The function of ezrin in stimulus-coupled acid secretion

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依赖的蛋白激酶(PKA)级联激活触发的，这 最终导致胃H，K-ATPase插入顶端质膜 细胞。H，K-ATPase的这种重新定位伴随着肌动蛋白的广泛重塑 细胞骨架位于顶膜，这也是激活胃酸分泌的必要步骤。 虽然壁细胞激活的这些方面都有很好的定义，但与壁细胞激活相关的分子机制 PKA信号转导通路对H，K-ATPase的动员和细胞骨架重构的影响尚不清楚。一个 偶联蛋白是Ezrin，一个80 kDa的磷酸蛋白，它需要被PKA磷酸化才能 壁细胞激活。然而，关于Ezrin的分子机制(S)知之甚少 在胃酸分泌中工作。我们研究的长期目标是描绘Ezrin如何 协调刺激偶联的胃酸分泌。要解决这个问题，有三个具体目标 建议：首先，我们将使用表位标签来评估磷酸化Ezrin如何与IQGAP2相互作用， 化学足迹和交联法。这些研究将涉及对 介导Ezrin-IQGAP2直接接触的结构决定因素。绑定域数据将用于 设计在体外结合试验中有效和特异性干扰Ezrin-IQGAP2相互作用的多肽。 这种相互作用的功能将由多肽对酸分泌的影响来确定。 使用通透性胃腺。其次，我们将确定Ezrin如何与Synaxin 3相互作用以 促进H，K-ATPase在刺激下插入顶膜。这些研究将 通过使用荧光记者对壁细胞激活进行实时显微分析，这一研究变得更加便利。 第三，我们计划确定Ezrin-PALS1相互作用在与以下相关的顶膜重塑中的作用 首先通过钉住它们的结合域来激活细胞。这种互动在中国的重要性 然后，将通过功能分析和超微结构分析来评估酸分泌。 研究壁细胞激活的分子和细胞机制具有重要意义 对了解肠道上皮细胞分泌调节的细胞生理学的意义，以及 有望在指导纠正异常胃酸的药物策略方面大有裨益 消化性溃疡和胃食道反流病等疾病的分泌物。

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 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 gastric acid secretion. To address this question, three Specific Aims are proposed: first, we will evaluate how phospho-ezrin interacts with IQGAP2 using epitope-tagging, chemical footprinting, and crosslinking approaches. These studies will involve a detailed analysis of the structural determinants that mediate a direct ezrin-IQGAP2 contact. Binding domain data will be used to design peptides that potently and specifically perturb ezrin-IQGAP2 interactions in in vitro binding assays. The function of this interaction will then be determined by the effects of the peptides on acid secretion using permeabilized gastric glands. Second, we will determine how ezrin interacts with syntaxin 3 to facilitate the insertion of H,K-ATPase into the apical membrane upon the stimulation. These studies will be facilitated by real time microscopic analyses of parietal cell activation using fluorescence reporters. Third, we plan to define the role of ezrin-PALSl interaction in the apical membrane remodeling related to the cell activation by first pin-pointing their binding domains. The importance of such an interaction in acid secretion will then be evaluated by functional assay coupled with ultrastructural analysis. Studying the molecular and cellular mechanisms underlying parietal cell activation is of substantial 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.