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）：我们的目标是了解脚手架蛋白在信号转导组织中的作用。脚手架通过控制细胞表面受体的位置并将其连接到下游效应子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，该信号传导介导兴奋性神经传递。谷氨酸受体信号通路是由膜相关的鸟苷酸激酶（MAGUKS）组织的。在兴奋性神经元中，有四个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扮演的特定作用的功能数据冲突。该提案研究了maguk结构和特异性的分子基础。我们在后肿块中有一个有效的重构受体 - 棍棒相互作用，以提供有关Maguk亲和力的丢失的定量数据和谷氨酸受体的选择性。受体细胞质结构域附着在平面磷脂双层上，形成一个官能化的表面，模仿突触后膜。 AIM 1将检验以下假设：四个PSD-Maguks之间的功能差异来自受体结合动力学的差异。单分子荧光和仿真将通过将已知结构放置在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，以定量MAGUK与NMDA和AMPA受体以及Stargazin的结合。 AIM 2将检验以下假设：Maguk第四纪结构的差异会导致对受体和其他配体的结合亲和力差异。目标3是确认体内PSD-Maguks的TE结构。在体外表征GFP标记的PSD-95（和活细胞量标尺）将构成定量解释活细胞FRET测量值的基础。 这些研究正在朝着PSD组装的物理和动力学描述前进。我们努力通过重建高阶系统来实现“细胞结构生物学”。该重构最终将作为结合其他邮政突触组件的平台。这些结果将表明突触中有多少可变信号行为归因于支架本身。描述兴奋性信号传导中的分子事件是神经科学的基本挑战，与大脑发育，记忆和学习直接相关，以及许多神经系统和神经精神疾病。