EZRIN IN STIMULUS-COUPLED GASTRIC ACID SECRETION

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介导的磷酸化与H，K-ATPase的动员和细胞骨架重塑的分子机制还不清楚。一个候选偶联蛋白Ezrin在壁细胞中被鉴定为一个80 kDa的磷酸蛋白，其被PKA的磷酸化与壁细胞的激活有关。Ezrin与肌动蛋白细丝的结合是胃酸分泌所必需的。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述Ezrin在刺激偶联上皮细胞分泌中的作用。为了解决这个问题，ALE提出了三个具体的目标：第一，我们将使用表位标记、化学足迹和交联法确定Ezrin与β-肌动蛋白结合所必需的区域。这些研究将涉及对介导Ezrin-肌动蛋白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-肌动蛋白相互作用的多肽。这种相互作用的功能将由这些多肽对使用通透性胃腺分泌的酸的影响来确定。其次，我们计划通过首次定位PKA介导的磷酸化位点来确定蛋白质磷酸化在Ezrin功能调节中的作用。然后，将通过在培养的壁细胞中表达非磷酸化的Ezrin突变体来确定Ezrin磷酸化在酸分泌中的功能。第三，我们将继续鉴定和表征新的Ezrin相互作用蛋白。这些研究将通过使用我们新产生的针对一组新的Ezrin结合蛋白的单抗来促进。研究壁细胞激活的分子和细胞机制对于了解肠道上皮细胞分泌调节的细胞生理学具有普遍意义，也有望对纠正胃和十二指肠溃疡以及胃食道反流病等疾病的胃酸异常分泌的药物策略具有重要意义。

Digestive function in 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, 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 PKA-mediated phosphorylation to mobilization of H,K-ATPases and cytoskeletal remodeling are not known. A candidate coupling protein, ezrin, was identified as an 80 kDa phosphoprotein in parietal cells whose phosphorylation by PKA was related to parietal cell activation. Binding of ezrin to actin filaments is essential for gastric acid secretion. 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 the role of ezrin in stimulus-coupled epithelial secretion. To address this question, three Specific Aims ale proposed: first, we will identify the regions of ezrin necessary for beta-actin association using epitope-tagging, chemical footprinting, and crosslinking approaches. These studies will involve a detailed analysis of the structural determinants that mediate a direct ezrin-actin contact. Binding domain data will be used to design peptides that potently and specifically perturb ezrin-actin 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 plan to determine the role of protein phosphorylation in the regulation of ezrin function by first mapping PKA-mediated phosphorylation sites. The function of ezrin phosphorylation in acid secretion will then be defined by expressing non-phosphorylatable ezrin mutants in cultured parietal cells. Third, we will continue to identify and characterize novel ezrin-interacting proteins. These studies will be facilitated by using our newly generated monoclonal antibodies directed against a novel set of ezrin-binding proteins. Studying the molecular and cellular mechanisms underlying parietal cell activation is of general importance in understanding cellular physiology of regulated epithelial secretion in 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 gastric and duodenal ulcers, and gastroesophageal reflux disease.