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Identification and functional analyses of novel proteins involved in the regulation of epithelial cell-cell adhesion

参与上皮细胞-细胞粘附调节的新型蛋白质的鉴定和功能分析

基本信息

批准号：
18370078
负责人：
FURUSE Mikio
金额：
$ 10.55万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18370078/
关键词：
epithelial cells cell adhesion tight junctions adherens junctions tricellular junctions GAP GFP

项目摘要

To clarify the molecular mechanism behind the regulation of epithelial cell-cell adhesion, we looked for novel proteins that are localized at tight and adherens junctions by a novel expression cloning method based on subcellular localization, namely the fluorescence localization-based retrovirus-mediated expression cloning (FL-REX). Initially, we confirmed that cDNAs for various known components associated with tight and adherens junctions were indeed identified in this screening by using cDNA libraries generated from various epithelial cell lines. Then, we found that ARHGAP12 and SPAL3, potential GTPase activating proteins for small G proteins identified in this method, are concentrated into epithelial adherens junctions by immunoflureoscence staining. When SPAL3, which has a Rap-GAP domain, was overexpressed in cultured fibroblasts, the cells remarkably elongated. Furthermore, cultured epithelial cells overexpressing SPAL3 exhibited retardation in cell-cell junction formation, suggesting that SPAL3 is involved in the regulation of epithelial cell-cell adhesion via actin filaments. In addition, by using the FL-REX method, we have succeeded in identification of a novel membrane protein TRCX, which is localized at tricellular junctions and is involved in the barrier function of the epithelial cellular sheet. This result is expected to contribute to the future analyses of the molecular mechanism behind the formation and physiological functions of tricellular junctions, which have not been well analyzed yet.

为了阐明调节上皮细胞-细胞粘附的分子机制，我们通过一种基于亚细胞定位的新型表达克隆方法，即基于荧光定位的逆转录病毒介导的表达克隆（FL-REX），寻找定位于紧密连接和粘附连接处的新型蛋白质。最初，我们证实，确实通过使用从各种上皮细胞系产生的cDNA文库在该筛选中鉴定了与紧密连接和粘附连接相关的各种已知组分的cDNA。然后，我们发现，ARHGAP 12和SPAL 3，在这种方法中鉴定的小G蛋白的潜在GT3激活蛋白，通过免疫荧光染色被浓缩到上皮粘附连接处。当具有Rap-GAP结构域的SPAL 3在培养的成纤维细胞中过表达时，细胞显著伸长。此外，培养的上皮细胞过表达SPAL 3表现出细胞连接形成的阻滞，表明SPAL 3参与调节上皮细胞通过肌动蛋白丝细胞粘附。此外，通过使用FL-REX方法，我们已经成功地鉴定了一种新的膜蛋白TRCX，它位于三细胞连接处，参与上皮细胞片的屏障功能。这一结果有望有助于未来分析三细胞连接的形成和生理功能背后的分子机制，这还没有得到很好的分析。