NHE3 Regulation and Signaling Complexes

NHE3 调节和信号传导复合物

• 批准号: 8277967
• 负责人: MARK DONOWITZ
• 金额: $ 54.47万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8277967
• 关键词: 1-Phosphatidylinositol 3-Kinase; Abbreviations; Acute; Affect; Area; Binding; Binding Proteins; Biological Assay; Brush Border; C-terminal; Ca(2+)-Calmodulin Dependent Protein Kinase; Caco-2 Cells; Cell Line; Clathrin; Complex; Detergents; Development; Disease; Dominant-Negative Mutation; Endocytosis; Epithelial; Goals; Grant; Health; Immunoblotting; Intestines; Knockout Mice; Lead; Lipid Binding; Lipids; Liposomes; Measurement; Membrane; Membrane Microdomains; Microscopy; Motor; Mutagenesis; N-terminal; PDPK1 gene; Pharmacotherapy; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Preparation; Principal Investigator; Proteins; Recruitment Activity; Regulation; Role; SHPS-1 protein; Signal Transduction; Signaling Molecule; System; Testing; Viral Vector; absorption; antiporter; base; calmodulin-dependent protein kinase II; casein kinase II; cellular microvillus; coated pit; ezrin; genetic regulatory protein; improved; insight; mouse model; mutant; myosin VI; phosphoinositide-3,4,5-triphosphate; programs; scaffold; small hairpin RNA; sodium-hydrogen exchanger regulatory factor; trafficking; two-photon

This proposal is to test the hypothesis that NHE3, the epithelial brush border Na/H antiporter, undergoes acute regulation by acting as a scaffold for some of its regulatory proteins and that the assembled regulatory complexes form in highly organized domains of the NHE3 C-terminus. The long-term goal is to understand how intestinal Na absorption is regulated as necessary background for developing drug therapy for diarrheal diseases based on the goal of pharmacologically stimulating NHE3 activity. The hypothesis underlying this grant will be tested by study of NHE3 regulation which centers on its C-terminal domain between aa 475 and 585, which is close to the N-terminal transport domain, and emphasizes the role of direct ezrin binding to NHE3, which was identified during the previous grant period as being necessary for multiple aspects of trafficking of NHE3. The Specific Aims of this grant are: Aim I: To understand how the C-terminal domain to which ezrin directly binds is involved in regulation NHE3 activity. We propose to define the role of direct ezrin binding to NHE3 on acute stimulation and inhibition of NHE3. We have identified that the domain of NHE3 which directly binds ezrin also interacts with multiple components of the PI-3K system and we will use mutagenesis to identify the role of each component on NHE3 activity. We will test the hypothesis that NHE3 trafficking is dependent on binding to the motor protein myosin VI. Aim II: We have identified that the phosphoinositides PIP2 and PIP3 appear to directly bind to NHE3 between aa 475 and 585. We will use mutagenesis and liposomal pull down assays to identify the domains of the NHE3 C-terminus through which interactions with the phosphoinositides occur and determine their role in NHE3 trafficking and basal and regulated activity. Studies will be carried out mostly in the polarized intestinal Na absorptive cell line, Caco-2 cells using mutagenesis, shRNA and dominant-negative constructs transiently infected via viral vectors, supplemented with commercially available knockout mice models of some of the NHE3 associating proteins with consequences determined using two-photon microscopy/SNARF-4F measurement of NHE3 activity in intact intestine.

该提议是为了检验以下假设：上皮刷边界Na/h抗植物的NHE3经历了 通过充当某些调节蛋白的脚手架来调节急性调节，并组装了调节 在NHE3 C末端高度组织的结构域中形成复合物。长期目标是了解如何 肠道NA的吸收是开发腹泻药物治疗的必要背景 基于药理学刺激NHE3活性的疾病。这是基础的假设 赠款将通过研究NHE3法规的研究，该法规以其C末端域为中心，在AA 475和 585，靠近N末端运输结构域，并强调直接Ezrin结合与 NHE3，在上一个赠款期间被确定为对于多个方面所必需的 NHE3的贩运。这笔赠款的具体目的是：目标I：了解C端领域如何 Ezrin直接结合的是调节NHE3活性。我们建议定义直接Ezrin的作用 在急性刺激和抑制NHE3时与NHE3结合。我们已经确定了NHE3的域 直接结合Ezrin也与PI-3K系统的多个组件相互作用，我们将使用 诱变以识别每个成分在NHE3活性中的作用。我们将测试NHE3的假设 运输取决于与运动蛋白肌球蛋白VI的结合。 AIM II：我们已经确定 磷酸肌醇PIP2和PIP3似乎直接与AA 475和585之间的NHE3结合。我们将使用 诱变和脂质体下拉测定法，以识别NHE3 C末端的域 与磷酸肌醇的相互作用发生并确定其在NHE3运输和基础上的作用 受调节活动。研究将主要在偏振肠吸收细胞系Caco-2中进行 细胞使用诱变，shRNA和显性阴性构建体瞬时通过病毒载体感染， 补充一些NHE3蛋白的市售敲除小鼠模型 使用两光子显微镜/SNARF-4F测量NHE3活性的后果 完整的肠。