Lipid raft localization regulates the cleavage specificity of protease activated receptor 1 in endothelial cells
Lipid raft localization regulates the cleavage specificity of protease activated receptor 1 in endothelial cells
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DOI：
10.1111/j.1538-7836.2008.02924.x
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发表时间：
2008-06-01
影响因子：
10.4
通讯作者：
Rezaie, A. R.
中科院分区：
文献类型：
--
作者：
Bae, J. -S.;Yang, L.;Rezaie, A. R.
背景：脂筏中的活化蛋白 C (APC) 或凝血酶对蛋白酶激活受体 1 (PAR-1) 进行内皮蛋白 C 受体 (EPCR) 依赖性裂解，从而启动内皮细胞中的保护性信号反应。目的：研究 APC 和凝血酶与脂筏中的 PAR-1 相互作用并裂解 PAR-1 的机制。方法：我们构建了两种类型的 PAR-1 裂解报告基因构建体，其中分泌型碱性磷酸酶 (ALP) 融合到 PAR-1 的胞外结构域。第一个构建体具有能够将融合蛋白均匀锚定到膜表面的跨膜结构域，而第二个构建体具有用于将融合蛋白靶向转染细胞中的脂筏/小凹的识别序列。结果：在用第一个构建体转染的 HUVEC 中，APC 和 Gla-domainless (GD)-APC 均以相似的效率切割 PAR-1 外域。与 APC 不同，GD-APC 不会在转染第二种构建体的细胞中裂解 PAR-1；然而，之前用蛋白 C 或凝血酶的 S195A 突变体处理细胞会导致 PAR-1 的 GD-APC 裂解具有相当或更高的催化效率。如果在 TNF-α 诱导的内皮细胞凋亡和通透性测定中监测 APC 和 GD-APC 的细胞信号传导特性，则会获得相同的结果。结论：脂筏定位使得 PAR-1 外域的易剪键无法与凝血蛋白酶相互作用。蛋白 C 的 Gla 结构域与 EPCR 或凝血酶的外切位点 1 与 PAR-1 的 C 端水蛭素样序列的结合改变了 PAR-1 外切结构域的膜定位和/或构象，以促进其识别和随后被这些蛋白酶切割。
Background: The endothelial protein C receptor (EPCR)-dependent cleavage of protease activated receptor 1 (PAR-1) by either activated protein C (APC) or thrombin in lipid rafts initiates protective signaling responses in endothelial cells. Objectives: To investigate the mechanism by which APC and thrombin interact with and cleave PAR-1 in lipid rafts. Methods: We constructed two types of PAR-1 cleavage reporter constructs in which a secreted alkaline phosphatase (ALP) was fused to the extracellular domain of PAR-1. The first construct has a transmembrane domain capable of uniformly anchoring the fusion protein to the membrane surface, while the second construct has the recognition sequence for targeting the fusion protein to lipid rafts/caveolae in transfected cells. Results: Both APC and the Gla-domainless (GD)-APC cleaved the PAR-1 exodomain with similar efficiency in HUVECs transfected with the first construct. Unlike APC, GD-APC did not cleave PAR-1 in cells transfected with the second construct; however, prior treatment of cells with S195A mutants of either protein C or thrombin led to the GD-APC cleavage of PAR-1 with a comparable or higher catalytic efficiency. The same results were obtained if the cellular signaling properties of APC and GD-APC were monitored in the TNF-alpha-induced endothelial cell apoptosis and permeability assays. Conclusions: The lipid raft localization renders the scissile bond of the PAR-1 exodomain unavailable for interaction with coagulation proteases. The binding of either the Gla-domain of protein C to EPCR or exosite-1 of thrombin to the C-terminal hirudin-like sequence of PAR-1 changes the membrane localization and/or the conformation of the PAR-1 exodomain to facilitate its recognition and subsequent cleavage by these proteases.