Regulation of AKAP79 Postsynaptic Membrane Targeting

描述（由申请人提供）：信号通路组织的核心是支架和锚定蛋白，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/钙调磷酸酶（CaN）锚定蛋白，通过PSD-95家族MAGUK支架连接NMDA和AMPA谷氨酸受体。这些组合被认为在调节受体活性、定位和发育过程中突触形成中的突触结构、学习和记忆中的突触可塑性以及神经病理如中风、神经变性、癫痫、慢性疼痛、精神分裂症和药物成瘾等方面发挥核心作用。特别是，akap79 /150锚定的PKA和CaN的突触后信号功能可能调节AMPA受体磷酸化和突触定位在NMDA受体依赖的LTP和LTD可塑性及其由去甲肾上腺素和多巴胺调节。AKAP79/150的突触后靶向是由结合PIP2、F-actin和cadherin粘附分子的n端区域介导的。AKAP79/150与MAGUKs和cadherins的定位被F-actin稳定，并被涉及AMPA受体调节的NMDA受体- can信号通路破坏。因此，PKA和CaN的锚定以及AKAP与钙粘蛋白-细胞骨架和maguk受体复合物的连锁可以调节突触的结构和功能。这些问题将通过Specific Aim 1: NMDAR调节AKAP79/150、AMPAR和F-actin通过PLC激活突触后定位来研究。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。这一假设将通过细胞转染、荧光成像、生化和电生理方法在培养的海马神经元和急性海马切片中进行检验。特异性目的2：锚定pka和CaN在NMDAR调控AKAP79/150和AMPAR突触后定位和活性中的作用。假设：NMDAR调节锚定的can和突触中AKAP79/150-PKA的缺失控制AMPAR的定位和活性。特异性目标3:AKAP79/150靶向结构域和MAGUK相互作用对突触发育的调控。假设：AKAP79的表达通过n端靶向结构域增加兴奋性突触数量，通过与MAGUKs和AMPAR募集的相互作用增加突触的大小和强度。目的2和3中的假设将通过RNAi敲除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。

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.