Molecular Mechanisms of Apical Membrane Traffic in Epithelial Cells

上皮细胞顶膜运输的分子机制

• 批准号: 6845089
• 负责人: Rytis Prekeris
• 金额: $ 20.46万
• 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 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6845089
• 关键词: MDCK cell; X ray crystallography; affinity chromatography; apical membrane; cellular polarity; epithelium; fluorescence microscopy; guanine nucleotide binding protein; intracellular transport; laboratory mouse; laboratory rat; mass spectrometry; membrane transport proteins; microtubules; phosphorylation; protein binding; protein protein interaction; protein structure function; protein transport; vesicle /vacuole; yeast two hybrid system

DESCRIPTION (provided by applicant): Epithelial cells establish and maintain the polarized distribution of membrane proteins and lipids by continuously sorting them during biogenesis and endocytic cycles. Protein targeting is critical for most epithelial functions. Consequently, its defects are known to lead to a variety of renal and digestive disorders. The main goal of my research is to investigate the mechanisms that regulate Rab 11-dependent apical protein targeting in epithelial cells. Targeting of proteins is achieved through the use of transport vesicles/tubules that fuse with specific target compartments. The vesicle protein, Rab 11, has been shown to be a key molecule involved in apical membrane traffic and may even function as a vesicle "address" tag. In general, Rabs function by recruiting effector proteins to transport vesicles. Recent studies have identified several novel Rab 11-binding proteins, including Rip 11, FIP2, and Eferin/FIP3, also known as FIPs. Based on my published and preliminary data, I hypothesize that (i) Rab 11/Rip11 complexes regulate apical membrane traffic by forming "targeting patches" on transport vesicles, (ii) Rab 11/Rip 11 "targeting patch" is regulated by Rip l 1 phosphorylation, and (iii) Rab 11/Rip 11 complex function by recruiting additional proteins, such as kinesin II to transport vesicles. Four specific aims are designed to further analyze the structure and function of Rab 11/effector complexes. First, the structural basis for Rab 11 interactions with Rip 11 will be investigated using domain mapping and X-ray crystallography. Second, we will use the structural information generated by Aim #1 to determine the role of Rip 11 as well as other FIPs in apical and basolateral targeting using in in vivo transport assays. Third, the role of Rip11 phosphorylation in polarized epithelial traffic will be investigated. Forth, identification and characterization of the proteins interacting with Rab 11/Rip 11 complexes. Characterization of Rab 11/Rip11 complex will provide critical mechanistic information for understanding protein targeting in epithelial cells.

描述（由申请人提供）：上皮细胞通过在生物发生和内吞周期中连续排序膜蛋白和脂质的极化分布。蛋白质靶向对于大多数上皮功能至关重要。因此，已知其缺陷导致各种肾脏和消化系统疾病。我的研究的主要目的是研究调节上皮细胞中RAB 11依赖性顶端蛋白的机制。通过使用融合特定目标隔室的运输囊泡/小管来实现蛋白质的靶向。囊泡蛋白Rab 11已被证明是参与顶膜流量的关键分子，甚至可能充当囊泡“地址”标签。通常，Rabs通过募集效应蛋白运输囊泡来发挥作用。最近的研究已经确定了几种新型的RAB 11结合蛋白，包括RIP 11，FIP2和Eferin/Fip3，也称为FIPS。根据我的发布和初步数据，我假设（i）RAB 11/RIP11复合物通过在传输囊泡上形成“靶向斑块”来调节顶端膜流量，（ii）RAB 11/RIP 11“靶向斑块”由RIP L 1 1磷酸化和（III）RAB 11/RIP 11/RIP 11/RIP 11/RIP 11/RIP 11/RIP 11/RIP 11/RIP的运输量来招募其他KOTIRE，例如招募其他K型kine kotiles，例如其他KORTICES，例如招募其他kine knotiles k kotiles k kotiles knotiles knotiles knotiles k k.设计了四个特定的目标，以进一步分析RAB 11/效应子络合物的结构和功能。首先，将使用域映射和X射线晶体学研究RAB 11与RIP 11的结构基础。其次，我们将使用AIM＃1生成的结构信息来确定RIP 11以及其他FIP在顶端和基底外侧靶向中使用体内运输分析的作用。第三，将研究RIP11磷酸化在极化上皮流量中的作用。第四，与RAB 11/RIP 11复合物相互作用的蛋白质的鉴定和表征。 RAB 11/RIP11复合物的表征将提供关键的机械信息，以了解上皮细胞中的蛋白质靶向。