Regulation of AKAP79 Postsynaptic Membrane Targeting

AKAP79 突触后膜靶向的调节

• 批准号: 7071237
• 负责人: MARK L DELL'ACQUA
• 金额: $ 34.29万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7071237
• 关键词: A kinase anchoring protein; AMPA receptors; NMDA receptors; RNA interference; actins; electrophysiology; enzyme activity; fluorescence microscopy; fluorescent dye /probe; hippocampus; infant animal; laboratory rat; neural plasticity; phospholipase C; phosphorylation; protein kinase A; protein localization; protein protein interaction; receptor expression; synapses; synaptogenesis; tissue /cell culture; transfection

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）：信号通路组织的核心是脚手架和锚定蛋白质，这些蛋白质介导了含有受体的蛋白质络合物的局部组装，这些蛋白质复合物，第二信使酶，激酶，磷酸酶和底物。 AKAP79/150是通过PSD-95系列Maguk脚手架链接到与NMDA和AMPA谷氨酸受体相关的蛋白质的兴奋性突触后PKA和蛋白质磷酸酶2B/CANINERIN（CAN）锚定蛋白。人们认为这些组件在调节受体活性，定位和突触结构中起着核心作用。特别是，AKAP79/150锚定的PKA的突触后信号传导功能可以调节NMDA受体依赖性LTP和LTD塑性中的AMPA受体磷酸化和突触定位，以及其对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。通过F-肌动蛋白稳定AKAP79/150，用Maguks和cadherin的定位被与AMPA受体调节LTD中有关的NMDA受体式信号传导途径而稳定。因此，PKA和可以安装的AKAP与Cadherin-Cytoskeletal和Maguk-topeptor络合物的链接可以调节突触结构和功能。这些问题将通过特定目标1：AKAP79/150的NMDAR调节，AMPAR和F-肌动蛋白后突触的定位通过PLC激活。假设：PLC介导的PIP2水解的NMDAR激活对于丧失AKAP79/150的损失和突触中的AMPAR是必需的，F-肌动蛋白的重塑和LTD诱导。该假设将在培养的海马神经元和急性海马切片中使用细胞转染，荧光成像，生化和电生理方法进行检验。具体目标2：锚定-PKA和CAN在AKAP79/150和AMPAR突触后定位和活动中的NMDAR调节中的作用。假设：锚定-can和AKAP79/150-PKA损失的NMDAR调控突触控制AMPAR定位和活性。特定目标3：通过AKAP79/150靶向域和Maguk相互作用来调节突触发展。假设：AKAP79表达通过与Maguks和AMPAR募集的相互作用，通过N末端靶向域以及突触的大小和强度增加兴奋性突触数。 AIM 2和3中的假设将在海马神经元中使用AKAP150的RNAi敲低，野生型和突变型AKAP79蛋白，荧光成像和电生理学的转染。