Apical Ezrin Assembly the Cytoskeleton and Diarrheal Disorders

肠上皮细胞两种顶膜蛋白是cAMP介导信号转导的重要效应分子 这会导致分泌性腹泻：NHE-3和CFTR。两者都通过PDZ相互作用连接到NHERF蛋白上， 它们依次附着在Ezrin和肌动蛋白上。该支架的完整性对于PKA/cAMP信号是必不可少的 在CFTR上，可能起到极化的根尖保留信号的作用。我们实验室以前发表的工作 强调了中间纤维(IP)在组织顶端结构域中的重要性 上皮细胞，提示IP在这些细胞中基于肌动蛋白的尖端支架的组织中发挥了作用 细胞。此外，IP介导Ezrin组装到上述支架中，以及PKCiota或α 可能对Ezrin的激活负责。假设是Ezrin与心尖中间体结合 细丝启动Ezrin在肠道细胞的顶端定位，并为 它被PKCi激活。这种基于Ezrin的支架的组装对于执行器的功能是必不可少的 分泌性腹泻(NHE-3和CFTR)。为了验证这一假设，我建议：1)确定分子 Ezrin和角蛋白之间的相互作用及其在将Ezrin定位到肠细胞顶端区域中的作用 T567D/T567A Ezrin突变体体外结合试验，或GFP-Ezrin体内构建。2)确定 刷状缘中Ezrin的生理激活剂及其与PKCi或α之间的分子相互作用 角蛋白，使用重组PKC异构体，表达抗PKCI shRNA，显性负PKC异构体， 药物阻滞剂，稳定的Tet诱导的击倒角蛋白8的CACO-2细胞系，以及K8基因缺失的小鼠。 3)检测心尖Ezrin复合体组装对cAMP依赖性CI“分泌的功能影响， PKA定位和激活，Ezrin下游信号和CFTR定位，使用实验 在Ussing小室实验的先前目标中创建和分析的变量和试剂。长的- 这个项目的学期目标是确定负责组装顶端Ezrin的分子相互作用- 肠道细胞中的肌动蛋白支架可被操纵以干扰腹泻患者的离子通道功能 精神错乱。Lay声明：分泌性腹泻在美国和世界范围内是一个严重的健康问题。所有的 它们通过肠道中常见的效应器发挥作用，这些效应器能够分泌氯化物(和水)。这个 支持这些膜蛋白的机械以细胞骨架组件为基础，并被组装 围绕着一种名为Ezrin的蛋白质。该项目试图测试Ezrin(及其相关氯化物)的假设 分泌蛋白质)需要来自细胞骨架分裂的空间线索，即中间丝，才能成为 在精确的位置组装，将水分分泌到肠腔。蛋白质-蛋白质的研究 导致Ezrin及其相关蛋白精确和充分定位的相互作用将提供 这一分子复合体的功能具有潜在的分子干预作用。

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.