应用荧光检测法分析AMPA受体N末端结构域的相互作用

发布时间：2018-09-01 17:06

【摘要】：AMPA受体介导了中枢神经系统绝大部分快速型的兴奋性突触传递。在海马中大部分AMPA受体是以含GluR2的异四聚体的形式存在。AMPA受体的装配以一种“二聚化后二聚化”的模型进行装配。虽然在离子型谷氨酸受体的首次装配过程中,N末端结构域(N terminal domain NTD)起了一定的作用,但是还不清楚在活细胞中和完整的受体中NTD的相互作用的情况。我们采用荧光检测法包括双分子荧光互补法(bimolecular fluorescence complementation BiFC)和荧光共振能量转移法(Fluorescence Resonance Energy Transfer FRET)。来研究NTD的相互作用。BiFC是荧光蛋白在合适的位点打开,并标记目标蛋白,当这两片段标记的蛋白两两相互作用时,荧光蛋白能正确折叠并重建荧光团。为了方便后续的工作,我们首先构建了胞浆型和内质网型(endoplasmic reticulium ER)的表达载体。在HEK293和COS7细胞内对游离的AMPA受体GluRl亚单位的NTD进行了双分子荧光互补检测,可见黄色的荧光。证实在活细胞中,无论在内质网中还是在胞浆中,它们都能形成二聚体。为了排除蛋白之间非特异性的相互作用,我们选用代谢性谷氨酸受体的配体结合结构域(metabotropicglutamate receptor ligand binding domain mGluRl-LBD)作为阴性对照,可见微弱的非特异的相互作用.。为了检测在完整的受体中GluRl NTD的相互作用,采
[Abstract]:AMPA receptors mediate most of the fast excitatory synaptic transmissions in the central nervous system. In the hippocampus, most of the AMPA receptors are assembled in the form of heteropolymer containing GluR2. The assembly of AMPA-receptor is carried out in a model of "dimerization after dimerization". Although the N-terminal domain (N terminal domain NTD) plays a role in the first assembly of ionized glutamate receptors, it is unclear how NTD interacts in living cells and intact receptors. We use fluorescence detection methods including bimolecular fluorescence complementary method (bimolecular fluorescence complementation BiFC) and fluorescence resonance energy transfer method (Fluorescence Resonance Energy Transfer FRET). To study the interaction of NTD. BiFC is a fluorescent protein opened at a suitable site and labeled with the target protein. When the two fragments of the labeled protein interact with each other, the fluorescent protein can fold and reconstruct the fluorescence clusters correctly. To facilitate further work, we first constructed cytoplasmic and endoplasmic reticulum type (endoplasmic reticulium ER) expression vectors. In HEK293 and COS7 cells, the NTD of the free AMPA receptor GluRl subunit was detected by bimolecular fluorescence complementary detection, and yellow fluorescence was observed. It is proved that in living cells, both in endoplasmic reticulum and in cytoplasm, they can form dimers. In order to eliminate the nonspecific interaction between proteins, we selected the ligand binding domain (metabotropicglutamate receptor ligand binding domain mGluRl-LBD) of metabolic glutamate receptor as negative control. In order to detect the interaction of GluRl NTD in intact receptors,
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R33

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