The extracellular domain of angulin-1 and palmitoylation of its cytoplasmic region are required for angulin-1 assembly at tricellular contacts
The extracellular domain of angulin-1 and palmitoylation of its cytoplasmic region are required for angulin-1 assembly at tricellular contacts
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DOI：
10.1074/jbc.ra119.010491
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发表时间：
2020-02
期刊：
影响因子：
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通讯作者：
Yukako Oda;Taichi Sugawara;Y. Fukata;Y. Izumi;Tetsuhisa Otani;T. Higashi;M. Fukata;M. Furuse
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文献类型：
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作者：
Yukako Oda;Taichi Sugawara;Y. Fukata;Y. Izumi;Tetsuhisa Otani;T. Higashi;M. Fukata;M. Furuse
三细胞紧密连接（tTJ）在三细胞接触（TC）处产生细胞旁屏障，其中三个多边形上皮细胞的顶点相遇。tTJ的标志是富集两种类型的膜蛋白，三纤维素和角蛋白家族蛋白。然而，如何TC的几何形状是公认的tTJ形成仍然未知。在本研究中，我们研究的分子机制，为大会的angulin-1在TC。我们发现，半胱氨酸残基簇的血管膜区域内的细胞质结构域的angulin-1是高度棕榈酰化。在这个区域的半胱氨酸残基的突变分析表明，棕榈酰化是必不可少的本地化的angulin-1在TC。因此，抑制EpH 4细胞中表达的含Asp-His-His-Cys基序的棕榈酰转移酶显著损害了angulin-1的TC定位。培养的上皮细胞的质膜的胆固醇消耗阻碍了在TC的angulin-1的本地化，这表明在TC的脂质膜微域的存在，吸引高度棕榈酰化angulin-1。此外，angulin-1的细胞外结构域也需要其TC定位，无论细胞内棕榈酰化。两者合计，我们的研究结果表明，这两个angulin-1的细胞外结构域和棕榈酰化的胞质区域是必需的，其组装在TC。
Tricellular tight junctions (tTJs) create paracellular barriers at tricellular contacts (TCs), where the vertices of three polygonal epithelial cells meet. tTJs are marked by the enrichment of two types of membrane proteins, tricellulin and angulin family proteins. However, how TC geometry is recognized for tTJ formation remains unknown. In the present study, we examined the molecular mechanism for the assembly of angulin-1 at the TCs. We found that clusters of cysteine residues in the juxtamembrane region within the cytoplasmic domain of angulin-1 are highly palmitoylated. Mutagenesis analyses of the cysteine residues in this region revealed that palmitoylation is essential for localization of angulin-1 at TCs. Consistently, suppression of Asp-His-His-Cys motif–containing palmitoyltransferases expressed in EpH4 cells significantly impaired the TC localization of angulin-1. Cholesterol depletion from the plasma membrane of cultured epithelial cells hampered the localization of angulin-1 at TCs, suggesting the existence of a lipid membrane microdomain at TCs that attracts highly palmitoylated angulin-1. Furthermore, the extracellular domain of angulin-1 was also required for its TC localization, irrespective of the intracellular palmitoylation. Taken together, our findings suggest that both angulin-1's extracellular domain and palmitoylation of its cytoplasmic region are required for its assembly at TCs.