Single Molecule Analysis of MAGUK Structure and Ligand Binding

描述(由申请人提供)：我们的目标是了解支架蛋白在信号转导组织中的作用。支架通过控制细胞表面受体的位置并将它们连接到下游效应器来决定信号转导的结果。这项建议的重点是突触后谷氨酸信号，它介导兴奋性神经传递。谷氨酸受体信号通路是由膜相关鸟苷酸激酶(MAGuKs)组成的。兴奋性神经元中有4种MAGuK(PSD-95、PSD-93、SAP102和SAP97/DLG)。现有的生化数据与关于每个蛋白质在突触可塑性中所起的具体作用的功能数据相冲突。这一建议研究了Maguk结构和特异性的分子基础。我们在突触后对受体-支架相互作用进行了工作重建，以提供关于谷氨酸受体的Maguk亲和力和选择性的缺失的定量数据。受体胞质结构域连接到一个平面磷脂双层上，创建了一个模仿突触后膜的功能化表面。目的1将验证四种PSD-MAGuK之间的功能差异是由于受体结合动力学的不同而产生的假设。单分子荧光和模拟将通过将已知结构放在上下文中来解决所有四个全长MAGuK的结构。我们可以实时观察单个结合事件，以定量Maguk与NMDA和AMPA受体以及Stargazin的结合。目的2将检验这一假说，即Maguk四级结构的差异会导致受体和其他配体结合亲和力的差异。目的3确定PSD-MAGuKs的体内结构。GFP标记的PSD-95(和活细胞FRET标尺)的体外特征将形成对活细胞FRET测量的定量解释的基础。这些研究正在朝着PSD组装的物理和动力学描述的方向发展。我们努力通过重组高阶系统来实现“细胞结构生物学”。这种重建最终将成为整合更多突触后组件的平台。这些结果将表明突触中的可变信号行为在多大程度上可归因于支架本身。描述兴奋性信号中的分子事件是神经科学中的一个基本挑战，与大脑发育、记忆和学习以及许多神经和神经精神障碍直接相关。

DESCRIPTION (provided by applicant): Our goal is to understand the role of scaffold proteins in the organization of signal transduction. Scaffolds determine the outcome of signal transduction by controlling the location of cell surface receptors and connecting them to downstream effectors. This proposal focuses on post-synaptic glutamate signaling, which mediates excitatory neurotransmission. Glutamate receptor signaling pathways are organized by the membrane-associated guanylate kinases (MAGuKs). In excitatory neurons there are four MAGuKs (PSD-95, PSD-93, SAP102 and SAP97/Dlg). Existing biochemical data is in conflict with functional data regarding the specific role each protein plays in synaptic plasticity. This proposal investigates the molecular basis of MAGuK structure and specificity. We have a working reconstitution of receptor-scaffold interactions in the post-synapse to provide the missing quantitative data on MAGuK affinity and selectivity for glutamate receptors. Receptor cytoplasmic domains are attached to a planar phospholipid bilayer, creating a functionalized surface that mimics the postsynaptic membrane. Aim 1 will test the hypothesis that functional differences between the four PSD-MAGuKs arises from differences in the kinetics of receptor binding. Single molecule fluorescence and simulations will solve the structure of all four, full-length MAGuKs by placing the known structures in context. We can watch individual binding events in real time to quantitate the MAGuK binding to both NMDA and AMPA receptors and also Stargazin. Aim 2 will test the hypothesis that differences in MAGuK quaternary structure give rise to differences in binding affinity for receptors and other ligands. Aim 3 is to confirm te structure of PSD-MAGuKs in vivo. Characterization of the GFP-tagged PSD-95 (and a live-cell FRET ruler) in vitro will form the basis for quantitative interpretation of live-cell FRET measurements. These studies are advancing towards a physical and kinetic description of PSD assembly. We strive to achieve a "cellular structural biology" by reconstituting higher-order systems. This reconstitution will eventually serve as a platform to incorporate additional post synaptic components. These results will indicate how much of the variable signaling behavior in the synapse is attributable to the scaffold itself. Describing the molecular events in excitatory signaling is a fundamental challenge in neuroscience with direct relevance to brain development, memory and learning, and many neurological and neuropsychiatric disorders.