞中，表达对SNX1 siRNA有抗性的突变蛋白可恢复激动剂促进的PAR1降解，这表明SNX1是PAR1溶酶体降解所必需的。已知SNX1与哺乳动物逆向转运复合物的成分以及Hrs（一种早期内体膜相关蛋白）相互作用。然而，在耗尽逆向转运复合物Vps26/Vps35亚基、Hrs或与Hrs相互作用的蛋白Tsg101的细胞中，激活的PAR1降解并未受到影响。我们进一步表明，与SNX1二聚化的SNX2对于PAR1的溶酶体分选并非必不可少，但在异位表达时，它可以通过破坏内源性SNX1的内体定位来调节PAR1降解。总之，我们的研究结果确定了内源性SNX1在将激活的PAR1分选至一条独立于逆向转运复合物、Hrs和Tsg101的独特溶酶体降解途径中的重要作用。
