Regulation of AKAP79 Postsynaptic Membrane Targeting

AKAP79 突触后膜靶向的调节

基本信息

批准号：
7472488
负责人：
MARK L DELL'ACQUA
金额：
$ 33.29万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-08-01 至 2009-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7472488
关键词：
A kinase anchoring protein AMPA Receptors Actins Acute Adaptor Signaling Protein Adrenergic Agents Antibodies Binding Biochemical Biological Assay Cadherins Calcineurin Cell Adhesion Molecules Cell Fractionation Cells Chromosome Pairing Complex Cyclic AMP-Dependent Protein Kinases Cytoskeleton DLG1 gene Dendritic Spines Development Disruption Dopamine Drug Addiction Electrophysiology (science)Enzymes Epilepsy Event Excision Excitatory Synapse F-Actin Family Fluorescence Resonance Energy Transfer Funding Glutamate Receptor Hippocampus (Brain)Human Hydrolysis Image In Vitro Lead Learning Link Localized Long-Term Depression Long-Term Potentiation Maps Mediating Memory Methods Morphology N-Methyl-D-Aspartate Receptors N-Methylaspartate N-terminal Nerve Degeneration Neurons Norepinephrine Numbers PH Domain Pathway interactions Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phospholipase C Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Play Positioning Attribute Postsynaptic Membrane Production Protein Dephosphorylation Protein Kinase Protein Overexpression Proteins RNA Interference Rattus Receptor Signaling Recruitment Activity Regulation Role Role playing therapy Scaffolding Protein Schizophrenia Second Messenger Systems Signal Pathway Signal Transduction Signaling Protein Slice Stroke Structure Synapses Synaptic plasticity Tertiary Protein Structure Testing Thinking Transfection Vertebral column Week Whole-Cell Recordings adrenergic alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid amino 3 hydroxy 5 methylisoxazole 4 propionate chronic pain day density excitotoxicity extracellular fluorescence imaging knock-down link protein membrane-associated guanylate kinase mutant neuropathology novel postsynaptic presynaptic density protein 95 receptor receptor structure function response scaffold second messenger size synaptogenesis trafficking transmission process

项目摘要

DESCRIPTION (provided by applicant): Central to organization of signaling pathways are scaffolding and anchoring proteins that mediate localized assembly of protein complexes containing receptors, second messenger enzymes, kinases, phosphatases, and substrates. AKAP79/150 is an excitatory postsynaptic PKA and protein phosphatase 2B/Calcineurin (CaN) anchoring protein linked to NMDA and AMPA glutamate receptors through PSD-95 family MAGUK scaffolds. These assemblies are thought to play central roles in regulating receptor activity, localization, and synaptic structure in synapse formation during development, synaptic plasticity in learning and memory, and neuropathologies such as excitotoxicity in stroke, neurodegeneration, epilepsy, chronic pain, schizophrenia and drug addiction. In particular, postsynaptic signaling functions of AKAP79/150-anchored PKA and CaN may regulate AMPA receptor phosphorylation and synaptic localization in NMDA receptor-dependent LTP and LTD plasticity and its modulation by norepinephrine and dopamine. Postsynaptic targeting of AKAP79/150 is mediated by an N-terminal region that binds PIP2, F-actin, and cadherin adhesion molecules. Localization of AKAP79/150 with MAGUKs and cadherins is stabilized by F-actin and disrupted by NMDA receptor-CaN signaling pathways implicated in AMPA receptor regulation in LTD. Thus, PKA and CaN anhcoring as well as linkage of the AKAP to cadherin-cytoskeletal and MAGUK-receptor complexes could regulate synaptic structure and function. These issues will be investigated through Specific Aim 1: NMDAR regulation of AKAP79/150, AMPAR, and F-actin postsynaptic localization through PLC activation. Hypothesis: NMDAR activation of PLC-mediated PIP2 hydrolysis is necessary for loss of AKAP79/150 and AMPARs from synapses, remodeling of F-actin, and induction of LTD. This hypothesis will be tested using cellular transfection, fluorescence imaging, biochemical and electrophysiological methods in cultured hippocampal neurons and acute hippocampal slices. Specific Aim 2: Role of anchored-PKA and CaN in NMDAR regulation of AKAP79/150 and AMPAR postsynaptic localization and activity. Hypothesis: NMDAR regulation of anchored-CaN and AKAP79/150-PKA loss from synapses control AMPAR localization and activity. Specific Aim 3: Regulation of synapse development by AKAP79/150 targeting domain and MAGUK interactions. Hypothesis: AKAP79 expression increases excitatory synapse number through the N-terminal targeting domain and synaptic size and strength through interactions with MAGUKs and AMPAR recruitment. The hypotheses in aims 2 and 3 will be tested in hippocampal neurons using RNAi knock-down of AKAP150, transfection of wild-type and mutant AKAP79 proteins, fluorescence imaging and electrophysiology.

描述（由申请人提供）：信号传导途径组织的核心是支架蛋白和锚定蛋白，它们介导含有受体、第二信使酶、激酶、磷酸酶和底物的蛋白质复合物的局部组装。 AKAP79/150 是一种兴奋性突触后 PKA 和蛋白磷酸酶 2B/钙调神经磷酸酶 (CaN) 锚定蛋白，通过 PSD-95 家族 MAGUK 支架与 NMDA 和 AMPA 谷氨酸受体连接。这些组件被认为在调节发育过程中突触形成的受体活性、定位和突触结构、学习和记忆中的突触可塑性以及中风、神经退行性疾病、癫痫、慢性疼痛、精神分裂症和药物成瘾中的兴奋性毒性等神经病理学方面发挥着核心作用。特别是，AKAP79/150 锚定的 PKA 和 CaN 的突触后信号传导功能可能调节 NMDA 受体依赖性 LTP 和 LTD 可塑性中的 AMPA 受体磷酸化和突触定位及其通过去甲肾上腺素和多巴胺的调节。 AKAP79/150 的突触后靶向是由结合 PIP2、F-肌动蛋白和钙粘蛋白粘附分子的 N 末端区域介导的。 AKAP79/150 与 MAGUK 和钙粘蛋白的定位由 F-肌动蛋白稳定，并被与 LTD 中 AMPA 受体调节有关的 NMDA 受体-CaN 信号通路破坏。因此，PKA 和 CaN 锚定以及 AKAP 与钙粘蛋白-细胞骨架和 MAGUK-受体复合物的连接可以调节突触结构和功能。这些问题将通过具体目标 1 进行研究：NMDAR 通过 PLC 激活对 AKAP79/150、AMPAR 和 F-肌动蛋白突触后定位进行调节。假设：NMDAR 激活 PLC 介导的 PIP2 水解对于突触中 AKAP79/150 和 AMPAR 的丢失、F-肌动蛋白的重塑和 LTD 的诱导是必要的。该假设将使用细胞转染、荧光成像、生化和电生理学方法在培养的海马神经元和急性海马切片中进行测试。具体目标 2：锚定 PKA 和 CaN 在 NMDAR 调节 AKAP79/150 和 AMPAR 突触后定位和活性中的作用。假设：NMDAR 对突触锚定 CaN 和 AKAP79/150-PKA 损失的调节控制 AMPAR 定位和活性。具体目标 3：通过 AKAP79/150 靶向结构域和 MAGUK 相互作用调节突触发育。假设：AKAP79 表达通过 N 端靶向结构域增加兴奋性突触数量，并通过与 MAGUK 和 AMPAR 募集的相互作用增加突触大小和强度。目标 2 和 3 中的假设将使用 RNAi 敲低 AKAP150、转染野生型和突变型 AKAP79 蛋白、荧光成像和电生理学在海马神经元中进行测试。