NHE3 Regulation and Signaling Complexes

NHE3 调节和信号传导复合物

• 批准号: 7901956
• 负责人: MARK DONOWITZ
• 金额: $ 8.67万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7901956
• 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; casein kinase II; cellular microvillus; coated pit; ezrin; genetic regulatory protein; improved; insight; mouse model; mutant; myosin VI; programs; public health relevance; scaffold; small hairpin RNA; sodium-hydrogen exchanger regulatory factor; trafficking

DESCRIPTION (provided by applicant): 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-photo microscopy/SNARF-4F measurement of NHE3 activity in intact intestine. PUBLIC HEALTH RELEVANCE: The long-term goal of this project 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, which is the epithelial brush border Na/H antiporter and is involved in intestinal Na absorption. We will study regulation of NHE3 by signaling complexes that form on NHE3 itself in highly organized intracellular domains. We suspect that this will provide insight in how normal Na absorption occurs as well as how Na absorption becomes abnormal in diarrheal diseases.

描述（由申请人提供）：该提案是为了检验以下假设：上皮刷边框Na/H抗抗剂NHE3通过充当其某些调节蛋白的支架，并在NHE3 C-Terminus的高度有组织的域中进行组装的调节络合物。长期的目标是了解如何根据药理刺激NHE3活性的目标来调节肠道疾病的必要背景，以开发腹泻疾病的药物治疗。该赠款的基本假设将通过研究NHE3法规的研究，该法规集中在其在AA 475和585之间的C末端域，该领域接近N端运输领域，并强调直接Ezrin与NHE3的直接结合的作用，该作用是在先前的赠款期间确定的，该授予期间是对nhe3 off nhe3 of nhe3 of nhe3 nhe3的必要时期所必需的。该赠款的具体目的是：目标I：了解Ezrin直接结合的C末端域与调节NHE3活性有关。我们建议定义直接Ezrin与NHE3结合在急性刺激和抑制NHE3中的作用。我们已经确定直接结合Ezrin的NHE3的域也与PI-3K系统的多个组件相互作用，我们将使用诱变来识别每个组件在NHE3活性中的作用。我们将检验以下假设：NHE3运输取决于与运动蛋白肌球蛋白VI的结合。 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.研究将主要在极化肠道Na吸收细胞系，使用诱变，SHRNA和显性阴性构建体中通过病毒载体瞬时感染，并补充了一些NHE3蛋白质的模型，并使用NHE3的模型，并使用两种量子的固定蛋白与HER-Photo-photo-photo-snarf-4f-4f-snarf-4f相连肠。公共卫生相关性：该项目的长期目标是了解如何根据药理刺激NHE3活性的目标来调节肠道疾病的必要背景，以开发用于腹泻疾病的药物治疗，这是上皮刷子NA/H的抗抗抑制剂，并参与肠内NA摄入。我们将通过在高度有组织的细胞内结构域中在NHE3上发出信号复合物来研究NHE3的调节。我们怀疑这将提供有关正常NA吸收的洞察力以及NA吸收在腹泻疾病中的异常。