Membrane Organization in Cell Signaling and Adhesion

细胞信号传导和粘附中的膜组织

• 批准号: 7015037
• 负责人: TIONE BURANDA
• 金额: $ 14.08万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7015037
• 关键词: binding sites; biological signal transduction; biophysics; cell adhesion; cell line; cell membrane; cell surface receptors; conformation; flow cytometry; fluorescence resonance energy transfer; green fluorescent proteins; integrins; lipid bilayer membrane; membrane lipids; protein localization; receptor expression

DESCRIPTION (provided by applicant): The overall objective of the proposed research is to understand processes and mechanisms involved in the organization and distribution of proteins and lipids in the plasma membrane of both resting and receptor activated cells. As model systems, we will focus on G-protein coupled receptors (GPCRs) and integrins. Chemokine/chemoattractant GPCRs, such as the N-Formyl peptide receptor (FPR), regulate numerous aspects of leukocyte function, including controlling hematopoiesis, chemotaxis and activation as in the event of physiological insult. GPCR activation has been suggested to lead to a redistribution of receptor into rafts in the plasma membrane. This activation also leads to alterations in the functions of integrins, resulting in clustering and conversion to a high affinity state. Integrin clustering has been suggested to be a critical determinant in regulating the avidity of its binding interactions and hence its physiological function. Thus, both the organization of the protein within the membrane (clustering) and the distribution of lipids about the protein (rafts) are likely to represent critical determinants in the proper functioning of membrane proteins. Although rafts are widely believed to play a critical role in mediating biological functions, it has proven exceedingly difficult to provide a robust characterization of rafts in terms of size, composition, and dynamics. It is noted that chemical composition of membranes is dynamic due to trafficking of vesicles, metabolism of lipids and insertion and removal of lipid components. We propose to use a combination of spectroscopic techniques to image real time changes in the lateral organization of lipid membranes in live cells by measuring energy transfer between fluorescently-labeled lipid probes and receptors bound to fluorescently labeled small molecule ligands. The data will be analyzed in terms of distances of closest approach between donors (on lipid probes or bound to proteins) and acceptors (on lipid probes or proteins). Distance of closest approach in membranes reflect the size of a protein, or conformation (integrins), the presence of boundary lipid which excludes the acceptor, or the distance of the donor above the membrane. The aims are: Aim 1. To characterize the structure function relationship associated with integrin activation and connection to membrane organization. Aim 2. To assess in real time the relationship between GPCR signaling and membrane reorganization. Aim 3. To generate integrin GFP chimerae and cytoplasmic tail alpha/L and beta2 mutants of alpha4beta1 and assess lateral organization in K562 cells. The proposed work provides a unique training opportunity to use biophysical tools and concepts combined with molecular and cell biological approaches to address problems of immense interest to cell biology. Completion of the proposed work will yield a better mechanistic grasp of the dynamics of membrane organization as a factor in signaling and cell adhesion.

描述（由申请人提供）：拟议研究的总体目标是了解静息和受体激活细胞质膜中蛋白质和脂质组织和分布所涉及的过程和机制。作为模型系统，我们将集中在G蛋白偶联受体（GPCR）和整合素。趋化因子/化学引诱物GPCR，如N-甲酰肽受体（FPR），调节白细胞功能的许多方面，包括在生理损伤的情况下控制造血、趋化性和活化。GPCR活化已被认为导致受体重新分布到质膜中的筏中。这种激活也导致整合素功能的改变，导致聚集和转化为高亲和力状态。整合素成簇已被认为是调节其结合相互作用的亲合力并因此调节其生理功能的关键决定因素。因此，蛋白质在膜内的组织（聚集）和脂质在蛋白质周围的分布（筏）可能是膜蛋白正常功能的关键决定因素。 虽然筏被广泛认为在介导生物功能中起着关键作用，但事实证明，在大小、组成和动力学方面提供筏的稳健表征是非常困难的。值得注意的是，由于囊泡的运输、脂质的代谢以及脂质组分的插入和去除，膜的化学组成是动态的。我们建议使用光谱技术的组合来成像真实的时间变化的横向组织的脂质膜在活细胞中通过测量荧光标记的脂质探针和受体之间的能量转移结合到荧光标记的小分子配体。将根据供体（在脂质探针上或与蛋白质结合）和受体（在脂质探针或蛋白质上）之间最接近的距离分析数据。膜中最接近的距离反映了蛋白质的大小或构象（整联蛋白），排除受体的边界脂质的存在，或供体在膜上方的距离。目标是：目标1。表征与整合素活化和与膜组织连接相关的结构功能关系。目标2.真实的评估GPCR信号传导与膜重组之间的关系。目标3.产生整合素GFP嵌合体和α 4 β 1的胞质尾区α/L和β 2突变体，并评估K562细胞中的横向组织。拟议的工作提供了一个独特的培训机会，使用生物物理工具和概念结合分子和细胞生物学方法，以解决细胞生物学的巨大利益的问题。完成拟议的工作将产生一个更好的机械把握膜组织的动态信号和细胞粘附的一个因素。