Regulation of AKAP79 Postsynaptic Membrane Targeting

描述（由申请人提供）：组织信号通路的核心是支架和锚定蛋白，其介导含有受体、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。AKAP 79/150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。这些组装体被认为在调节受体活性、定位和发育期间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学如中风中的兴奋性毒性、神经变性、癫痫、慢性疼痛、精神分裂症和药物成瘾中发挥核心作用。特别是，AKAP 79/150锚定PKA和CaN的突触后信号传导功能可以调节AMPA受体磷酸化和NMDA受体依赖性LTP和LTD可塑性的突触定位以及去甲肾上腺素和多巴胺对其的调节。AKAP 79/150的突触后靶向由结合PIP 2、F-肌动蛋白和钙粘蛋白粘附分子的N-末端区域介导。AKAP 79/150与MAGUK和钙粘蛋白的定位被F-肌动蛋白稳定，并被参与LTD中AMPA受体调节的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：NMDAR调节AKAP 79/150，AMPAR，和F-肌动蛋白突触后定位通过PLC激活进行研究。假设：NMDAR激活PLC介导的PIP 2水解是必要的损失AKAP 79/150和AMPAR从突触，重塑F-肌动蛋白，诱导LTD。这一假设将使用细胞转染，荧光成像，生物化学和电生理方法在培养的海马神经元和急性海马切片进行测试。具体目标2：PKA和CaN在AKAP 79/150和AMPAR突触后定位和活性的NMDAR调节中的作用。假设：NMDAR调节突触中的钙调素和AKAP 79/150-PKA的丢失控制AMPAR的定位和活性。具体目标3：通过AKAP 79/150靶向结构域和MAGUK相互作用调节突触发育。假设：AKAP 79表达通过N-末端靶向结构域增加兴奋性突触数量，并通过与MAGUKs和AMPAR募集的相互作用增加突触大小和强度。将使用AKAP 150的RNAi敲低、野生型和突变体AKAP 79蛋白的转染、荧光成像和电生理学在海马神经元中测试目标2和3中的假设。

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.