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其与下游效应器连接来决定信号转导的结果。这一建议的重点是突触后谷氨酸信号，介导兴奋性神经传递。谷氨酸受体信号通路由膜相关鸟苷酸激酶（MAGuKs）组织。在兴奋性神经元中存在4种MAGuKs （PSD-95、PSD-93、SAP102和SAP97/Dlg）。关于每种蛋白质在突触可塑性中的具体作用，现有的生化数据与功能数据存在冲突。本课题研究了MAGuK结构和特异性的分子基础。我们对突触后受体-支架相互作用进行了重建，以提供MAGuK对谷氨酸受体的亲和力和选择性缺失的定量数据。受体细胞质结构域附着在平面磷脂双分子层上，形成一个模拟突触后膜的功能化表面。目的1将检验四种PSD-MAGuKs之间的功能差异是由受体结合动力学的差异引起的假设。单分子荧光和模拟将通过将已知结构置于上下文中来解决所有四个全长MAGuKs的结构。我们可以实时观察单个结合事件，量化MAGuK与NMDA和AMPA受体以及Stargazin的结合。Aim 2将验证MAGuK四元结构的差异导致对受体和其他配体结合亲和力的差异的假设。目的3是在体内确认PSD-MAGuKs的结构。表征gfp标记的PSD-95（和活细胞FRET尺子）在体外将形成活细胞FRET测量定量解释的基础。这些研究正朝着PSD组装的物理和动力学描述迈进。我们努力通过重构高阶系统来实现“细胞结构生物学”。这种重构最终将作为一个平台来整合额外的突触后组件。这些结果将表明突触中的可变信号行为有多少可归因于支架本身。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疾病直接相关。

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.