Analysis of Protein-Protein Interactions of Eps15 Using Fluorescence Resonance Energy Transfer (FRET)

使用荧光共振能量转移 (FRET) 分析 Eps15 的蛋白质-蛋白质相互作用

• 批准号: 9904802
• 负责人: Alexander Sorkin
• 金额: $ 10万
• 依托单位: University of Colorado at Denver
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9904802&HistoricalAwards=false
• 关键词: Analysis; Protein; Interactions; Eps15; Using

Endocytosis, the process whereby cells internalize regions of their cell surfaces (plasma membranes), is a critical property of living cells. For single-celled organisms, endocytosis is an important mechanism for sampling the environment and for ingesting microbial food. Cells that are part of a multicellular organism must communicate with one another in order to coordinate their activities. Often, such communication is in the form of signaling molecules, such as hormones, that are produced by one cell type and that bind to specific receptors on other (target) cell types. Endocytosis of the receptor-signal complex is generally then a necessary second step in order for the target cell to respond appropriately to the signal. Endocytosis is also the means whereby cell surface components that are no longer needed are removed from the surface. Because of the critical importance of endocytosis to the lives of cells and organisms, the mechanism of endocytosis has been an important focus of cell biological research. This project addresses a very specific molecular aspect of the endocytic machinery of cells.Eps15 is a recently identified component of the cellular internalization machinery. Eps15 is constitutively associated with the a-adaptin subunit of clathrin adaptor protein AP-2, and is located in the plasma membrane clathrin-coated pits and vesicles. The importance of Eps15 in the clathrin-mediated process has been demonstrated in functional studies that used dominant-negative Eps15 mutants and microinjection techniques. However, the precise function of Eps15 in clathrin vesicle formation is unknown. Eps15 is a prototype of the large family of proteins containing the EH (for Eps15 Homology) domain, conserved protein-protein interaction modules that are found in mammals, frog, fly, nematode and yeast proteins. EH domains of Eps15 bind several proteins containing Asp-Pro-Phe (NPF) sequence motifs. The role of EH domain interactions in endocytosis remains to be determined. Eps15 is also constitutively dimerized/tetramerized, although the mechanism of Eps15 oligomerization is not fully understood. Elucidation of the molecular details of Eps15 interactions is essential for understanding the function of this protein.The goal of the project is to analyze the Eps15 interactions that are important for the steps of endocytosis regulated by Eps15. New technical approaches will be developed to study the dynamics of Eps15 complexes in living cells. A high-resolution microscopic technique called Fluorescence Resonance Energy Transfer (FRET) will be used to investigate protein interactions. FRET is based on the transfer of energy between two proximate fluorochromes. Recent development of green fluorescent proteins (GFP) with different spectral characteristics allows utilization of chimeric proteins tagged with such molecules as tools for the analysis of near-neighbor protein interactions in live cells. Dr. Sorkin has already constructed and characterized a GFP-Eps15 chimera for use in a FRET-based assay. Additional chimeric Eps15 constructs tagged with yellow (YFP) or cyan fluorescent protein (CFP) will be constructed and co-expressed in the same cells to study the mechanisms of dimerization and tetramerization of Eps15. To analyze the interactions of Eps15 with cargo receptors and the components of the internalization machinery, Eps15-CFP will be co-expressed with YFP-tagged epidermal growth factor (EGF) receptor, clathrin adaptors, clathrin assembly lymphoid myeloid (CALM) protein or intersectin. In FRET experiments, CFP, when excited by low energy light of an appropriate wavelength, can transfer the energy directly to YFP if the YFP is located in close proximity, thus allowing the emission of higher energy photons by YFP. The measurements of FRET efficiency on a pixel-by-pixel basis will permit monitoring the protein-protein interactions of Eps15 in live cells in real time, and will also permit the precise determination of where in the cell these interactions occur.In addition to providing important specific information about Eps15 interactions with other Eps15 molecules and other proteins, the development of this sensitive assay to study protein:protein interactions in living cells using Eps15 macromolecular complexes as a model system may lead to a breakthrough in the analysis of the interactions involving various other protein complexes and cellular machineries in living cells.

内吞作用是细胞内化其细胞表面区域（质膜）的过程，是活细胞的关键特性。 对于单细胞生物来说，内吞作用是一种重要的环境取样和摄取微生物食物的机制。 作为多细胞生物体一部分的细胞必须相互交流以协调它们的活动。 通常，这种通信是以信号分子的形式，如激素，由一种细胞类型产生，并与其他（靶）细胞类型上的特定受体结合。 受体-信号复合物的内吞作用通常是为了使靶细胞适当地响应信号所必需的第二步。 内吞作用也是将不再需要的细胞表面成分从表面去除的手段。 由于内吞作用对细胞和生物体的生命至关重要，因此内吞作用的机制一直是细胞生物学研究的一个重要焦点。 本项目主要研究细胞内吞机制的一个非常特殊的分子方面，Eps 15是最近发现的细胞内化机制的一个组成部分。Eps 15与网格蛋白衔接蛋白AP-2的α-衔接素亚基组成型相关，并且位于质膜网格蛋白包被的凹坑和囊泡中。Eps 15在网格蛋白介导的过程中的重要性已在使用显性负性Eps 15突变体和显微注射技术的功能研究中得到证实。 然而，Eps 15在网格蛋白囊泡形成中的确切功能尚不清楚。 Eps 15是包含EH（对于Eps 15同源）结构域的蛋白质大家族的原型，EH结构域是在哺乳动物、青蛙、苍蝇、线虫和酵母蛋白中发现的保守蛋白质-蛋白质相互作用模块。Eps 15的EH结构域结合几种含有Asp-Pro-Phe（NPF）序列基序的蛋白质。 EH结构域相互作用在内吞作用中的作用仍有待确定。 Eps 15也是组成型二聚体/四聚体，尽管Eps 15寡聚化的机制尚未完全理解。 阐明Eps 15相互作用的分子细节对于理解这种蛋白质的功能至关重要。该项目的目标是分析Eps 15相互作用，这些相互作用对于Eps 15调节的内吞作用步骤至关重要。将开发新的技术方法来研究活细胞中Eps 15复合物的动力学。一种称为荧光共振能量转移（FRET）的高分辨率显微技术将用于研究蛋白质相互作用。FRET是基于两个相邻的荧光染料之间的能量转移。具有不同光谱特性的绿色荧光蛋白（GFP）的最新发展允许利用标记有此类分子的嵌合蛋白作为分析活细胞中近邻蛋白相互作用的工具。 Sorkin博士已经构建并表征了用于FRET检测的GFP-Eps 15嵌合体。 将构建标记有黄色（YFP）或青色荧光蛋白（CFP）的另外的嵌合Eps 15构建体并在相同的细胞中共表达以研究Eps 15的二聚化和四聚化的机制。为了分析Eps 15与货物受体和内化机制的组分的相互作用，Eps 15-CFP将与YFP标记的表皮生长因子（EGF）受体、网格蛋白衔接子、网格蛋白组装淋巴髓样（CALM）蛋白或interstin共表达。在FRET实验中，当CFP被适当波长的低能量光激发时，如果YFP位于非常接近的位置，则CFP可以将能量直接转移到YFP，从而允许YFP发射更高能量的光子。在逐像素的基础上测量FRET效率将允许真实的监测活细胞中Eps 15的蛋白质-蛋白质相互作用，并且还将允许精确确定这些相互作用在细胞中的何处发生。用Eps 15大分子复合物作为模型系统，研究活细胞中蛋白质的相互作用，可能会在分析活细胞中各种其他蛋白质复合物和细胞机械的相互作用方面取得突破。