Tissue factor (TF) activity is stringently regulated through processes termed encryption. Post-translational modification of TF and its interactions with various protein and lipid moieties allows for a multi-step de-encryption of TF and procoagulant activation. Membrane-associated guanylate kinase-with inverted configuration (MAGI) proteins are known to regulate the localisation and activity of a number of proteins including cell-surface receptors. The interaction of TF with MAGI1 protein was examined as a means of regulating TF activity. MDA-MB-231 cell line was used which express TF and MAGI1, and respond well to protease activated receptor (PAR)2 activation. Proximity ligation assay (PLA), co-immunoprecipitation and pull-down experiments were used to examine the interaction of TF with MAGI1-3 proteins and to investigate the influence of PAR2 activation. Furthermore, by cloning and expressing the PDZ domains from MAGI1, the TF-binding domain was identified. The ability of the recombinant PDZ domains to act as competitors for MAGI1, allowing the induction of TF procoagulant and signalling activity was then examined. PLA and fluorescence microscopic analysis indicated that TF predominantly associates with MAGI1 and less with MAGI2 and MAGI3 proteins. The interaction of TF with MAGI1 was also demonstrated by both co-immunoprecipitation of TF with MAGI1, and co-immunoprecipitation of MAGI1 with TF. Moreover, activation of PAR2 resulted in reduction in the association of these two proteins. Pull-down assays using TF-cytoplasmic domain peptides indicated that the phosphorylation of Ser253 within TF prevents its association with MAGI1. Additionally, the five HA-tagged PDZ domains of MAGI1 were overexpressed separately, and the putative TF-binding domain was identified as PDZ1 domain. Expression of this PDZ domain in cells significantly augmented the TF activity measured both as thrombin-generation and also TF-mediated proliferative signalling. Our data indicate a stabilising interaction between TF and the PDZ-1 domain of MAGI1 and demonstrate that the activation of PAR2 disrupts this interaction. The release of TF from MAGI1 appears to be an initial step in TF de-encryption, associated with increased TF-mediated procoagulant and signalling activities. This mechanism is also likely to lead to further interactions and modifications leading to further enhancement of procoagulant activity, or the release of TF.

中文翻译：

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通过与 MAGI1 的第一个 PDZ 结构域相互作用调节组织因子活性

组织因子 (TF) 活动通过称为加密的过程受到严格监管。TF 的翻译后修饰及其与各种蛋白质和脂质部分的相互作用允许对 TF 和促凝血激活进行多步骤解密。已知膜相关鸟苷酸激酶反转构型 (MAGI) 蛋白可调节包括细胞表面受体在内的许多蛋白的定位和活性。TF 与 MAGI1 蛋白的相互作用作为调节 TF 活性的手段进行了检查。使用表达 TF 和 MAGI1 的 MDA-MB-231 细胞系，并对蛋白酶激活受体 (PAR)2 激活反应良好。采用邻近连接实验(PLA)、免疫共沉淀和pull-down实验检测TF与MAGI1-3蛋白的相互作用，并研究PAR2激活的影响。此外，通过克隆和表达 MAGI1 的 PDZ 结构域，鉴定出了 TF 结合结构域。然后检查了重组 PDZ 结构域作为 MAGI1 竞争者的能力，从而诱导 TF 促凝血和信号传导活性。PLA 和荧光显微镜分析表明 TF 主要与 MAGI1 蛋白相关，较少与 MAGI2 和 MAGI3 蛋白相关。TF 与 MAGI1 的相互作用也通过 TF 与 MAGI1 的免疫共沉淀以及 MAGI1 与 TF 的免疫共沉淀得到证实。此外，PAR2 的激活导致这两种蛋白质的结合减少。使用 TF 胞质结构域肽的 Pull-down 测定表明，TF 内 Ser253 的磷酸化阻止了其与 MAGI1 的关联。此外，MAGI1 的 5 个 HA 标记的 PDZ 结构域分别过表达，假定的 TF 结合结构域被鉴定为 PDZ1 结构域。该 PDZ 结构域在细胞中的表达显着增强了 TF 活性（通过凝血酶生成和 TF 介导的增殖信号传导来测量）。我们的数据表明 TF 和 MAGI1 的 PDZ-1 结构域之间存在稳定的相互作用，并证明 PAR2 的激活会破坏这种相互作用。MAGI1 中 TF 的释放似乎是 TF 解密的第一步，与 TF 介导的促凝血和信号传导活性的增加相关。这种机制也可能导致进一步的相互作用和修饰，从而进一步增强促凝血活性或 TF 的释放。