Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals
Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals
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DOI：
10.1074/jbc.m111.299776
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发表时间：
2011-11-25
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：
Chen, Buxin;Dores, Michael R.;Trejo, JoAnn
凝血酶的G蛋白偶联受体（GPCR）蛋白酶激活受体1（PAR 1）的信号传导受脱敏和内化的调节。PAR 1脱敏由β-抑制蛋白介导，与大多数经典GPCR一样。相反，PAR 1的内化通过独立于β-抑制蛋白的网格蛋白和动力蛋白依赖性途径发生。PAR 1表现出两种内化模式。未活化的PAR 1的组成性内化由网格蛋白衔接蛋白复合物-2（AP-2）介导，其中μ 2-衔接蛋白亚基直接结合到位于受体C-尾结构域内的基于酪氨酸的基序。然而，AP-2消耗仅部分抑制激动剂诱导的PAR 1内化，表明其它网格蛋白衔接子在该过程中的功能。在这里，我们现在报告AP-2和epsin-1都是激动剂刺激的PAR 1内化的关键介质。我们表明PAR 1的泛素化和epsin-1的泛素相互作用基序是激活的PAR 1的epsin-1依赖性内化所必需的。此外，PAR 1的活化促进epsin-1去泛素化，这可能增加其内吞衔接子活性以促进受体内化。AP-2还通过识别远端C-尾磷酸化位点而不是典型的基于酪氨酸的基序来调节活化的PAR 1内化。因此，AP-2和epsin-1都需要促进激活的PAR 1的有效内化和识别离散的受体分选信号。本研究为哺乳动物GPCR的内化确定了一条新的途径。
Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by beta-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of beta-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the mu 2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR!, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization, We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-l-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs.