N-Linked Glycosylation of Protease-activated Receptor-1 Second Extracellular Loop A CRITICAL DETERMINANT FOR LIGAND-INDUCED RECEPTOR ACTIVATION AND INTERNALIZATION
N-Linked Glycosylation of Protease-activated Receptor-1 Second Extracellular Loop A CRITICAL DETERMINANT FOR LIGAND-INDUCED RECEPTOR ACTIVATION AND INTERNALIZATION
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DOI：
10.1074/jbc.m110.111088
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发表时间：
2010-06-11
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：
Soto, Antonio G.;Trejo, JoAnn
蛋白酶激活受体1（PAR 1）含有5个N-连接糖基化共有位点，如下所示：3个位于N末端，2个位于第二胞外环（ECL 2）表面。为了研究N-连接的糖基化在PAR 1信号传导和运输调节中的作用，我们产生了突变体，其中共有位点的关键天冬酰胺发生突变。在这里，我们报告，PAR 1 N末端和ECL 2作为N-连接糖基化的网站，但在受体信号转导和贩运的调节有不同的功能。PAR 1 N末端的N-连接糖基化对于转运至细胞表面是重要的，而在ECL 2处缺乏糖基化的PAR 1突变体（NA ECL 2）像野生型受体一样转运至细胞表面。然而，激活PAR 1 NA ECL 2突变体的内化与野生型受体相比受损，而未激活的受体的组成性内化保持完整。值得注意的是，凝血酶激活的PAR 1 NA ECL 2突变体与野生型受体相比显示出增强的最大信号传导反应。增加的PAR 1 NA ECL 2突变体信号传导不是由于凝血酶切割受体的能力或信号终止机制的缺陷。相反，PAR 1 NA ECL 2突变体在凝血酶刺激的G蛋白信号传导中显示出更大的功效。因此，PAR 1细胞外表面的N-连接糖基化可能影响配体对接相互作用和活性受体构象的稳定性。总之，这些研究强烈表明，PAR 1在N末端的N-连接糖基化与ECL 2表面的N-连接糖基化具有不同的功能，对于受体运输的适当调节和凝血酶信号传导的保真度至关重要。
Protease-activated receptor-1 (PAR1) contains five N-linked glycosylation consensus sites as follows: three residing in the N terminus and two localized on the surface of the second extracellular loop (ECL2). To study the effect of N-linked glycosylation in the regulation of PAR1 signaling and trafficking, we generated mutants in which the critical asparagines of the consensus sites were mutated. Here, we report that both the PAR1 N terminus and ECL2 serve as sites for N-linked glycosylation but have different functions in the regulation of receptor signaling and trafficking. N-Linked glycosylation of the PAR1 N terminus is important for transport to the cell surface, whereas the PAR1 mutant lacking glycosylation at ECL2 (NA ECL2) trafficked to the cell surface like the wild-type receptor. However, activated PAR1 NA ECL2 mutant internalization was impaired compared with wild-type receptor, whereas constitutive internalization of unactivated receptor remained intact. Remarkably, thrombin-activated PAR1 NA ECL2 mutant displayed an enhanced maximal signaling response compared with wild-type receptor. The increased PAR1 NA ECL2 mutant signaling was not due to defects in the ability of thrombin to cleave the receptor or signal termination mechanisms. Rather, the PAR1 NA ECL2 mutant displayed a greater efficacy in thrombin-stimulated G protein signaling. Thus, N-linked glycosylation of the PAR1 extracellular surface likely influences ligand docking interactions and the stability of the active receptor conformation. Together, these studies strongly suggest that N-linked glycosylation of PAR1 at the N terminus versus the surface of ECL2 serves distinct functions critical for proper regulation of receptor trafficking and the fidelity of thrombin signaling.