Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins
Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins
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DOI：
10.1074/jbc.m115.702993
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发表时间：
2016-01-29
影响因子：
4.8
通讯作者：
Trejo, JoAnn
中科院分区：
文献类型：
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作者：
Grimsey, Neil J.;Coronel, Luisa J.;Trejo, JoAnn
蛋白酶激活受体-1（PAR 1）是一种G蛋白偶联受体，可被凝血和抗凝蛋白酶不可逆地蛋白水解激活。鉴于PAR 1的不可逆活化，受体的信号传导通过脱敏和细胞内运输受到严格调节。PAR 1显示组成性和激动剂诱导的内化。PAR 1的组成性内化对于产生补充细胞表面并促进再敏感的幼稚受体的内部池是重要的，而激动剂诱导的PAR 1的内化对于终止G蛋白信号传导是关键的。我们发现PAR 1的组成性内化是由衔接蛋白复合物-2（AP-2）介导的，而AP-2和epsin控制激动剂诱导的PAR 1内化。然而，调节PAR 1再循环的机制尚不清楚。在本研究中，我们筛选了140种不同的膜运输蛋白的siRNA文库，以确定PAR 1细胞内运输的关键调节因子。除了已知的PAR 1内吞作用的介质，我们确定Rab 11B作为PAR 1运输的关键调节因子。我们发现，siRNA介导的Rab 11B而不是Rab 11 A的耗竭阻断了PAR 1的再循环，这增强了受体溶酶体的降解。尽管Rab 11 A不是PAR 1回收所需的，但Rab 11 A的耗尽通过破坏受体的基础溶酶体降解而导致PAR 1的细胞内积累。此外，在Rab 11B缺陷细胞中观察到的PAR 1的增强降解被Rab 11 A和自噬相关-5蛋白的耗尽所阻断，表明PAR 1在Rab 11B再循环的情况下穿梭于自噬降解途径。总之，这些发现表明，Rab 11 A和Rab 11B差异调节PAR 1通过不同的内体分选机制的细胞内运输。
Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that undergoes proteolytic irreversible activation by coagulant and anti-coagulant proteases. Given the irreversible activation of PAR1, signaling by the receptor is tightly regulated through desensitization and intracellular trafficking. PAR1 displays both constitutive and agonist-induced internalization. Constitutive internalization of PAR1 is important for generating an internal pool of naive receptors that replenish the cell surface and facilitate resensitization, whereas agonist-induced internalization of PAR1 is critical for terminating G protein signaling. We showed that PAR1 constitutive internalization is mediated by the adaptor protein complex-2 (AP-2), whereas AP-2 and epsin control agonist-induced PAR1 internalization. However, the mechanisms that regulate PAR1 recycling are not known. In the present study we screened a siRNA library of 140 different membrane trafficking proteins to identify key regulators of PAR1 intracellular trafficking. In addition to known mediators of PAR1 endocytosis, we identified Rab11B as a critical regulator of PAR1 trafficking. We found that siRNA-mediated depletion of Rab11B and not Rab11A blocks PAR1 recycling, which enhanced receptor lysosomal degradation. Although Rab11A is not required for PAR1 recycling, depletion of Rab11A resulted in intracellular accumulation of PAR1 through disruption of basal lysosomal degradation of the receptor. Moreover, enhanced degradation of PAR1 observed in Rab11B-deficient cells is blocked by depletion of Rab11A and the autophagy related-5 protein, suggesting that PAR1 is shuttled to an autophagic degradation pathway in the absence of Rab11B recycling. Together these findings suggest that Rab11A and Rab11B differentially regulate intracellular trafficking of PAR1 through distinct endosomal sorting mechanisms.