The function of ezrin in stimulus-coupled acid secretion

描述（由申请人提供）：胃的消化功能取决于胃腔的酸化。胃酸分泌进入管腔是由camp依赖性蛋白激酶（PKA）级联激活触发的，最终导致胃H， k - atp酶插入壁细胞的顶质膜。H， k - atp酶的这种重新定位与顶端膜肌动蛋白细胞骨架的广泛重塑同时发生，这也是激活酸分泌的重要步骤。虽然壁细胞活化的这些方面都很清楚，但将PKA信号级联到H、k - atp酶的动员和细胞骨架重塑的分子机制尚不清楚。偶联蛋白是ezrin，一种80 kDa的磷酸化蛋白，其Ser66位点被PKA磷酸化是壁细胞激活所必需的。然而，关于ezrin在胃酸分泌中起作用的分子机制知之甚少。我们研究的长期目标是描述ezrin如何协调刺激偶联的酸分泌。为了解决这个问题，我们提出了三个具体目标：首先，我们将利用表位标记、化学足迹和交联方法评估phospho-ezrin如何与ACAP4相互作用。这些研究将包括对介导ezrin-ACAP4直接接触的结构决定因素的详细分析。结合结构域数据将用于设计在体外结合试验中有效且特异性地干扰ezrin-ACAP4相互作用的肽。这种相互作用的功能将通过多肽对渗透胃腺分泌酸的影响来评估。其次，我们将明确ARF6-ACAP4-ezrin顶端信号复合物在顶叶细胞激活中的作用。这种相互作用在酸分泌中的重要性将通过功能测定和超分辨率成像分析进行评估。第三，我们计划阐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的光学成像来促进。

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.