IQGAP蛋白在极化上皮细胞中的功能研究
摘要
肌动蛋白骨架在上皮细胞极化形成中起着重要作用，Rho GTPase家族的成员Cdc42通过IQGAPs等下游底物调节着肌动蛋白骨架动力学。胃壁细胞是一种典型的极化上皮细胞，其酸分泌过程为研究cAMP介导的胞吐过程及顶膜肌动蛋白骨架动力学提供了一个极佳的模式系统。我们早先的研究表明，肌动蛋白的两种亚型在胃壁细胞中呈极化分布，同时肌动蛋白骨架的完整性对胃酸分泌非常重要。为探索肌动蛋白骨架在上皮细胞极化形成过程中的分子机理，我们利用亲和树脂结合质谱分析发现胃壁细胞含有两种IQGAP亚型(IQGAP1和IQGAP2)。进一步的激光共聚焦显微镜分析发现：Cdc42主要定位于壁细胞的顶膜，同时Cdc42的两个底物IQGAP1和IQGAP2也在胃壁细胞中呈极化分布，其中IQGAP1主要定位于胃壁细胞的底膜，IQGAP2主要定位于胃壁细胞的项膜。人工合成的IQGAP来源的与Cdc42相互作用的小肽段可以竞争型的结合Cdc42，通过破坏IQGAPs、Cdc42和肌动蛋白骨架的相互作用，阻止刺激所引起顶膜肌动蛋白骨架重排从而达到抑制壁细胞酸分泌的作用。我们认为：IQGAP2可以把Cdc42与顶膜肌动蛋白骨架联系起来从而保证了胃壁细胞极化酸分泌的顺利进行。目前我们正利用生物光谱学技术手段来探寻“顶膜信号复合体”的蛋白质作用网络在实时胃酸分泌过程中的时空调控规律。相信在不久的将来，借助于“顶膜信号复合体”联结的蛋白质作用网络，我们可以描绘出胃酸分泌的系统生物学模型。
Actin cytoskeleton plays an important role in the establishment and maintenance of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. Previous studies show shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. To dissert the molecular mechanisms underlying polarized actin cytoskeleton network between apical and basolateral membranes, I have carried out antibody screen to identify actin cytoskeletal regulators that are polarized to apical membrane of the gastric parietal cells. My study had revealed that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells while IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in SLO-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose
Actin cytoskeleton plays an important role in the establishment and maintenance of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. Previous studies show shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. To dissert the molecular mechanisms underlying polarized actin cytoskeleton network between apical and basolateral membranes, I have carried out antibody screen to identify actin cytoskeletal regulators that are polarized to apical membrane of the gastric parietal cells. My study had revealed that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells while IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in SLO-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose
引文
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