The function of ezrin in stimulus-coupled acid secretion

埃兹蛋白在刺激耦合酸分泌中的功能

• 批准号: 7327779
• 负责人: XUEBIAO YAO
• 金额: $ 28.67万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 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 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7327779
• 关键词: Acids; Actin-Binding Protein; Actins; Address; Am 80; Apical; Binding; Biological Assay; Cell Polarity; Cell membrane; Cell physiology; Cells; Chemicals; Complex; Coupled; Couples; Coupling; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Data; Disease; Docking; Electrons; Epithelial; Epithelial Cells; Epitopes; Fluorescence; Fluorescence Microscopy; Gastric Acid; Gastric Glands; Gastric Parietal Cells; Gastroesophageal reflux disease; Gland; Goals; Green Fluorescent Proteins; H(+)-K(+)-Exchanging ATPase; Hormones; In Vitro; Mediating; Membrane; Microscopic; Molecular; Monitor; Optical reporter; Peptic Ulcer; Peptides; Phosphoproteins; Phosphorylation; Phosphorylation Site; Process; Protein Kinase; Proteins; Regulation; Reporter; Research; Role; Signal Transduction; Signaling Protein; Stimulus; Stomach; Time; Vesicle; apical membrane; chemical genetics; crosslink; design; ezrin; syntaxin 3

DESCRIPTION (provided by the 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 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.

描述（由申请方提供）：胃的消化功能取决于胃腔的酸化。通过cAMP依赖性蛋白激酶（PKA）级联的激活触发酸分泌到腔中，其最终导致胃H，K-ATP酶插入壁细胞的顶端质膜中。H，K-ATP酶的这种重新定位伴随着顶端膜处肌动蛋白细胞骨架的广泛重塑而发生，这也是酸分泌激活的重要步骤。虽然壁细胞活化的这些方面是明确的，但将PKA信号级联与H，K-ATP酶的动员和细胞骨架重塑偶联的分子机制尚不清楚。偶联蛋白是ezrin，一种80 kDa的磷蛋白，其被PKA磷酸化是壁细胞活化所需的。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埃兹林如何协调刺激耦合的胃酸分泌。为了解决这个问题，提出了三个具体目标：首先，我们将评估磷酸埃兹蛋白如何使用表位标记，化学足迹和交联方法与IQGAP 2相互作用。这些研究将涉及介导直接ezrin-IQGAP 2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干扰ezrin-IQGAP 2相互作用的肽。 这种相互作用的功能将通过使用透化的胃腺的肽对酸分泌的影响来确定。其次，我们将确定ezrin如何与syntaxin 3相互作用，以促进H，K-ATP酶在刺激后插入顶端膜。这些研究将通过使用荧光报告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，我们计划通过首先精确定位它们的结合结构域来确定ezrin-PALSl相互作用在与细胞活化相关的顶膜重塑中的作用。这种相互作用在酸分泌中的重要性将通过功能测定结合超微结构分析进行评价。 研究壁细胞激活的分子和细胞机制对于理解肠道中调节上皮分泌的细胞生理学具有重要意义，并且还预期对于导致用于纠正诸如消化性溃疡和胃食管反流病等疾病中的异常胃酸分泌的药理学策略具有很大益处。