EZRIN IN STIMULUS-COUPLED GASTRIC ACID SECRETION

Ezrin 在刺激耦合胃酸分泌中的作用

• 批准号: 2898639
• 负责人: XUEBIAO YAO
• 金额: --
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-12-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2898639
• 关键词: actins; cytoskeletal proteins; gastric acid; gastrointestinal epithelium; hydrogen potassium exchanging ATPase; laboratory mouse; laboratory rabbit; laboratory rat; phosphoproteins; phosphorylation; protein kinase A; protein protein interaction; secretion; tissue /cell culture

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.

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pka介导的磷酸化与H、k - atp酶的动员和细胞骨架重塑之间的分子机制尚不清楚。一个候选偶联蛋白ezrin在壁细胞中被鉴定为一个80 kDa的磷酸化蛋白，其被PKA磷酸化与壁细胞活化有关。ezrin与肌动蛋白丝的结合是胃酸分泌所必需的。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在刺激偶联上皮分泌中的作用。为了解决这个问题，提出了三个具体目标：首先，我们将使用表位标记、化学足迹和交联方法确定β -肌动蛋白结合所必需的ezrin区域。这些研究将包括对介导ezrin-actin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在体外结合试验中设计有效且特异性地干扰ezrin-actin相互作用的肽。这种相互作用的功能将通过多肽对胃腺渗透分泌酸的影响来确定。其次，我们计划通过首先定位pka介导的磷酸化位点来确定蛋白磷酸化在ezrin功能调控中的作用。通过在培养的壁细胞中表达不可磷酸化的ezrin突变体，可以确定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生成的针对一组新的ezrin结合蛋白的单克隆抗体来促进。研究壁细胞活化的分子和细胞机制对于理解肠道上皮分泌调控的细胞生理学具有重要意义，也有望对纠正胃和十二指肠溃疡、胃食管反流病等疾病中胃酸分泌异常的药理学策略有很大好处。