Membrane Organization in Cell Signaling and 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的细胞质膜中蛋白质和脂类的组织和分布过程和机制。作为模型系统，我们将重点研究G蛋白偶联受体(GPCRs)和整合素。趋化因子/趋化因子GPCRs，如N-甲酰肽受体(FPR)，调节白细胞功能的许多方面，包括在生理损伤时控制造血、趋化和激活。GPCR的激活被认为是导致受体重新分布到质膜上的筏子中的原因。这种激活也导致整合素功能的改变，导致聚集和转换到高亲和力状态。整合素聚集被认为是调节其结合相互作用亲和力的关键决定因素，从而调节其生理功能。因此，膜内蛋白质的组织(聚集性)和蛋白质周围的脂类分布(筏)都可能是膜蛋白正常功能的关键决定因素。虽然人们普遍认为木筏在调节生物功能方面起着关键作用，但事实证明，从大小、组成和动力学方面提供木筏的可靠特征是非常困难的。值得注意的是，膜的化学成分是动态的，这是由于囊泡的运输、脂类的新陈代谢以及脂类成分的插入和移除。我们建议使用光谱技术的组合来通过测量荧光标记的脂类探针和与荧光标记的小分子配体结合的受体之间的能量转移来实时成像活细胞中脂膜的横向组织的实时变化。这些数据将根据供体(在脂类探针上或与蛋白质结合)和受体(在脂类探针或蛋白质上)之间的最接近距离进行分析。膜上最接近的距离反映了蛋白质或构象(整合素)的大小，不包括受体的边界脂的存在，或供体在膜上的距离。目的：1.研究整合素活化与膜组织连接的结构与功能关系。目的2.实时评价GPCR信号转导与膜重组的关系。目的3.构建整合素GFP嵌合体和α4β1胞质尾部α/L和β2突变体，并对K562细胞的侧向组织进行检测。拟议的工作提供了一个独特的培训机会，利用生物物理工具和概念与分子和细胞生物学方法相结合，解决细胞生物学极感兴趣的问题。这项拟议工作的完成将更好地从机械上把握作为信号和细胞黏附因素的膜组织的动力学。

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.