Subunit-Specific Regulation Of Glutamate Receptors

谷氨酸受体的亚基特异性调节

• 批准号: 8940060
• 负责人: Katherine Roche
• 金额: $ 177.26万
• 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/8940060
• 关键词: AMPA Receptors; Affect; Animals; Binding; Biochemical; Bioinformatics; Biological Assay; Brain; Brain region; Calcium; Cell membrane; Cells; Collaborations; Complement; Complex; Core Facility; Couples; Data; Dendritic Spines; Development; Dynamin; Enzymes; Excitatory Synapse; Family; Frequencies; G-Protein-Coupled Receptors; Glutamate Receptor; Glutamates; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hippocampus (Brain); In Vitro; Individual; Kainic Acid Receptors; Knock-in Mouse; Knockout Mice; Laboratory Study; Ligands; Long-Term Depression; Long-Term Potentiation; Maintenance; Mediating; Metabotropic Glutamate Receptors; Molecular; Mono-S; Mus; Mutate; N-Methyl-D-Aspartate Receptors; Names; National Institute of Mental Health; Neuraxis; Neurons; Neurotransmitter Receptor; Neurotransmitters; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Point Mutation; Post-Translational Protein Processing; Process; Protein Overexpression; Proteins; Regulation; Role; Serine; Site-Directed Mutagenesis; Slice; Stimulus; Surface; Synapses; Synaptic Transmission; Synaptic plasticity; Techniques; Transgenic Organisms; Ubiquitination; Vertebral column; calmodulin-dependent protein kinase II; casein kinase II; density; experience; in vivo; inorganic phosphate; mutant; neurotransmission; neurotransmitter release; postnatal; postsynaptic; protein protein interaction; receptor; response; small hairpin RNA; synaptic function; synaptogenesis; tool; 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. 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; NR2A-D; NR3A-B). We have made significant progress in the detailed characterization of the synaptic expression of NMDARs and the role of NR2A and NR2B 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 (NR2B to NR2A), 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. 
However, how synaptic activity drives this process remains unclear because CK2 is a constitutively active kinase, which is not directly regulated by calcium. We recently demonstrated that activated CaMKII couples GluN2B and CK2 to form a tri-molecular complex and increase CK2-mediated phosphorylation of GluN2B S1480. In addition, a GluN2B mutant, which contains an insert to mimic the GluN2A sequence and cannot bind to CaMKII, displays reduced S1480 phosphorylation and increased surface-expression. Importantly, we find that although disrupting GluN2B/CaMKII binding reduces synapse number, it increases synaptic-GluN2B content. Therefore, the GluN2B/CaMKII association controls synapse density and PSD composition in an activity-dependent manner, including recruitment of CK2 to remove GluN2B from synapses. 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. In collaboration with the NIMH Transgenic Core Facility 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. Because it is anticipated that these animals will show an impaired developmental GluN2 subunit switch (Sanz-Clemente et al, 2010), they will be a valuable tool for understanding how this process contributes to the refinement of neuronal connections. We have also investigated the role of posttranslational modifications, such as ubiquitination and phosphorylation, on AMPA receptor trafficking. We found that the first intracellular loop domain (Loop1) of GluA1, a previously overlooked region within AMPA receptors, is critical for receptor targeting to synapses, but not for delivery of receptors to the plasma membrane. We identified a CaMKII phosphorylation site (S567) in the GluA1 Loop1, which is phosphorylated in vitro and in vivo. Furthermore, we show that S567 is a key residue that regulates Loop1-mediated AMPA receptor trafficking, revealing a unique mechanism for targeting AMPA receptors to synapses to mediate synaptic transmission. Because this S567 is a relatively weak CaMKII substrate in contrast to its substrate residue in the GluA1 C-terminus (Ser831), and that the first half of the region is moderately conserved between subunits, we sought to identify other putative kinases. We performed a bioinformatics analysis of AMPARs and found that CK2 was a good candidate to phosphorylate the intracellular loop1 region of AMPAR subunits GluA1 and GluA2. Using in vitro kinase assays, we determined that CK2 phosphorylates the GluA1 and GluA2 intracellular loop1 region, but not their C-termini. Site-directed mutagenesis combined with an in vitro kinase assays revealed the presence of two CK2-phosphorylated serine residues in the GluA1 intracellular loop1 region, including S567 and a more robust substrate for CK2, S579. To investigate a role for CK2 in AMPAR trafficking, we reduced the endogenous expression of CK2 using an shRNA against the regulatory subunit CK2 beta, and we detected a reduction of GluA1 surface expression, whereas GluA2 was unchanged. Importantly, the expression of GluA1 phosphodeficient mutant (S579A) in hippocampal neurons displayed reduced surface expression. Therefore, our study identifies CK2 as a regulator of GluA1 surface expression by phosphorylating the intracellular loop1 region. Ubiquitination is a post-translational modification that dynamically regulates the synaptic expression of many proteins. However, very few of the ubiquitinating enzymes implicated in the process have been identified. In a screen to identify transmembrane RING domain-containing E3 ubiquitin ligases that regulate surface expression of AMPARs, we identified RNF167 and RNF112. Previously, we have demonstrated that RNF167 regulates excitatory synaptic transmission. Interestingly, we now find that RNF112 is a brain-specific functional GTPase, as well as E3 ligase. We have now named it neurolastin (RNF112/Znf179) because it is most closely related to the dynamin superfamily GTPase, atlastin. Neurolastin is the first identified protein with a unique domain organization harboring both GTPase and RING domains. We have demonstrated that neurolastin has the ability to hydrolyze GTP to mono-phosphate (GMP) and the GTPase activity is involved in the maintenance of dendritic spine density. In addition neurolastin leads to an increase in the density of dendritic spines on hippocampal neurons, whereas expression of the GTPase activity mutant did not affect the spine density. Subsequently, we also observed a significant decrease in the frequency of mEPSCs in hippocampal slices from knockout mice indicating a marked reduction in the number of functional synapses in the absence of neurolastin. These results indicate that neurolastin affects synaptic function by regulating synaptogenesis and spine maintenance.

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；NR2A-D；NR3A-B）。我们在 NMDAR 突触表达的详细表征以及 NR2A 和 NR2B 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（NR2B 到 NR2A）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。 
然而，突触活动如何驱动这一过程仍不清楚，因为 CK2 是一种组成型活性激酶，不受钙直接调节。我们最近证明，激活的 CaMKII 与 GluN2B 和 CK2 偶联形成三分子复合物，并增加 CK2 介导的 GluN2B S1480 磷酸化。此外，GluN2B 突变体包含模拟 GluN2A 序列的插入片段，并且不能与 CaMKII 结合，显示出 S1480 磷酸化减少和表面表达增加。重要的是，我们发现虽然破坏 GluN2B/CaMKII 结合会减少突触数量，但会增加突触 GluN2B 含量。因此，GluN2B/CaMKII 关联以活性依赖性方式控制突触密度和 PSD 组成，包括招募 CK2 以从突触中去除 GluN2B。 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。目前，我们与 NIMH 转基因核心设施合作，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。由于预计这些动物将表现出发育受损的 GluN2 亚基转换（Sanz-Clemente 等，2010），因此它们将成为了解这一过程如何促进神经元连接细化的宝贵工具。 我们还研究了翻译后修饰（例如泛素化和磷酸化）对 AMPA 受体运输的作用。我们发现 GluA1 的第一个细胞内环结构域 (Loop1) 是 AMPA 受体内以前被忽视的区域，对于受体靶向突触至关重要，但对于将受体递送到质膜并不重要。我们在 GluA1 Loop1 中发现了一个 CaMKII 磷酸化位点 (S567)，该位点在体外和体内均被磷酸化。此外，我们发现S567是调节Loop1介导的AMPA受体运输的关键残基，揭示了将AMPA受体靶向突触以介导突触传递的独特机制。由于与 GluA1 C 末端 (Ser831) 中的底物残基相比，该 S567 是相对较弱的 CaMKII 底物，并且该区域的前半部分在亚基之间适度保守，因此我们试图鉴定其他假定的激酶。我们对 AMPAR 进行了生物信息学分析，发现 CK2 是磷酸化 AMPAR 亚基 GluA1 和 GluA2 细胞内 Loop1 区域的良好候选者。使用体外激酶测定，我们确定 CK2 磷酸化 GluA1 和 GluA2 细胞内 Loop1 区域，但不磷酸化它们的 C 末端。定点诱变与体外激酶测定相结合揭示了 GluA1 细胞内 Loop1 区域中存在两个 CK2 磷酸化丝氨酸残基，包括 S567 和 CK2 更强大的底物 S579。为了研究 CK2 在 AMPAR 运输中的作用，我们使用针对调节亚基 CK2 beta 的 shRNA 减少了 CK2 的内源表达，并且检测到 GluA1 表面表达减少，而 GluA2 没有变化。重要的是，海马神经元中 GluA1 磷酸缺陷突变体 (S579A) 的表达显示出表面表达减少。因此，我们的研究确定 CK2 通过磷酸化细胞内的 Loop1 区域来调节 GluA1 表面表达。 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。然而，很少有与该过程相关的泛素化酶被鉴定出来。在筛选含有调节 AMPAR 表面表达的跨膜 RING 结构域的 E3 泛素连接酶时，我们鉴定出了 RNF167 和 RNF112。之前，我们已经证明 RNF167 调节兴奋性突触传递。有趣的是，我们现在发现RNF112是一种大脑特异性功能性GTP酶，以及E3连接酶。我们现在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。 Neurolastin 是第一个鉴定出的具有独特结构域组织的蛋白质，其中包含 GTPase 和 RING 结构域。我们已经证明神经拉斯汀具有将 GTP 水解为单磷酸盐 (GMP) 的能力，并且 GTP 酶活性参与维持树突棘密度。此外，neurlastin 导致海马神经元树突棘密度增加，而 GTPase 活性突变体的表达并不影响树突棘密度。随后，我们还观察到基因敲除小鼠海马切片中 mEPSC 的频率显着降低，表明在缺乏神经拉斯汀的情况下功能性突触数量显着减少。这些结果表明神经拉斯汀通过调节突触发生和脊柱维持来影响突触功能。