Protein interactions at the tight junction - Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3
Protein interactions at the tight junction - Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3
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DOI：
10.1074/jbc.274.49.35179
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发表时间：
1999-12-03
影响因子：
4.8
通讯作者：
Stevenson, BR
中科院分区：
文献类型：
--
作者：
Wittchen, ES;Haskins, J;Stevenson, BR
确定紧密连接的分子组分如何相互作用是理解紧密连接生理学的先决条件，我们利用纯化的重组蛋白的体外结合测定和免疫沉淀分析来确定ZO-1、ZO-2、ZO-3、occludin和肌动蛋白细胞骨架之间的相互作用。肌动蛋白共沉降研究表明ZO-2、ZO-3和occludin都在体外直接与F-肌动蛋白相互作用，表明肌动蛋白在紧密连接处参与了多种相互作用。低速沉降分析表明，无论是ZO-2，ZO-3，也不是occludin作为F-肌动蛋白交联蛋白，进一步的证据表明，这些蛋白质不结合肌动蛋白丝端。ZO-2，ZO-3，和occludin的结合相互作用在体内得到证实，免疫荧光共定位实验表明，所有三种蛋白共定位与肌动蛋白聚集在细胞松弛素D处理的Madin-Darby犬肾细胞的细胞边界。其他紧密连接蛋白相互作用的探索表明，ZO-2直接结合ZO-1和occludin，与以前的信念相反，我们的免疫沉淀结果表明，ZO-1，ZO-2，ZO-3存在于原位主要是作为独立的ZO-1 ZO-2和ZO-1 ZO-3复合物，而不是一个三聚体ZO-1 ZO-2 ZO-3分组。这些研究阐明了紧密连接相关蛋白之间的直接结合相互作用，使人们深入了解它们作为一个多分子结构的组织。
Defining how the molecular constituents of the tight junction interact is a prerequisite to understanding tight junction physiology, We utilized in vitro binding assays with purified recombinant proteins and immunoprecipitation analyses to define interactions between ZO-1, ZO-2, ZO-3, occludin, and the actin cytoskeleton, Actin cosedimentation studies showed that ZO-2, ZO-3, and occludin all interact directly with F-actin in vitro, indicating that actin is engaged in multiple interactions at the tight junction. Low speed sedimentation analyses demonstrated that neither ZO-2, ZO-3, nor occludin act as F-actin cross-linking proteins, and further evidence indicates that these proteins do not bind to actin filament ends. The binding interactions of ZO-2, ZO-3, and occludin were corroborated in vivo by immunofluorescence colocalization experiments which showed that all three proteins colocalized with actin aggregates at cell borders in cytochalasin D-treated Madin-Darby canine kidney cells. Exploration of other tight junction protein interactions demonstrated that ZO-2 binds directly to both ZO-1 and occludin, Contrary to previous beliefs, our immunoprecipitation results indicate that ZO-1, ZO-2, and ZO-3 exist in situ primarily as independent ZO-1 ZO-2 and ZO-1 ZO-3 complexes rather than a trimeric ZO-1 ZO-2 ZO-3 grouping. These studies elucidate direct binding interactions among tight junction-associated proteins, giving insight into their organization as a multimolecular structure.