Apical Ezrin Assembly the Cytoskeleton and Diarrheal Disorders

描述（由申请人提供）：肠上皮细胞中的两（2）种顶端膜蛋白是cAMP介导的信号传导的重要效应物，可导致分泌性胰腺炎：NHE-3和CFTR。两者都通过PDZ相互作用连接到NHERF蛋白，NHERF蛋白又连接到埃兹蛋白和肌动蛋白。该支架的完整性对于CFTR上的PKA/cAMP信号传导是必不可少的，并且可以作为极化的顶端保留信号发挥作用。我们实验室以前发表的工作强调了中间丝（IP）在简单上皮细胞顶端结构域组织中的重要性，并表明IP在这些细胞中顶端肌动蛋白支架的组织中发挥作用。此外，IP介导ezrin组装成上述支架，并且PKC 10或α可能负责ezrin的活化。该假设是，结合埃兹蛋白顶端中间丝启动埃兹蛋白在肠细胞的顶端定位，并提供了一个微域限制的网站，其激活PKCi。这种基于埃兹蛋白的支架的组装对于分泌型EGFR的效应子（NHE-3和CFTR）的功能是必不可少的。为了验证这一假设，我建议：1）确定埃兹蛋白和角蛋白之间的分子相互作用和它们的作用，本地化埃兹蛋白的肠上皮细胞的顶端域，使用T567 D/T567 A埃兹蛋白突变体在体外结合试验，或GFP埃兹蛋白构建体在体内。2)鉴定刷状缘中ezrin的生理激活剂以及PKCi或a与角蛋白之间的分子相互作用，使用重组PKC同种型、抗PKCi shRNA的表达、显性阴性PKC同种型、药理学阻断剂、敲低角蛋白8的稳定TET诱导的CACO-2细胞系和K8缺失小鼠。和3）使用在使用室实验中的先前目的中创建和分析的实验变量和试剂，测试顶端ezrin复合物组装对cAMP依赖性Cl-分泌、PKA定位和活化、ezrin下游信号传导和CFTR定位的功能后果。该项目的长期目标是确定负责在肠细胞中组装顶端ezrin-肌动蛋白支架的分子相互作用，该分子相互作用可以被操纵以干扰肠道疾病中离子通道的功能。非专业人士声明：分泌性支气管炎在美国和全世界都是一个严重的健康问题。所有这些都通过肠道中的共同效应器起作用，使氯化物（和水）分泌。支持这些膜蛋白的机制是基于细胞骨架成分，并围绕一种称为埃兹蛋白的蛋白质组装。该项目旨在测试以下假设：ezrin（及其相关的氯化物分泌蛋白）需要来自细胞骨架（中间丝）的分裂的空间线索，以便在精确的位置组装以将水分泌到肠腔。蛋白质-蛋白质相互作用的研究，导致在精确和充分的本地化ezrin及其相关蛋白质将提供潜在的点，对这种分子复合物的功能的分子干预。

DESCRIPTION (provided by applicant): Two (2) apical membrane proteins in intestinal epithelial cells are important effectors of cAMP-mediated signaling that result in secretory diarrheas: NHE-3 and CFTR. Both are attached via PDZ interactions to NHERF proteins, which, in turn, are attached to ezrin and actin. The integrity of this scaffold is essential for PKA/cAMP signaling on CFTR and may play a role as an apical retention signal for polarization. Previous published work in our lab has highlighted the importance of intermediate filaments (IPs) in the organization of the apical domain in simple epithelial cells, and suggest that IPs play a role in the organization of the apical actin-based scaffold in these cells. Furthermore, IPs mediate the assembly of ezrin into the above-mentioned scaffold, and PKCiota or alpha may be responsible for the activation of ezrin. The hypothesis is that binding of ezrin to apical intermediate filaments initiates the apical localization of ezrin in intestinal cells and provides a microdomain-restricted site for its activation by PKCi. The assembly of this ezrin-based scaffold is essential for the function of the effectors of secretory diarrheas (NHE-3 and CFTR). To test this hypothesis I propose to: 1) Identify the molecular interactions between ezrin and keratins and their role to localize ezrin to the apical domain of enterocytes, using T567D/T567A ezrin mutants in vitro binding assays, or GFP-ezrin constructs in vivo. 2) Identify the physiological activator of ezrin in the brushborder and the molecular interactions between PKCi or a and keratins, using recombinant PKC isoforms, expression of anti-PKCi shRNA, dominant negative PKCisoforms, pharmacological blockers, stable TET-inducible CACO-2 cell lines that knock down keratin 8, and K8 null mice. And 3) Test the functional consequences of apical ezrin complex assembly on cAMP-dependent CI" secretion, PKA localization and activation, signaling downstream of ezrin and CFTR localization, using the experimental variables and reagents created and analyzed in the previous aims in using chamber experiments. The long-term goal of this project is to identify the molecular interactions responsible for the assembly of the apical ezrin-actin scaffold in intestinal cells that can be manipulated to interfere with the function of ion channels in diarrheal disorders. Lay statement: Secretory diarrheas are a severe health problem in the U.S. and worldwide. All of them operate through common effectors in the intestine that enable the secretion of chloride (and water). The machinery that supports these membrane proteins is based on cytoskeletal components and is assembled around a protein known as ezrin. This project seeks to test the hypothesis that ezrin (and its associated chloride secreting proteins) require spatial cues from a division of the cytoskeleton, the intermediate filaments, to become assembled in the precise location to secrete water to the lumen of the intestine. The study of protein-protein interactions that result in the precise and adequate localization of ezrin and its associated proteins will provide potential points for molecular intervention on the function of this molecular complex.