Subunit-Specific Regulation Of Glutamate Receptors

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。我的实验室研究谷氨酸受体运输和本地化的生物化学和分子技术相结合的调节。我们专注于定义亚单位的具体机制，调节不同亚型的谷氨酸受体。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。 该实验室的一个主要重点是研究调节NMDA受体贩运的分子机制，这些受体是多亚基复合物（NR 1; NR 2A-D; NR 3A-B）。在过去的一年里，我们在NR 2A与NR 2B的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，NR 2B亚基，而不是NR 2A，是专门磷酸化酪蛋白激酶2（CK 2）的一个关键残基在NR 2B的C-末端结构域。出生后第二周，NR 2B的CK 2磷酸化增加，在亚基转换（NR 2B到NR 2A）中很重要，这在发育期间和对活动的反应中发生在许多皮质区域。这些数据支持独特的贡献，个别NMDA受体亚基的NMDA受体的贩运和本地化。 我们还研究了PSD-95蛋白家族（PSD-95、PSD-93、SAP 97、SAP 102）对NR 2A和NR 2B的特异性调节。我们的结果支持SAP 102在调节含NR 2B的NMDA受体中的独特作用。SAP 102在发育早期高度表达，并在突触发生期间介导NMDA受体和AMPA受体的运输。 我们发现，NR 2B与SAP 102，而不是PSD-95，通过二级PDZ独立的结合域相互作用。 NR 2B结合位点位于SAP 102 N-末端结构域内，并受SAP 102的选择性剪接调节。我们发现，SAP 102，具有一个N-末端插入发育调控在mRNA和蛋白质水平。此外，SAP 102的选择性剪接调节树突棘形态。含有N-末端插入物的SAP 102的表达促进树突棘的延长，而相同SAP 102剪接变体的短发夹RNA敲低导致棘收缩。此外，阻断NMDA受体活性可防止由SAP 102的N-末端剪接变体诱导的脊柱延长。据报道，人类SAP 102的突变会导致精神发育迟滞，这通常伴随着树突棘的异常。然而，SAP 102在调节突触形成或棘形态中的作用知之甚少。我们的研究结果提供了第一个证据表明，SAP 102连接NMDA受体激活脊柱形态的改变。 我们还研究了介导NMDA受体表面表达和运输的突触后机制，包括突触后SNARE，SNAP 23。我们发现SNAP-23调节NMDA受体的表面表达和膜再循环。我们通过同源重组产生了Snap 23缺失小鼠。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小鼠不能存活。我们无法获得新生的SNAP-23缺陷小鼠，并且对来自Snap 23 +/-交配的植入前胚胎的分析显示，Snap 23无效胚泡在胚胎日E3.5在植入前死亡。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，如泛素化和磷酸化，对AMPA受体运输的作用。我们发现GluA 1的第一个胞内环结构域（Loop 1）是AMPA受体中以前被忽视的区域，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GluA 1 Loop 1中发现了一个CaMKII磷酸化位点（S567），该位点在体外和体内都被磷酸化。此外，我们表明，S567是一个关键的残基，调节环1介导的AMPA受体贩运，揭示了一个独特的机制，针对AMPA受体的突触介导的突触传递。此外，我们已经描述了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。

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.