The function of ezrin in stimulus-coupled acid secretion

胃的消化功能取决于胃腔的酸化。酸分泌 通过激活cAMP依赖性蛋白激酶（PKA）级联反应触发， 最终导致胃H，K-ATP酶插入壁细胞的顶端质膜， 细胞这种重新定位的H，K-ATP酶发生伴随着广泛的重塑的肌动蛋白 细胞骨架在顶膜，这也是一个重要的步骤，在激活酸分泌。 虽然壁细胞激活的这些方面是明确定义的，但将细胞激活与细胞凋亡相结合的分子机制仍然是未知的。 PKA信号级联到H，K-ATP酶的动员和细胞骨架重塑尚不清楚。一 偶联蛋白是ezrin，一种80 kDa的磷蛋白，其被PKA磷酸化是 壁细胞激活然而，很少有人知道的分子机制（S），埃兹林 作用于胃酸分泌。我们研究的长期目标是描述埃兹林 协调刺激耦合胃酸分泌。为了解决这个问题，有三个具体目标： 建议：首先，我们将使用表位标记来评估磷酸-埃兹蛋白如何与IQGAP 2相互作用， 化学足迹法和交联方法。这些研究将涉及对 介导ezrin-IQGAP 2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且特异性干扰ezrin-IQGAP 2相互作用的肽。 这种相互作用的功能将由肽对酸分泌的影响决定 使用透化的胃腺。其次，我们将确定ezrin如何与syntaxin 3相互作用， 促进H，K-ATP酶在刺激时插入顶膜。这些研究将 通过使用荧光报道分子对壁细胞活化进行真实的时间显微镜分析来促进。 第三，我们计划确定ezrin-PALSl相互作用在与以下相关的根尖膜重塑中的作用： 通过首先精确定位它们的结合域来激活细胞。这种相互作用的重要性， 然后通过结合超微结构分析的功能测定来评价酸分泌。 研究壁细胞激活的分子和细胞机制具有重要意义 在理解调节肠道上皮分泌的细胞生理学方面具有重要意义， 预期在导致纠正异常胃酸的药理学策略方面有很大益处 消化性溃疡和胃食管反流病等疾病中的分泌物。

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.