Subunit-Specific Regulation Of Glutamate Receptors

谷氨酸受体的亚基特异性调节

• 批准号: 10017630
• 负责人: Katherine Roche
• 金额: $ 191.81万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10017630
• 关键词: AMPA Receptors; Affect; Animals; Behavioral; Binding; Biochemical; Biological; Brain; Brain region; C-terminal; Calcium; Cells; Complex; Corpus striatum structure; Data; Databases; Defect; Dendritic Spines; Development; Dynamin; Endocytosis; Endosomes; Epilepsy; Excitatory Synapse; Exclusion; Family; Family member; Functional disorder; G-Protein-Coupled Receptors; Glutamate Receptor; Glutamates; Guanosine Triphosphate Phosphohydrolases; Hippocampus (Brain); Human Genetics; Impairment; Individual; Kainic Acid Receptors; Knock-in Mouse; Knock-out; Knockout Mice; Laboratory Study; Ligands; Literature; Long-Term Depression; Long-Term Potentiation; Mental disorders; Metabotropic Glutamate Receptors; Missense Mutation; Mitochondria; Modeling; Molecular; Mus; Mutate; Mutation; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; Names; Neuraxis; Neurons; Neurotransmitter Receptor; Neurotransmitters; Paper; Patients; Phenotype; Phosphorylation; Play; Point Mutation; Post-Translational Protein Processing; Protein Dephosphorylation; Protein Overexpression; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Proto-Oncogene Proteins c-fyn; Publications; Publishing; Regulation; Reporting; Research; Role; Scaffolding Protein; Stimulus; Structure; Structure-Activity Relationship; Surface; Synapses; Synaptic Transmission; Synaptic plasticity; Techniques; Testing; Tyrosine; Ubiquitination; Variant; Vertebral column; autism spectrum disorder; base; bench to bedside; casein kinase II; deep sequencing; density; experience; experimental study; in vivo; knock-down; knockout animal; multicatalytic endopeptidase complex; nervous system disorder; neurotransmission; neurotransmitter release; novel strategies; postnatal; postsynaptic; postsynaptic density protein; protein protein interaction; receptor; receptor expression; response; synaptic function; trafficking; ubiquitin-protein ligase

The unique distribution of neurotransmitter receptors and their subtypes within a single cell and throughout the brain requires highly selective intracellular targeting mechanisms. My laboratory studies the regulation of glutamate receptor trafficking and localization using a combination of biochemical and molecular techniques. Glutamate receptors are the major excitatory neurotransmitter receptors in the mammalian brain and are a diverse family with many different subtypes. The ionotropic glutamate receptors include AMPA, NMDA, and kainate receptor subtypes, each of which are formed from a variety of subunits. The metabotropic glutamate receptors (mGluR1-8) are G protein-coupled receptors (GPCRs), which are assembled as homodimers. We focus on defining subunit-specific mechanisms that regulate the synaptic localization and functional regulation of glutamate receptors as well as synaptic scaffolding proteins. These mechanisms include posttranslational modifications such as phosphorylation and ubiquitination, as well as protein-protein interactions. A major focus of the lab is the study of the molecular mechanisms regulating the trafficking of NMDA receptors, which are multi-subunit complexes (GluN1; GluN2A-D; GluN3A-B). We have made significant progress in the detailed characterization of the synaptic expression of NMDARs and the role of GluN2A and GluN2B in receptor trafficking and synaptic expression. NMDA receptors are removed from synapses in an activity- and calcium-dependent manner, via casein kinase 2 (CK2) phosphorylation of the PDZ-ligand of the GluN2B subunit (S1480). We find that the NR2B subunit, and not NR2A, is specifically phosphorylated by CK2 and phosphorylation of NR2B increases in the second postnatal week and is important in the subunit switch (GluN2B to GluN2A), which takes place in many cortical regions during development and in response to activity. These data support unique contributions of the individual NMDA receptor subunits to NMDA receptor trafficking and localization. Our studies have shown that a single point mutation in the GluN2B C-terminus (E1479Q) totally blocks CK2 phosphorylation of S1480 and results in significant increases in synaptic GluN2B. We are currently generating a line of genetically-altered mice: a knock-in mouse expressing a point-mutated non-phosphorylatable GluN2B subunit (GluN2B E1479Q). This knock-in mouse will allow us to examine the precise regulation of GluN2B S1480 phosphorylation in neurons, in vivo, and without the requirement of exogenous protein overexpression. It is anticipated that these animals would show an impaired developmental GluN2 subunit switch (Sanz-Clemente et al, 2010). In addition, we are generating a knock-in mouse, GluN2B S1480E, which mimics phosphorylation of that key residue and constitutively blocks binding to PSD-95. Our experiments have shown us this mutation results in very low surface and synaptic expression of GluN2B receptors, thus we expect the mouse to have synaptic deficits as well. We are also exploring the role of tyrosine kinases and phosphatases on the regulation of synaptic NMDARs. GluN2B contains a classic tyrosine-based endocytic motif (-YEKL) that is a strong regulator of NMDAR surface expression. Both the tyrosine kinase Fyn and the tyrosine phosphatase striatal-enriched protein tyrosine phosphatase (STEP) target Y1472, which affects endocytosis and synaptic expression of receptors. In particular, STEP reduces the surface expression of NMDARs by promoting dephosphorylation of GluN2B Y1472, whereas the synaptic scaffolding protein postsynaptic density protein 95 (PSD-95) stabilizes the surface expression of NMDARs via direct binding to the C-terminal PDZ ligand (-ESDV). We have discovered that STEP61 binds to PSD-95 but not to other PSD-95 family members. In addition, PSD-95 expression triggers the degradation of STEP61 via ubiquitination and degradation by the proteasome. Surprisingly, we found that STEP61 is not enriched in the PSD fraction. However, STEP61 expression in the PSD is increased upon knockdown of PSD-95 or in vivo as detected in PSD-95-KO mice, demonstrating that PSD-95 excludes STEP61 from the PSD. An important consequence of STEP having low abundance at the PSD is that only extrasynaptic NMDAR expression and currents were increased upon STEP knock-down. Therefore, our findings support a dual role for PSD-95 in stabilizing synaptic NMDARs by binding directly to GluN2B but also by promoting synaptic exclusion and degradation of the negative regulator STEP61. More recently, we have characterized the effect of STEP expression on AMPARs. We find that STEP regulates the synaptic expression of GluA2 and GluA3 subunits of AMPARs and we are currently exploring the molecular underpinnings of this effect in detail. Our data support a model in which STEP regulates the synaptic and extrasynaptic organization of AMPA and NMDA receptors. It is intriguing that removing STEP from neurons by knock-down or knock-out strategies results in increased extrasynaptic NMDA receptors, but increased synaptic AMPA receptors. And, the effects are subunit-specific. Over the last few years, we have focused on a new approach to studying structure/function of NMDARs. We are trying a "bedside-to-bench" approach to help guide us in testing receptor domains that are important for synaptic function. In particular, we used information from published papers and public databases that report variants identified by deep sequencing of patients with neurological or psychiatric disorders. We then began conducting experiments on missense variants identified in the intracellular C-terminal domain of the GluN2B NMDAR subunit. We found that one mutation in particular, identified in a patient with autism, reduced the surface expression of GluN2B as well as the binding to PSD-95. This variant, GluN2B S1415L (S1413L in mouse), showed a deficit in rescue of synaptic NMDAR currents and fewer dendritic spines. This phenotype is interesting because there are many examples in the literature of spine abnormalities being associated with autism. More broadly, this research shows that using patient data is an effective approach to probing the structure/function relationship of NMDARs. We have now generated a knock-in mouse of GluN2B S1413L and we are characterizing its phenotype. Our biochemical studies already reveal a region-specific effect, with hippocampus showing deficits in synaptic proteins. We are conducting behavioral analyses as well. Ubiquitination is a post-translational modification that dynamically regulates the synaptic expression of many proteins. Years ago, we performed a screen to identify transmembrane RING domain-containing E3 ubiquitin ligases that regulate surface expression of AMPARs, and identified two candidates. One of these, RNF112, is a brain-specific protein that we have characterized using a variety of approaches. We find that it is a functional GTPase, as well as an E3 ligase. We named it neurolastin (RNF112/Znf179) because it is most closely related to the dynamin superfamily GTPase, atlastin. We generated a knock-out line of mice and in our initial publication, we showed that neurolastin regulates endosome size and spine density in vivo. Neurolastin requires both an intact RING and GTPase domain to maintain spine density. Interestingly, mutations in the RING domain result in mistargeting of neurolastin from a primarily endosomal to primarily mitochondrial localization. We have now identified a mitochondrial targeting sequence on neurolastin. Furthermore, we have analyzed knock-out animals using EM and find mitochondrial defects.

神经递质受体及其亚型在单个细胞内和整个大脑中的独特分布需要高度选择性的细胞内靶向机制。我的实验室结合生化和分子技术研究谷氨酸受体运输和定位的调节。谷氨酸受体是哺乳动物大脑中主要的兴奋性神经递质受体，是一个具有许多不同亚型的多样化家族。离子型谷氨酸受体包括AMPA、NMDA和红藻氨酸受体亚型，每种受体亚型均由多种亚基形成。代谢型谷氨酸受体 (mGluR1-8) 是 G 蛋白偶联受体 (GPCR)，以同型二聚体形式组装。我们专注于定义调节谷氨酸受体以及突触支架蛋白的突触定位和功能调节的亚基特异性机制。这些机制包括翻译后修饰，例如磷酸化和泛素化，以及蛋白质-蛋白质相互作用。 该实验室的一个主要重点是研究调节 NMDA 受体运输的分子机制，这些受体是多亚基复合物（GluN1；GluN2A-D；GluN3A-B）。我们在 NMDAR 突触表达的详细表征以及 GluN2A 和 GluN2B 在受体运输和突触表达中的作用方面取得了重大进展。通过 GluN2B 亚基 (S1480) 的 PDZ 配体的酪蛋白激酶 2 (CK2) 磷酸化，NMDA 受体以活性和钙依赖性方式从突触中去除。我们发现 NR2B 亚基（而不是 NR2A）被 CK2 特异性磷酸化，并且 NR2B 的磷酸化在出生后第二周增加，并且在亚基转换（GluN2B 到 GluN2A）中很重要，亚基转换发生在发育过程中和响应活动的许多皮质区域。这些数据支持单个 NMDA 受体亚基对 NMDA 受体运输和定位的独特贡献。我们的研究表明，GluN2B C 末端 (E1479Q) 的单点突变完全阻断 S1480 的 CK2 磷酸化，并导致突触 GluN2B 显着增加。我们目前正在培育一系列基因改造小鼠：表达点突变非磷酸化 GluN2B 亚基 (GluN2B E1479Q) 的敲入小鼠。这种敲入小鼠将使我们能够在体内检查神经元中 GluN2B S1480 磷酸化的精确调节，并且不需要外源蛋白过度表达。预计这些动物会表现出发育受损的 GluN2 亚基转换（Sanz-Clemente 等，2010）。此外，我们正在生成一种敲入小鼠 GluN2B S1480E，它模拟该关键残基的磷酸化并组成型阻断与 PSD-95 的结合。我们的实验表明，这种突变导致 GluN2B 受体的表面和突触表达非常低，因此我们预计小鼠也会出现突触缺陷。 我们还在探索酪氨酸激酶和磷酸酶对突触 NMDAR 调节的作用。 GluN2B 包含经典的基于酪氨酸的内吞基序 (-YEKL)，它是 NMDAR 表面表达的强调节因子。酪氨酸激酶 Fyn 和纹状体富含酪氨酸磷酸酶的蛋白酪氨酸磷酸酶 (STEP) 都以 Y1472 为靶标，Y1472 影响受体的内吞作用和突触表达。特别是，STEP 通过促进 GluN2B Y1472 的去磷酸化来减少 NMDAR 的表面表达，而突触支架蛋白突触后密度蛋白 95 (PSD-95) 通过直接结合到 C 端 PDZ 配体 (-ESDV) 来稳定 NMDAR 的表面表达。我们发现 STEP61 与 PSD-95 结合，但不与其他 PSD-95 家族成员结合。 In addition, PSD-95 expression triggers the degradation of STEP61 via ubiquitination and degradation by the proteasome.令人惊讶的是，我们发现 STEP61 并未在 PSD 部分中富集。然而，PSD 中的 STEP61 表达在 PSD-95 敲低后或在 PSD-95-KO 小鼠体内检测到增加，表明 PSD-95 将 STEP61 排除在 PSD 之外。在 PSD 处具有低丰度的 STEP 的一个重要后果是，在 STEP 敲低后，只有突触外 NMDAR 表达和电流增加。因此，我们的研究结果支持 PSD-95 通过直接与 GluN2B 结合以及促进突触排斥和负调节因子 STEP61 的降解来稳定突触 NMDAR 的双重作用。最近，我们描述了 STEP 表达对 AMPAR 的影响。我们发现 STEP 调节 AMPAR 的 GluA2 和 GluA3 亚基的突触表达，目前我们正在详细探索这种效应的分子基础。我们的数据支持一个模型，其中 STEP 调节 AMPA 和 NMDA 受体的突触和突触外组织。有趣的是，通过敲除或敲除策略从神经元中去除 STEP 会导致突触外 NMDA 受体增加，但突触 AMPA 受体也会增加。而且，效果是特定于亚基的。 在过去的几年里，我们专注于研究 NMDAR 结构/功能的新方法。我们正在尝试一种“从床边到工作台”的方法来帮助指导我们测试对突触功能很重要的受体域。特别是，我们使用了已发表的论文和公共数据库中的信息，这些论文和公共数据库报告了通过对神经或精神疾病患者进行深度测序鉴定出的变异。然后，我们开始对 GluN2B NMDAR 亚基胞内 C 端结构域中发现的错义变体进行实验。我们发现，在一名自闭症患者中发现的一种突变降低了 GluN2B 的表面表达以及与 PSD-95 的结合。这种变体 GluN2B S1415L（小鼠中的 S1413L）在突触 NMDAR 电流的救援方面表现出缺陷，并且树突棘较少。这种表型很有趣，因为文献中有很多脊柱异常与自闭症相关的例子。更广泛地说，这项研究表明，使用患者数据是探索 NMDAR 结构/功能关系的有效方法。我们现在已经生成了 GluN2B S1413L 的敲入小鼠，并且正在表征其表型。我们的生化研究已经揭示了区域特异性效应，海马体显示出突触蛋白的缺陷。我们也在进行行为分析。 泛素化是一种翻译后修饰，可动态调节许多蛋白质的突触表达。几年前，我们进行了筛选，以确定调节 AMPAR 表面表达的包含 E3 泛素跨膜 RING 结构域的连接酶，并确定了两种候选物。其中之一 RNF112 是一种大脑特异性蛋白质，我们使用多种方法对其进行了表征。我们发现它是一种功能性 GTP 酶，也是一种 E3 连接酶。我们将其命名为神经拉斯汀 (RNF112/Znf179)，因为它与动力蛋白超家族 GTP 酶阿拉斯汀 (atlastin) 最密切相关。我们产生了小鼠的敲除系，并在最初的出版物中表明，神经拉斯汀在体内调节内体大小和脊柱密度。 Neurolastin 需要完整的 RING 和 GTPase 结构域来维持脊柱密度。有趣的是，RING 结构域的突变导致神经拉斯汀从主要内体定位到主要线粒体定位的错误定位。我们现在已经确定了神经拉斯汀的线粒体靶向序列。此外，我们还使用 EM 分析了基因敲除动物并发现了线粒体缺陷。