Single Molecule Analysis of MAGUK Structure and Ligand Binding

MAGUK 结构和配体结合的单分子分析

• 批准号: 8370963
• 负责人: Mark E Bowen
• 金额: $ 37.92万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-10 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8370963
• 关键词: AMPA Receptors; Address; Affinity; Autistic Disorder; Behavior; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biosensor; Brain; Cell Surface Receptors; Cell membrane; Cells; Chimeric Proteins; Communication; Complex; Conflict (Psychology); Crowding; Cytoplasmic Tail; DLG1 gene; Data; Development; Dimensions; Disease; Epilepsy; Event; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Glutamate Receptor; Glutamates; Goals; Guanylate kinase; Heterogeneity; Higher Order Chromatin Structure; In Vitro; Individual; Isomerism; Kinetics; Lateral; Learning; Length; Life; Ligand Binding; Ligands; Link; Location; Measurement; Measures; Mediating; Membrane; Memory; Methodology; Methods; Molecular; Molecular Conformation; Mutagenesis; N-Methyl-D-Aspartate Receptors; Nature; Nerve Degeneration; Neurologic; Neurons; Neurosciences; Outcome; Peptides; Phospholipids; Physiological; Play; Positioning Attribute; Postsynaptic Membrane; Property; Protein Binding; Protein Isoforms; Proteins; Receptor Signaling; Role; SH3 Domains; Scaffolding Protein; Schizophrenia; Signal Pathway; Signal Transduction; Solutions; Specificity; Stroke; Structure; Surface; Synapses; Synaptic plasticity; System; Tertiary Protein Structure; Testing; Time; Video Microscopy; Work; base; density; excitatory neuron; in vivo; membrane-associated guanylate kinase; molecular dynamics; mutant; neuropsychiatry; neurotransmission; physical model; protein structure; public health relevance; receptor; receptor binding; reconstitution; restraint; scaffold; simulation; single molecule; single-molecule FRET; stargazin; structural biology; trafficking

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.

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连接到下游效应器来确定信号转导的结果。该提案的重点是突触后谷氨酸信号传导，它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 (MAGuK) 组织。兴奋性神经元中有四种 MAGuK（PSD-95、PSD-93、SAP102 和 SAP97/Dlg）。现有的生化数据与有关每种蛋白质在突触可塑性中所发挥的特定作用的功能数据相冲突。该提案研究了 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有效重建，以提供有关 MAGuK 亲和力和谷氨酸受体选择性的缺失定量数据。受体细胞质结构域附着在平面磷脂双层上，形成模仿突触后膜的功能化表面。 目标 1 将检验以下假设：四种 PSD-MAGuK 之间的功能差异源于受体结合动力学的差异。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 MAGuK 的结构。我们可以实时观察各个结合事件，以定量 MAGuK 与 NMDA 和 AMPA 受体以及 Stargazin 的结合。目标 2 将检验 MAGuK 四级结构的差异导致受体和其他配体的结合亲和力差异的假设。目标 3 是确认 PSD-MAGuK 的体内结构。 GFP 标记的 PSD-95（和活细胞 FRET 标尺）的体外表征将构成活细胞 FRET 测量定量解释的基础。 这些研究正在朝着 PSD 组装的物理和动力学描述方向发展。我们努力通过重构高阶系统来实现“细胞结构生物学”。这种重建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传导行为有多少可归因于支架本身。描述兴奋性信号传导中的分子事件是神经科学中的一项基本挑战，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。