NR2亚单位C末端介导的NMDA受体亚型分布式样以及与PSD-MAGUK家族蛋白的相互作用

发布时间：2018-03-24 11:48

本文选题：NMDA受体　切入点：NR2A　出处：《浙江大学》2007年博士论文

【摘要】： NMDA受体是离子型谷氨酸受体的一种亚型,在许多神经系统的生理功能和病理机制中起着至关重要的作用,如突触可塑性、突触发生、疼痛和兴奋性神经毒性等。研究表明,NMDA受体主要是由2个NR1和2个NR2亚单位装配而成的异四聚体。其中,NR1亚单位有8种转录剪接变体,是形成功能性NMDA受体通道的必需组分,而NR2亚单位则起调节亚单位的作用,包括NR2A-NR2D等4个相关基因。NMDA受体亚单位的组成方式限定了许多对于突触可塑性非常重要的受体属性,特别是NR2亚单位在调节NMDA受体通道的镁离子阻滞的电压依赖性、决定受体通道开放时程的长短等都起着非常重要的作用,并且还决定了NMDA受体可以与不同的细胞内蛋白相互作用,而这些胞内蛋白恰恰在介导NMDA受体的突触定位、受体成簇以及细胞信号转导等诸多属性上都相当重要。NMDA受体的膜表面分布式样和突触定位与突触强度以及不同类型突触可塑性的调节都具有密切关系,而这些受体定位和分布的性质同样也是由组成NMDA受体的NR2亚单位的不同属性决定的,正如以往的研究提示,在成熟神经元中,与含有NR2B亚单位的NMDA受体不同,含有NR2A亚单位的NMDA受体几乎只定位在突触部位,尽管近来也有文献指出突触外也存在含有NR2A亚单位的NMDA受体。另外,有研究提示在突触形成以前,神经元发育的早期膜表面的NMDA受体就以受体簇的形式沿着树突干运输了,因此基于形态描述的受体簇概念并不能完全等同于突触部位的受体,这样既便是利用电生理的方法可能也无法检测到突触外的可能存在的受体簇。由于电生理检测的方法学局限性,对于含有NR2A和NR2B亚单位的NMDA受体膜表面分布式样的系统而全面的形态学分析是非常必要的。在本研究中,我们制备了抗绿色荧光蛋白(green fluorescence protein,GFP)多克隆抗体的Fab片段,并标记上Cy3荧光基团,可以用来直接标记表达在细胞表面的具有GFP标签的NR2A或NR2B亚单位蛋白。由于未与NR1亚单位装配的NR2亚单位滞留在ER上而不能表达于细胞膜表面,我们通过活细胞表面荧光染色转染的NR2亚单位,显示的是分布于细胞或培养海马神经元表面的含有NR2A或NR2B的NMDA受体亚型。结果显示,含有NR2A亚单位的NMDA受体的膜表面分布比含有NR2B亚单位的NMDA受体更倾向于成簇,并且通过相互掉换NR2A和NR2B亚单位的整个胞内C末端,嵌合NR2A胞内C端的NR2B亚单位的膜表面分布变得更容易成簇,而嵌合NR2B胞内段的NR2A亚单位分布却变得弥散。并且,这种NR2A亚单位胞内C末端决定的表面NMDA受体成簇特征并非完全依赖于NR2亚单位C末端的PDZ结合功能域。进而,我们还发现在成熟神经元中突触后支架蛋白PSD-95选择性地与含有NR2A的NMDA受体簇共定位,而同一家族的另外一种蛋白SAP-102与含有NR2A亚单位或NR2B亚单位的NMDA受体簇共定位却不存在选择性。然而,NR2亚单位中与这些支架蛋白相结合的功能区却无法直接解释NR2A与PSD-95蛋白之间的这种特异性的相互作用。另外,在神经元发育的早期,NR2A亚单位的提前过表达可以促进PSD-95蛋白的提前成簇。综上所述,我们的结果显示,含NR2A的NMDA受体亚型成簇分布,而含NR2B的NMDA受体亚型则以弥散分布为主,并且NMDA受体成簇分布的特征是由NR2A亚单位的胞内段决定的,且并没有明显的C末端PDZ结合域的依赖型。同时,与NR2B-NMDA受体亚型相比,突触后支架蛋白PSD-95与膜表面NR2A-NMDA受体亚型具有一定的选择选择性共定位关系,但似乎不依赖于NR2A与PSD-95之间通过PDZ的结合,而SAP102与NR2A-和NR2B-NMDA受体的共定位却无明显的选择性。最后我们的结果显示NR2A的表达可以诱导PSD-95的成簇分布。
[Abstract]:NMDA receptor is a subtype of ionotropic glutamate receptor, plays a crucial role in many physiological and pathological mechanisms of the nervous system, such as synaptic plasticity, synaptogenesis, pain and excitotoxicity. Research shows that the NMDA receptor is ISO four dimers composed of 2 NR1 and 2 NR2 subunit assembly. Among them, there are 8 kinds of transcription splice variants of NR1 subunit, is essential for the formation of group NMDA receptor channel function, while the NR2 subunit is the regulatory subunit composition including NR2A-NR2D function, 4 genes related to.NMDA receptor subunit defines many for synaptic plasticity. Receptor properties are important, especially the dependence of the NR2 subunit of voltage in the magnesium ion regulation of NMDA receptor channel block, determine channel open duration and plays a very important role, and also determines the NMDA receptor Can the protein with different intracellular localization and synaptic interactions, these intracellular proteins that guide NMDA receptor in dielectric, receptor clustering and cell signal transduction and many other attributes are very important.NMDA receptor membrane distribution patterns and synaptic localization and synaptic strength and regulation of the same type of synaptic plasticity are closely related the nature of these receptors, localization and distribution is also by the different properties of the NR2 subunit of the NMDA receptor decision, previous studies as that in mature neurons, and containing NR2B subunit of NMDA receptor, containing the NR2A subunit of the NMDA receptor was only located at the synapses, although recently the extrasynaptic also exists with NR2A subunit of the NMDA receptor. In addition, studies suggest that synapse formation in neurons before the early development of NMDA receptor on the membrane surface Receptor clusters form transport along the dendrite, so based on the morphological description of the concept of receptor clusters are not exactly the same as the synapses of receptors, even this is a possible method of using electrophysiological also unable to detect the possible existence of extrasynaptic receptor clusters. Because the electric physiological detection method for distribution system study limitations. NMDA receptor membrane surface patterns containing NR2A and NR2B subunit and comprehensive morphological analysis is very necessary.
In this study, we prepared anti green fluorescent protein synthesis (green fluorescence protein, GFP) polyclonal antibody Fab fragment and Cy3 labeled fluorophores can be used directly labeled with GFP tag expression in NR2A or NR2B cell surface protein subunit and NR1 subunit. The assembly of NR2 the unit stuck in ER and not expressed on the cell surface, we through the NR2 subunit of living cell surface immunofluorescence transfection, shows the distribution of cells or in cultured hippocampal neurons on the surface of NR2B containing NR2A or NMDA receptor subtypes. The results showed that the membrane containing NR2A subunit of NMDA receptor than containing the NR2B subunit of the NMDA receptor is more inclined to Yu Chengcu, and through mutual exchange of NR2A and NR2B subunits of the intracellular C terminus, membrane surface distribution of NR2B subunit of chimeric NR2A intracellular C end becomes more and more easily into clusters, and The NR2A subunit of chimeric NR2B intracellular distribution became dispersed. And surface NMDA receptor clustering characteristic is not end this NR2A subunit of intracellular C decision depends entirely on the ends of NR2 subunit C PDZ binding domain. Furthermore, we also found in mature neuron support protein PSD-95 selectively co localization of NMDA receptor clusters containing NR2A, and the same family of SAP-102 and another protein subunit containing NR2A or NR2B subunits of NMDA receptor clusters co localization does not exist selectivity. However, the functional region of NR2 subunit in combination with the scaffold protein but not directly explain between NR2A and PSD-95 protein the interaction of this specificity. In addition, in the early development of neuron, the NR2A subunit of PSD-95 protein expression in advance can promote the early clustering. In summary, our results show that NR2A containing NMDA receptor Subtype clustering distribution, while the NMDA receptor subtype containing NR2B is diffused, and the characteristics of NMDA receptor clustering distribution is determined by the NR2A subunit of intracellular segment, and there were no obvious C terminal PDZ binding domain dependent. At the same time, compared with the NR2B-NMDA receptor subtype, postsynaptic the surface of NR2A-NMDA scaffold protein PSD-95 and membrane receptor subtype selective with certain Co located, but does not seem to depend on between NR2A and PSD-95 by binding to PDZ, and the co localization of SAP102 and NR2A- and NR2B-NMDA receptors had no obvious selectivity. Finally, our results show that clustered NR2A can induce expression of PSD-95.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R33

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