Subunit-Specific Regulation Of Glutamate Receptors

神经递质受体及其亚型在单个细胞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。我们专注于定义调节不同亚型谷氨酸受体的亚基特异性机制。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。 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，这些受体是多亚单位复合体(NR1；NR2A-D；NR3A-B)。在过去的一年里，我们在详细描述NR2A与NR2B的贩运和突触表达方面取得了重大进展。我们发现NR2B亚基，而不是NR2A，是被NR2B C-末端结构域关键残基上的酪蛋白激酶2(CK2)特异性磷酸化的。NR2B的CK2磷酸化在出生后第二周增加，并且在亚单位转换(NR2B到NR2A)中是重要的，亚单位转换发生在发育过程中的许多皮质区域，并对活动做出反应。这些数据支持单个NMDA受体亚单位对NMDA受体运输和定位的独特贡献。 我们还在研究PSD-95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受体中具有独特的作用。SAP102在发育早期高度表达，并在突触发生过程中调节NMDA受体和AMPA受体的运输。我们发现NR2B通过一个不依赖PDZ的二级结合域与SAP102相互作用，而不是PSD-95。NR2B结合位点位于SAP102的N-末端结构域内，并受SAP102的选择性剪接调控。我们发现，具有N-末端插入的SAP102在mRNA和蛋白质水平上都受到发育调节。此外，SAP102的选择性剪接调节树突棘的形态。含有N-末端插入的SAP102的表达促进树突棘的延长，而同一SAP102剪接变异体的短发夹状RNA敲除会导致脊柱收缩。此外，阻断NMDA受体活性可防止由SAP102的N端剪接变异体引起的脊柱延长。据报道，人类SAP102基因突变会导致智力低下，通常伴随着树突棘的异常。然而，SAP102在调节突触形成或脊柱形态中的作用知之甚少。我们的发现首次提供了SAP102将NMDA受体激活与脊柱形态改变联系起来的证据。 我们还研究了介导NMDA受体表面表达和运输的突触后机制，包括突触后陷阱SNAP23。我们发现SNAP-23调控NMDA受体的表面表达和膜循环。我们通过同源重组获得了Snap23缺失的小鼠。证明SNAP-23功能在小鼠发育中的重要性，我们发现SNAP-23 KO小鼠是不能存活的。我们无法获得新生的SNAP-23缺陷小鼠，对Snap23+/-配对的植入前胚胎的分析显示，Snap23-空的囊胚在胚胎3.5天着床前死亡。这些数据揭示了SNAP-23在胚胎发育过程中的关键作用。 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，GluA1的第一个细胞内环区(Loop1)是AMPA受体中一个以前被忽视的区域，它对于受体靶向突触至关重要，但对于将受体运送到质膜并不关键。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都被磷酸化。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，我们已经描述了AMPA受体的活性依赖泛素化，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.