Isolation of 2000-kDa complexes of N-methyl-D-aspartate receptor and postsynaptic density 95 from mouse brain
Isolation of 2000-kDa complexes of N-methyl-D-aspartate receptor and postsynaptic density 95 from mouse brain
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DOI：
10.1046/j.1471-4159.2001.t01-1-00248.x
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发表时间：
2001-04-01
影响因子：
4.7
通讯作者：
Grant, SGN
中科院分区：
文献类型：
--
作者：
Husi, H;Grant, SGN
体内的神经递质受体与细胞内适配器蛋白和驱动下游通路的信号分子相连。物理分离的方法对于回答有关体内复合体的大小、结构和组成的基本问题以及对广泛使用的酵母双杂交方法的补充至关重要。N-甲基-D-天冬氨酸受体(NMDAR)与突触后密度95(PSD-95)蛋白结合；两者都是突触可塑性和学习所必需的，并参与其他重要的病理生理功能。在这里，我们描述了从小鼠脑中大规模分离NMDAR-PSD-95复合体的新方法的发展和优化，包括免疫亲和、免疫沉淀、配基亲和和固定化PSD-95结合肽。模拟NMDAR亚基的短PDZ结合肽被证明可以分离NMDAR复合体。凝胶过滤表明天然的NMDAR-PSD-95复合体为2000 kDa，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示了一个复杂的网络，表明结构成分和信号酶都是一个巨大的网络结构。这些方法可以用来确定不同突触和携带基因突变的小鼠的复合体的结构，以及药物发现的新工具。
Neurotransmitter receptors in vivo are linked to intracellular adaptor proteins and signalling molecules driving downstream pathways. Methods for physical isolation are essential to answer fundamental questions about the size, structure and composition of in vivo complexes and complement the widely used yeast 2-hybrid method. The N-methyl-D-aspartate receptor (NMDAR) binds postsynaptic density 95 (PSD-95) protein; both are required for synaptic plasticity and learning and participate in other important pathophysiological functions. Here we describe the development and optimization of novel methods for large-scale isolation of NMDAR-PSD-95 complexes from mouse brain including immunoaffinity, immunoprecipitation, ligand-affinity and immobilized PSD-95 binding peptides. Short PDZ binding peptides, modelled on NMDAR subunits were shown to isolate NMDAR complexes. Gel filtration indicated the native NMDAR-PSD-95 complexes were 2000 kDa, and sodium dodecyl sulfate polyacrylamide gel e[electrophoresis (SDS-PAGE) revealed a complexity suggesting a huge network of both structural components and signalling enzymes. These methods can be used to define the structure of the complexes at different synapses and in mice carrying gene mutations as well as new tools for drug discovery.