Regulation of AKAP79 Postsynaptic Membrane Targeting

描述(申请人提供)：信号通路组织的中心是支架和锚定蛋白质，它们介导包含受体、第二信使酶、激酶、磷酸酶和底物的蛋白质复合体的局部组装。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中的受体活性、定位和突触结构，学习和记忆中的突触可塑性，以及中风、神经变性、癫痫、慢性疼痛、精神分裂症和药物成瘾等神经病理中发挥核心作用。特别是，AKAP79/150锚定的PKA和CaN在NMDA受体依赖的LTP和LTD可塑性中可能调节AMPA受体的磷酸化和突触定位，以及去甲肾上腺素和多巴胺对其调节。AKAP79/150的突触后靶向是由一个N端区域介导的，该区域结合了PIP2、F-肌动蛋白和钙粘附素黏附分子。在LTD中，AKAP79/150结合MAGUKs和钙粘蛋白的定位被F-肌动蛋白稳定，并被参与AMPA受体调控的NMDA受体-CaN信号通路破坏。因此，PKA和CaN的核心以及AKAP与钙粘素细胞骨架和Maguk受体复合体的连接可以调节突触的结构和功能。这些问题将通过特定目标1：通过激活PLC对AKAP79/150、AMPAR和F-肌动蛋白突触后定位的NMDAR调节来研究。假设：PLC介导的PIP2水解酶的NMDAR激活是突触失去AKAP79/150和AMPAR、F-肌动蛋白重塑和诱导LTD所必需的。这一假说将通过细胞转染、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片中得到验证。具体目标2：Anchored-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：突触中锚定-CaN和AKAP79/150-PKA缺失的NMDAR调节控制AMPAR的定位和活性。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的表达通过N端靶向结构域增加兴奋性突触的数量，并通过与MAGUKs和AMPAR招募的相互作用增加突触的大小和强度。AIMS 2和AIMS 3中的假设将通过AKAP150的RNAi敲除、野生型和突变型AKAP79蛋白的转染、荧光成像和电生理学在海马神经元中得到验证。

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.