Subunit-Specific Regulation Of Glutamate Receptors

谷氨酸受体的亚基特异性调节

• 批准号: 8557030
• 负责人: Katherine Roche
• 金额: $ 168.29万
• 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/8557030
• 关键词: AMPA Receptors; Alternative Splicing; Binding; Binding Sites; Biochemical; Brain; Brain region; C-terminal; Cell membrane; Cells; Complement; Complex; DLG1 gene; Data; Dendritic Spines; Development; Embryo; Embryonic Development; Excitatory Synapse; Glutamate Receptor; Glutamates; Human; In Vitro; Individual; Knockout Mice; Laboratory Study; Ligands; Link; Long-Term Depression; Long-Term Potentiation; Mediating; Membrane; Mental Retardation; Messenger RNA; Molecular; Morphology; Mus; Mutation; N-Methyl-D-Aspartate Receptors; N-terminal; NR1 gene; Neuraxis; Neurotransmitter Receptor; Neurotransmitters; Newborn Infant; Phosphorylation; Phosphorylation Site; Play; Post-Translational Protein Processing; Protein Family; Proteins; RNA Splicing; Receptor Activation; Recycling; Regulation; Reporting; Role; S-nitro-N-acetylpenicillamine; SNAP receptor; Stimulus; Surface; Synapses; Synaptic Transmission; Synaptic plasticity; Techniques; Ubiquitination; Variant; Vertebral column; blastocyst; calmodulin-dependent protein kinase II; casein kinase II; discs, large (Drosophila) homolog 2 protein, rat; experience; homologous recombination; implantation; in vivo; mouse development; neurotransmission; neurotransmitter release; postnatal; postsynaptic; preimplantation; prevent; protein protein interaction; receptor; response; small hairpin RNA; synaptogenesis; 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. We focus on defining subunit-specific mechanisms that regulate different subtypes 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 (NR1; NR2A-D; NR3A-B). Over the last year, we have made significant progress in the detailed characterization of NR2A vs. NR2B trafficking and synaptic expression. We find that the NR2B subunit, and not NR2A, is specifically phosphorylated by casein kinase 2 (CK2) on a critical residue in the NR2B C-terminal domain. CK2 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. We are also studying the specific regulation of NR2A and NR2B by the PSD-95 family of proteins (PSD-95, PSD-93, SAP97, SAP102) Our results support a unique role for SAP102 in regulating NR2B-containing NMDA receptors. SAP102 is highly expressed early in development and mediates the trafficking of both NMDA receptors and AMPA receptors during synaptogenesis. We find that NR2B interacts with SAP102, not PSD-95, via a secondary PDZ-independent binding domain. The NR2B binding site is located within the SAP102 N-terminal domain and is regulated by alternative splicing of SAP102. We find that SAP102 that possesses an N-terminal insert is developmentally regulated at both mRNA and protein levels. In addition the alternative splicing of SAP102 regulates dendritic spine morphology. Expression of SAP102 that contains the N-terminal insert promotes lengthening of dendritic spines, whereas a short hairpin RNA knockdown of the same SAP102 splice variant causes spine shrinkage. In addition, blocking NMDA receptor activity prevents the spine lengthening induced by the N-terminal splice variant of SAP102. It has been reported that mutations in human SAP102 cause mental retardation, which is often accompanied by abnormalities in dendritic spines. However, little is known about the role of SAP102 in regulating synapse formation or spine morphology. Our findings provide the first evidence that SAP102 links NMDA receptor activation to alterations in spine morphology. We have also examined the postsynaptic machinery that mediates NMDA receptor surface expression and trafficking, including the postsynaptic SNARE, SNAP23. We found that SNAP-23 regulated the surface expression and membrane recycling of NMDA receptors. We generated Snap23-null mice by homologous recombination. Attesting to the importance of SNAP-23 function in mouse development, we found the SNAP-23 KO mice were not viable. We were unable to obtain newborn SNAP-23-deficient mice, and analysis of pre-implantation embryos from Snap23+/- matings revealed that Snap23-null blastocysts were dying prior to implantation at embryonic day E3.5. These data reveal a critical role for SNAP-23 during embryogenesis. 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. In addition, we have described activity-dependent ubiquitination of AMPA receptors and are currently investigating specific E3 ligases that regulate AMPA receptor ubiquitination and trafficking.

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。我们专注于定义调节谷氨酸受体不同亚型的亚基特异性机制。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（NR1；NR2A-D；NR3A-B）。去年，我们在 NR2A 与 NR2B 运输和突触表达的详细表征方面取得了重大进展。我们发现 NR2B 亚基（而非 NR2A）被酪蛋白激酶 2 (CK2) 在 NR2B C 末端结构域的关键残基上特异性磷酸化。 NR2B 的 CK2 磷酸化在出生后第二周增加，并且对于亚基转换（NR2B 到 NR2A）很重要，亚基转换发生在发育过程中和对活动的反应中的许多皮质区域。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。 我们还在研究 PSD-95 蛋白家族（PSD-95、PSD-93、SAP97、SAP102）对 NR2A 和 NR2B 的特异性调节。我们的结果支持 SAP102 在调节含有 NR2B 的 NMDA 受体方面具有独特的作用。 SAP102 在发育早期高度表达，并在突触发生过程中介导 NMDA 受体和 AMPA 受体的运输。 我们发现 NR2B 通过第二个独立于 PDZ 的结合域与 SAP102（而不是 PSD-95）相互作用。 NR2B 结合位点位于 SAP102 N 末端结构域内，并受 SAP102 的选择性剪接调节。我们发现具有 N 末端插入的 SAP102 在 mRNA 和蛋白质水平上受到发育调节。此外，SAP102 的选择性剪接可调节树突棘形态。包含 N 末端插入片段的 SAP102 的表达促进树突棘的延长，而同一 SAP102 剪接变体的短发夹 RNA 敲除会导致树突棘收缩。此外，阻断 NMDA 受体活性可防止 SAP102 N 端剪接变体诱导的脊柱延长。据报道，人类SAP102突变会导致智力低下，往往伴有树突棘异常。然而，人们对 SAP102 在调节突触形成或脊柱形态方面的作用知之甚少。我们的研究结果提供了第一个证据，证明 SAP102 将 NMDA 受体激活与脊柱形态的改变联系起来。 我们还检查了介导 NMDA 受体表面表达和运输的突触后机制，包括突触后 SNARE、SNAP23。我们发现 SNAP-23 调节 NMDA 受体的表面表达和膜回收。我们通过同源重组产生了 Snap23-null 小鼠。为了证明 SNAP-23 功能在小鼠发育中的重要性，我们发现 SNAP-23 KO 小鼠无法存活。我们无法获得新生的 SNAP-23 缺陷小鼠，对 Snap23+/- 交配的植入前胚胎的分析表明，Snap23 缺失的囊胚在胚胎 E3.5 天植入前就已经死亡。这些数据揭示了 SNAP-23 在胚胎发生过程中的关键作用。 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。此外，我们描述了 AMPA 受体的活性依赖性泛素化，目前正在研究调节 AMPA 受体泛素化和运输的特定 E3 连接酶。