Apical Ezrin Assembly the Cytoskeleton and Diarrheal Disorders

肠上皮细胞中的两种顶端膜蛋白是cAMP介导的信号传导的重要效应子 导致分泌型肾小球：NHE-3和CFTR。两者都通过PDZ相互作用连接到NHERF蛋白， 它们依次连接到埃兹蛋白和肌动蛋白上。这种支架的完整性对PKA/cAMP信号传导至关重要 在CFTR上，并且可能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（IPs）在简单的顶域组织中的重要性， 上皮细胞，并建议，IP发挥的作用，在组织的顶端肌动蛋白为基础的支架在这些 细胞此外，IP介导埃兹蛋白组装成上述支架，而PKC 10或α介导埃兹蛋白组装成上述支架。 可能是激活埃兹蛋白的原因假设ezrin与顶端中间体的结合 细丝启动了ezrin在肠细胞中的顶端定位，并提供了一个微区限制位点， 其被PKCi激活。这种基于埃兹蛋白的支架的组装对于细胞因子的效应器的功能是必不可少的。 分泌型嗜酸性粒细胞（NHE-3和CFTR）。为了验证这一假设，我建议：1）确定分子 埃兹蛋白和角蛋白之间的相互作用以及它们将埃兹蛋白定位于肠上皮细胞顶端区域的作用， T567 D/T567 A埃兹蛋白突变体在体外结合测定中，或GFP-埃兹蛋白构建体在体内。2)识别 刷状缘中Ezrin的生理激活剂以及PKCi或a与 角蛋白，使用重组PKC亚型，表达抗PKC 1 shRNA，显性阴性PKC亚型， 药理学阻断剂、敲低角蛋白8的稳定TET诱导型CACO-2细胞系和K8缺失小鼠。 和3）测试顶端埃兹蛋白复合物组装对cAMP依赖性C1-分泌的功能后果， PKA定位和激活，ezrin和CFTR定位的下游信号传导，使用实验性的 变量和试剂创建和分析在以前的目标在尤辛室实验。很长的- 该项目的长期目标是确定负责顶端ezrin组装的分子相互作用， 肠细胞中的肌动蛋白支架，可被操纵以增强肠细胞中离子通道的功能 紊乱非专业人士声明：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 它们通过肠中的共同效应器起作用，该效应器使得能够分泌氯化物（和水）。的 支持这些膜蛋白的机制是基于细胞骨架成分， 一种叫做埃兹蛋白的蛋白质该项目旨在测试埃兹林（及其相关氯化物） 分泌蛋白质）需要来自细胞骨架（中间丝）分裂的空间线索， 在精确的位置组装以将水分泌到肠腔。蛋白质-蛋白质研究 导致ezrin及其相关蛋白精确和充分定位的相互作用将提供 潜在的点，分子干预的功能，这种分子复合物。

Two 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 PKCisoforms, 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 Ussing 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 interferewith 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 hypothesisthat 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.