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: 8103826
• 负责人: XUEBIAO YAO
• 金额: $ 30.44万
• 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 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8103826
• 关键词: Acids; Actin-Binding Protein; Actins; Activity Cycles; Address; Am 80; Apical; Binding; Biochemical; Biological Assay; Cell membrane; Cell model; Cell physiology; Cells; Chemicals; Complex; Coupled; Couples; Coupling; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Data; Disease; Electrons; Epithelial; Epitopes; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; Gastric Acid; Gastric Glands; Gastric Parietal Cells; Gastroesophageal reflux disease; Goals; Guanosine Triphosphate Phosphohydrolases; H(+)-K(+)-Exchanging ATPase; Hormones; Hydrochloric Acid; Image; Image Analysis; In Vitro; Life; Mediating; Membrane; Microscope; Microscopic; Modeling; Molecular; Monitor; Optical reporter; Peptic Ulcer; Peptides; Phosphoproteins; Phosphorylation; Phosphorylation Site; Process; Protein Kinase; Proteins; Proteomics; Recruitment Activity; Regulation; Research; Resolution; Role; SNAP receptor; Signal Transduction; Stimulus; Stomach; Time; Vesicle; apical membrane; crosslink; design; ezrin; gastrointestinal; in vitro activity; molecular dynamics; novel; optical imaging; protein complex; public health relevance; spatiotemporal; syntaxin 3

DESCRIPTION (provided by 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 at Ser66 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 acid secretion. To address this question, three Specific Aims are proposed: first, we will evaluate how phospho-ezrin interacts with ACAP4 using epitope-tagging, chemical footprinting, and crosslinking approaches. These studies will involve a detailed analysis of the structural determinants that mediate a direct ezrin-ACAP4 contact. Binding domain data will be used to design peptides that potently and specifically perturb ezrin-ACAP4 interactions in in vitro binding assays. The function of this interaction will then be evaluated by the effects of the peptides on acid secretion using permeabilized gastric glands. Second, we will define the role of ARF6-ACAP4-ezrin apical signaling complex in parietal cell activation. The importance of such an interaction in acid secretion will then be evaluated by functional assay and supra-resolution imaging analysis. Third, we plan to illustrate the spatiotemporal dynamics of SNARE assembly during parietal cell acid secretion. These studies will be facilitated by biochemical and functional characterization coupled with optical imaging in live cells. PUBLIC HEALTH RELEVANCE: Studying the molecular mechanisms underlying parietal cell activation is of great 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（由申请人提供）：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磷蛋白，其通过PKA在Ser 66处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绘埃兹林如何协调刺激耦合的酸分泌。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与ACAP 4相互作用，使用表位标记，化学足迹和交联方法。这些研究将涉及介导直接ezrin-ACAP 4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测定中有效且特异性地干扰ezrin-ACAP 4相互作用的肽。然后使用透化的胃腺通过肽对酸分泌的影响来评价这种相互作用的功能。其次，我们将确定ARF 6-ACAP 4-ezrin顶端信号复合物在壁细胞活化中的作用。这种相互作用在酸分泌中的重要性将通过功能测定和超分辨率成像分析来评估。第三，我们计划说明的时空动态陷阱组装在壁细胞酸分泌。这些研究将通过生物化学和功能表征以及活细胞中的光学成像来促进。 公共卫生相关性： 研究壁细胞激活的分子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。