Molecular mechanisms of polarized endocytic traffic in epithelial cells

上皮细胞极化内吞运输的分子机制

• 批准号: 7805482
• 负责人: Rytis Prekeris
• 金额: $ 32.74万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-15 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7805482
• 关键词: Actins; Apical; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Calorimetry; Canis familiaris; Carrier Proteins; Cell membrane; Cells; Complex; Data; Defect; Disease; Drug Delivery Systems; Endosomes; Ensure; Epithelial; Epithelial Cells; Epithelium; Family; Glutathione; Goals; Guanosine Triphosphate Phosphohydrolases; Immunoprecipitation; In Vitro; KRP protein; Kidney; Laboratories; Lead; Lipids; Mediating; Membrane; Membrane Protein Traffic; Molecular; Mutagenesis; Nephrogenic Diabetes Insipidus; Pathway interactions; Process; Protein Family; Proteins; Publishing; Pulmonary Cystic Fibrosis; Recruitment Activity; Recycling; Role; Site-Directed Mutagenesis; Small Interfering RNA; Sorting - Cell Movement; Techniques; Testing; Tetanus Helper Peptide; Titrations; Transmembrane Transport; Work; Yeasts; base; basolateral membrane; cell motility; design; fluorescence imaging; in vivo; insight; kidney epithelial cell; knock-down; member; monolayer; nexin; novel therapeutic intervention; polarized cell; polymerization; protein complex; protein transport; public health relevance; rab GTP-Binding Proteins; research study; scaffold; small hairpin RNA; trafficking; yeast two hybrid system

DESCRIPTION (provided by applicant): Epithelia consist from polarized cells that are capable of selectively transporting substances across epithelial monolayer. This selective transport is achieved by the partitioning of the plasma membrane into distinct domains: apical and basolateral, with both of these plasma membrane compartments having distinct lipid and protein compositions. Since the fidelity of trans-epithelial protein transport is crucial to a variety of epithelial functions, epithelial cells have developed complicated mechanisms to ensure correct transport of proteins. Rab11 GTPases are the members of small monomeric GTPase super-family that has been implicated in regulating endocytic membrane transport. Rab GTPase work by recruiting various effector proteins to the distinct cellular compartments. Thus, understanding the role of these effector proteins is a key step in understanding the function of epithelial cell. In the last six years several Rab11-binding proteins have been identified, which include Rab11 family interacting proteins, also known as FIPs. Works from several laboratories, including ours, have shown that Rip11/FIP5 member of FIP family regulate polarized protein transport in epithelial cells. Furthermore, it was shown that FIPs act as scaffolding factors allowing the assembly of specific sorting/transport complexes required for the epithelial protein traffic. Based on recently published results and on preliminary data, we propose the following hypotheses. First, that Rip11/FIP5 mediates protein transport to apical plasma membrane. Second, that Rip11/FIP5 regulates apical protein transport by sequential recruiting of sortin nexin 18 (SNX18) and kinesin II to the endocytic membranes. Thus, the main goal of this proposal is to further characterize Rip11/FIP5-SNX18 and Rip11/FIP5-Kinesin II complex formation and determine their role on epithelial transport. I propose three different aims designed to test these hypotheses. In the aim #1 we will analyze the role of Rip11/FIP5 in regulating apical protein transport. We will use combination of tet-inducible protein knock-down, fluorescence imaging and in vivo transport assays. In the aim #2 and aim #3 we will characterize the binding of Rip11/FIP5 to SNX18 and Kinesin II. We will use the combination of immunoprecipitations, glutathione bead pull-down, yeast two-hybrid and isothermal calorimetry assays. In addition, in aim #2 and aim #3 we will determine the roles of Rip11/FIP5-binding proteins in regulating apical and basolateral membrane traffic. To that end we will use the combination of tet- inducible protein knock-down, fluorescence imaging, site-directed mutagenesis and in vivo transport assays. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand the molecular mechanisms of Rab11-dependent polarized membrane traffic and define the membrane traffic steps that are regulated by each Rab11-Rip11/FIP5 protein complex. Furthermore, this project will also identify the molecular machinery mediating Rab11-dependent endocytic protein sorting. These data will advance our understanding, both conceptually and mechanistically, of the machinery governing protein targeting in epithelial cells. To elucidate the mechanisms of protein targeting is of major importance because the defects in this process cause a variety of pulmonary (cystic fibrosis) and renal (nephrogenic diabetes insipidus) disorders. Thus, new insights into the mechanisms of polarized membrane traffic may lead to the identification of new drug targets as well as new therapeutic approaches aimed at the identification and treatment of membrane traffic disorders.

描述（由申请人提供）：上皮由极化细胞组成，能够选择性地在上皮单层上运输物质。这种选择性运输是通过将质膜划分为不同的区域来实现的：顶端和基底外侧，这两个质膜区室具有不同的脂质和蛋白质组成。由于跨上皮蛋白运输的保真度对多种上皮功能至关重要，上皮细胞已经发展出复杂的机制来确保蛋白质的正确运输。Rab11 GTPase是小单体GTPase超家族的成员，参与调节内吞膜运输。Rab GTPase通过向不同的细胞区室招募各种效应蛋白来起作用。因此，了解这些效应蛋白的作用是了解上皮细胞功能的关键一步。在过去的六年中，已经发现了几种Rab11结合蛋白，其中包括Rab11家族相互作用蛋白，也称为FIPs。包括我们在内的几个实验室的研究表明，FIP家族的Rip11/FIP5成员调节上皮细胞中极化蛋白的转运。此外，研究表明，FIPs作为支架因子，允许上皮蛋白运输所需的特定分选/运输复合物的组装。根据最近发表的研究结果和初步数据，我们提出以下假设。首先，Rip11/FIP5介导蛋白向顶质膜的转运。第二，Rip11/FIP5通过向内吞膜连续募集分选蛋白连接蛋白18 （SNX18）和运动蛋白II来调节顶端蛋白的运输。因此，本研究的主要目标是进一步表征Rip11/FIP5-SNX18和Rip11/FIP5-Kinesin II复合物的形成，并确定它们在上皮运输中的作用。我提出了三个不同的目标来检验这些假设。在目的1中，我们将分析Rip11/FIP5在调节根尖蛋白运输中的作用。我们将结合使用诱导蛋白敲除、荧光成像和体内转运试验。在目标2和目标3中，我们将描述Rip11/FIP5与SNX18和Kinesin II的结合。我们将使用免疫沉淀，谷胱甘肽头拉下，酵母双杂交和等温量热测定相结合的方法。此外，在目标#2和目标#3中，我们将确定Rip11/ fip5结合蛋白在调节根尖和基底侧膜运输中的作用。为此，我们将结合tet诱导蛋白敲除、荧光成像、定点诱变和体内转运试验。公共卫生相关性：本项目的目标是了解rab11依赖的极化膜传输的分子机制，并定义由每个Rab11-Rip11/FIP5蛋白复合物调节的膜传输步骤。此外，本项目还将确定介导rab11依赖性内吞蛋白分选的分子机制。这些数据将促进我们在概念和机制上对上皮细胞中蛋白质靶向机制的理解。阐明蛋白靶向的机制具有重要意义，因为这一过程中的缺陷会导致多种肺部（囊性纤维化）和肾脏（肾源性尿崩症）疾病。因此，对极化膜传输机制的新认识可能会导致新的药物靶点的识别以及新的治疗方法，旨在识别和治疗膜传输障碍。