Subunit-Specific Regulation Of Glutamate Receptors

谷氨酸受体的亚基特异性调节

• 批准号: 10691965
• 负责人: Katherine Roche
• 金额: $ 163.59万
• 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/10691965
• 关键词: AMPA Receptors; Affect; Amino Acids; Biochemical; Biological; Brain; C-terminal; Central Nervous System; Complex; Data; Defect; Development; Diagnosis; Disease; Epilepsy; Event; Excitatory Synapse; Eye; Family; Functional disorder; G-Protein-Coupled Receptors; Genes; Glutamate Receptor; Glutamates; Goals; Hippocampus (Brain); Human Genetics; Individual; Intellectual functioning disability; Kainic Acid Receptors; Laboratory Study; Ligands; Mental disorders; Metabotropic Glutamate Receptors; Missense Mutation; Molecular; Mus; Mutation; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; Neurodevelopmental Disorder; Neurons; Neurotransmitter Receptor; Neurotransmitters; Paper; Pathogenicity; Patients; Phosphorylation; Play; Post-Translational Protein Processing; Proteins; Publishing; Regulation; Reporting; Research; Role; Scaffolding Protein; Serine; Site; Stimulus; Stretching; Structure; Surface; Synapses; Techniques; Testing; Therapeutic; Ubiquitination; Variant; Vertebral column; autism spectrum disorder; bench to bedside; calmodulin-dependent protein kinase II; cohort; de novo mutation; deep sequencing; density; experience; experimental study; interest; nervous system disorder; novel strategies; protein protein interaction; public database; rare variant; receptor; receptor function; receptor structure function; response; sorting nexins; synaptic function; trafficking

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. We study of the molecular mechanisms regulating NMDA receptors, and the goal is to better understand NMDA receptor function under normal circumstances and the specific dysfunction underlying some neurodevelopmental disorders. NMDA receptors are multi-subunit complexes (tetramers) composed of homologous subunits (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. We primarily focus on GluN2A and GluN2B because these subunits are highly expressed in hippocampus and cortex and are known to undergo activity- and developmentally-regulated trafficking events. Over the last decade, we have shifted to a new approach to studying structure/function of NMDARs using human genetics to inform our research. We began with this "bedside-to-bench" strategy to help guide us in testing receptor domains that are important for synaptic function. 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 GRIN genes (that encode NMDA receptor subunits. Specifically, we are examining variants causing mutations in the intracellular C-terminal domain of the GluN2 NMDAR subunits (GluN2A and GluN2B). As the human genetics data have accumulated, it has become clear that many de novo mutations in NMDA receptor subunits are highly associated with neurodevelopmental disorders including autism spectrum disorder, intellectual disability and epilepsy. Therefore, our goal is to better understand the synaptic dysfunction caused by these disease-associated rare variants with an eye towards developing therapeutics. Because of our expertise in studying receptor trafficking and protein interactions, we primarily focus on rare variants identified in the intracellular C-termini of NMDA receptor subunits, although we are also embarking on studies using mice with GRIN2B haploinsufficiency. In a recently published study, we characterized a rare variant in GluN2A (S1459G) identified in an epilepsy cohort (Mota Vieira et al., 2020). The patient also was diagnosed with intellectual disability. This de novo mutation is within the extreme C-terminal domain near the PDZ ligand. We found that this serine is a CaMKII site and phosphorylation of this residue dictates the receptor interactions with PSD-95 and sorting nexin 27 (SNX27). Thus we identified a regulatory site that determines the trafficking and synaptic expression of GluN2A-containing NMDA receptors. We are currently finishing up a related study on a GRIN2A rare variant (identified in a patient with neurodevelopmental disorders) encoding a frameshift that results in a truncation of half of the C-terminal domain as well as encoding a unique stretch of amino acids. We find that the receptor has increased surface expression, but specifically enriched at extrasynaptic sites. There are defects in protein interactions, spine density, and synapse number. Again, our approach reveals new findings in NMDA receptor structure/function. We hope to use the information from the analyses of different pathogenic rare variants to better understand NMDA receptor trafficking and localization and help test for more precise therapeutics.

我的实验室使用生化和分子技术相结合的方法研究谷氨酸受体的运输和定位的调节。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性家族。离子型谷氨酸受体包括AMPA、NMDA和海人藻酸受体亚型，每种亚型都由不同的亚基组成。代谢型谷氨酸受体(mGluR1-8)是G蛋白偶联受体，以同源二聚体的形式组装而成。我们专注于确定调节谷氨酸受体和突触支架蛋白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，如磷酸化和泛素化，以及蛋白质-蛋白质相互作用。 我们研究NMDA受体调控的分子机制，目的是为了更好地了解正常情况下NMDA受体的功能以及一些神经发育障碍背后的特定功能障碍。NMDA受体是由同源亚基(GluN1、GluN2A-D、GluN3A-B)组成的多亚单位复合体(四聚体)。我们在NMDAR突触表达的详细特征以及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。我们主要关注GluN2A和GluN2B，因为这些亚基在海马体和皮质中高度表达，并已知经历活动和发育调节的贩运事件。 在过去的十年里，我们已经转向了一种新的方法来研究NMDAR的结构/功能，使用人类遗传学来指导我们的研究。我们从这种“床边到长凳”的策略开始，以帮助指导我们测试对突触功能重要的受体区域。我们使用了已发表的论文和公共数据库中的信息，这些信息报告了通过对神经或精神障碍患者进行深度测序而确定的变异。然后，我们开始对在GRIN基因(编码NMDA受体亚基的基因)中识别的错义变体进行实验。具体地说，我们正在检查导致GluN2NMDAR亚单位(GluN2A和GluN2B)细胞内C-末端区域突变的变异。随着人类遗传学数据的积累，NMDA受体亚单位的许多从头突变与包括自闭症谱系障碍、智能障碍和癫痫在内的神经发育障碍高度相关。因此，我们的目标是更好地了解这些与疾病相关的罕见变异引起的突触功能障碍，着眼于开发治疗方法。由于我们在研究受体运输和蛋白质相互作用方面的专业知识，我们主要专注于在NMDA受体亚单位的细胞内C末端发现的罕见变异，尽管我们也开始使用GRIN2B单倍体不足的小鼠进行研究。 在最近发表的一项研究中，我们描述了在癫痫队列中发现的一种罕见的GluN2A(S1459G)变异(Mota Vieira等人，2020)。这名患者还被诊断为智力残疾。这种从头突变位于PDZ配体附近的极端C-末端区域内。我们发现这个丝氨酸是一个CaMKII位点，这个残基的磷酸化决定了受体与PSD-95和分类连接蛋白27(SNX27)的相互作用。因此，我们确定了一个调控位点，它决定了含有GluN2A的NMDA受体的运输和突触表达。我们目前正在完成一项关于GRIN2A罕见变体(在一名神经发育障碍患者中发现)编码移码的相关研究，该移码导致C末端结构域的一半截断，以及编码一段独特的氨基酸。我们发现，该受体的表面表达增加，但在突触外部位特异性地丰富。在蛋白质相互作用、棘突密度和突触数量方面存在缺陷。我们的方法再次揭示了NMDA受体结构/功能的新发现。我们希望利用从不同致病罕见变异的分析中获得的信息来更好地了解NMDA受体的运输和定位，并帮助测试更精确的治疗方法。