Apical Ezrin Assembly the Cytoskeleton and Diarrheal Disorders

描述（申请人提供）：肠上皮细胞中的两(2)种顶膜蛋白是camp介导的导致分泌性腹泻的信号传导的重要效应器：NHE-3和CFTR。两者都通过PDZ相互作用连接到NHERF蛋白上，而NHERF蛋白又连接到ezrin和actin上。该支架的完整性对于CFTR上的PKA/cAMP信号传导至关重要，并可能作为极化的根尖保留信号发挥作用。我们实验室之前发表的研究强调了中间丝（ipps）在简单上皮细胞顶端结构域组织中的重要性，并表明ipps在这些细胞的顶端肌动蛋白支架的组织中发挥作用。此外，IPs介导ezrin组装到上述支架中，PKCiota或alpha可能负责ezrin的激活。假设ezrin与肠细胞顶端中间丝的结合启动了ezrin在肠细胞的顶端定位，并为PKCi激活ezrin提供了一个微结构域限制位点。这种基于ezrin的支架的组装对于分泌性腹泻效应物（NHE-3和CFTR）的功能至关重要。为了验证这一假设，我提出：1)利用T567D/T567A ezrin突变体体外结合试验或GFP-ezrin构建体内结合试验，确定ezrin和角蛋白之间的分子相互作用及其在将ezrin定位到肠细胞顶端结构域中的作用。2)利用重组PKC异构体、抗PKCi shRNA表达、显性阴性PKCi异构体、药理学阻阻剂、稳定的tet诱导cco -2敲低角蛋白8细胞系和K8缺失小鼠，鉴定刷缘ezrin的生理激活因子以及PKCi或a与角蛋白之间的分子相互作用。3)利用前文在室内实验中创建和分析的实验变量和试剂，检测顶端ezrin复合物组装对camp依赖性CI分泌、PKA定位和激活、ezrin下游信号和CFTR定位的功能影响。该项目的长期目标是确定肠细胞中负责顶端ezrin-actin支架组装的分子相互作用，这种相互作用可以被操纵来干扰腹泻疾病中离子通道的功能。Lay声明：秘书腹泻在美国和全世界都是一个严重的健康问题。它们都是通过肠道中共同的效应器起作用，使氯化物（和水）的分泌成为可能。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ezrin的蛋白质组装。该项目旨在测试ezrin（及其相关的氯分泌蛋白）需要细胞骨架分裂的空间线索，即中间细丝，以便在精确的位置组装以向肠腔分泌水。蛋白质-蛋白质相互作用的研究导致ezrin及其相关蛋白的精确和充分定位，将为分子干预该分子复合物的功能提供潜在的点。

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.