The function of ezrin in stimulus-coupled acid secretion

描述(申请人提供)：胃的消化功能取决于胃腔的酸化。胃酸分泌到管腔是由cAMP依赖的蛋白激酶(PKA)级联反应触发的，最终导致胃H，K-ATPase插入壁细胞顶端质膜。H，K-ATPase的这种重新定位伴随着顶膜上肌动蛋白细胞骨架的广泛重塑，这也是激活酸分泌的一个重要步骤。虽然壁细胞激活的这些方面已经被很好地定义，但PKA信号级联与H，K-ATPase的动员和细胞骨架重构的分子机制尚不清楚。Ezrin是一种偶联蛋白，是一种80 kDa的磷酸蛋白，它在Ser66处被PKA磷酸化，是壁细胞激活所必需的。然而，对于Ezrin在胃酸分泌中的分子机制(S)知之甚少。我们研究的长期目标是描绘Ezrin如何协调刺激耦合的酸分泌。为了解决这个问题，我们提出了三个具体的目标：第一，我们将使用表位标记、化学足迹和交联法来评估磷酸化Ezrin如何与ACAP4相互作用。这些研究将涉及对介导Ezrin-ACAP4直接接触的结构决定因素的详细分析。结合结构域数据将被用来设计在体外结合试验中有效和特异地干扰Ezrin-ACAP4相互作用的多肽。这种相互作用的功能将通过使用通透性胃腺的多肽对酸分泌的影响来评估。其次，我们将确定ARF6-ACAP4-Ezrin顶端信号复合体在壁细胞激活中的作用。这样的相互作用在酸分泌中的重要性将通过功能分析和超分辨率成像分析来评估。第三，我们计划说明在壁细胞酸分泌过程中SNARE组装的时空动态。这些研究将通过生物化学和功能表征以及活细胞中的光学成像来促进。 公共卫生相关性： 研究壁细胞激活的分子机制对于了解肠道上皮细胞分泌调节的细胞生理学具有重要意义，也有望有助于指导纠正消化性溃疡和胃食道反流病等疾病的胃酸异常分泌的药物策略。

DESCRIPTION (provided by applicant): The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. This relocation of the H,K-ATPase occurs concomitantly with extensive remodeling of the actin cytoskeleton at the apical membrane, which is also an essential step in the activation of acid secretion. While these aspects of parietal cell activation are well defined, the molecular mechanisms that couple the PKA signaling cascade to mobilization of H,K-ATPases and cytoskeletal remodeling are not known. A coupling protein is ezrin, an 80 kDa phosphoprotein, whose phosphorylation at Ser66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. The long-term goal of our research is to delineate how ezrin orchestrates stimulus-coupled acid secretion. To address this question, three Specific Aims are proposed: first, we will evaluate how phospho-ezrin interacts with ACAP4 using epitope-tagging, chemical footprinting, and crosslinking approaches. These studies will involve a detailed analysis of the structural determinants that mediate a direct ezrin-ACAP4 contact. Binding domain data will be used to design peptides that potently and specifically perturb ezrin-ACAP4 interactions in in vitro binding assays. The function of this interaction will then be evaluated by the effects of the peptides on acid secretion using permeabilized gastric glands. Second, we will define the role of ARF6-ACAP4-ezrin apical signaling complex in parietal cell activation. The importance of such an interaction in acid secretion will then be evaluated by functional assay and supra-resolution imaging analysis. Third, we plan to illustrate the spatiotemporal dynamics of SNARE assembly during parietal cell acid secretion. These studies will be facilitated by biochemical and functional characterization coupled with optical imaging in live cells. PUBLIC HEALTH RELEVANCE: Studying the molecular mechanisms underlying parietal cell activation is of great significance in understanding the cellular physiology of regulated epithelial secretion in the gut, and is also expected to be of great benefit in leading to pharmacological strategies for correcting abnormal gastric acid secretion in disorders such as peptic ulcers, and gastroesophageal reflux disease.