NR2A亚单位胞内C末端区对NMDA受体装配、运输以及表面表达的影响
发布时间：2018-08-27 12:51
【摘要】：NMDA受体是由NR1和NR2亚单位组成的异聚体复合物,亚单位之间的装配对于NMDA受体通道的形成和表面表达至关重要。以往的研究提示,NR2亚单位胞内C末端区可能参与调节NMDA受体的表面表达和突触定位。本研究的第一部分通过一系列C末端截短的NR2A亚单位突变体的构建以及NMDA受体膜表面表达和功能的检测,在HEK293细胞和培养海马神经元研究了NR2A亚单位的C末端在NMDA受体装配和表面表达中的作用。结果发现,当NR2A亚单位的C末端截短后其TM4下游分别剩下59、5、3个氨基酸残基时(2AΔC59、2AΔC5和2AΔC3),它们均能与NR1-1a形成有功能的受体通道并表达于细胞膜表面,但是与未截短的NR2A相比其表面表达均有所下降;并且,当2AΔC5的TM4下游仅存的5个氨基酸EHLFY突变成EAAAA(2AΔC5_(EAAAA))时,仍可与NR1-1a形成受体复合物并表达于细胞膜表面。若将NR2A的C末端截短仅保留TM4后的1个氨基酸时(2AΔC1),该突变体与NR1-1a共转染时,就不再获得受体复合物的细胞膜表面表达,也不能记录到谷氨酸诱发的电流。有趣的是,当NR1-4a与2AΔC1共转染时,NR1-4a/2AΔ1受体复合物仍然能表达于HEK293细胞表面,并能检测到通道功能。在此,与NR1-1a不同的是NR1-4a不含有内质网滞留基序。以上结果提示,NR2A亚单位TM4后的3个氨基酸是NR1-1a/NR2A受体复合物细胞膜表面表达所必需的,然而,NR2A亚单位的C末端并不是NR2A与NR1装配和形成功能性通道所必需的,但能帮助NR1-1a克服内质网滞留作用使NMDA受体复合物表达于细胞膜表面。 本研究的第二部分研究了NR2A亚单位单独转染时膜表面表达。以往研究
[Abstract]:NMDA receptor is an heteropolymer complex composed of NR1 and NR2 subunits. The assembly between subunits is very important for the formation and surface expression of NMDA receptor channels. Previous studies suggest that the C-terminal region of NR2 subunit may be involved in regulating the surface expression and synaptic localization of NMDA receptor. In the first part of this study, a series of C-terminal truncated NR2A subunit mutants were constructed and the surface expression and function of NMDA receptor were detected. The role of C-terminal of NR2A subunit in the assembly and surface expression of NMDA receptor was studied in HEK293 cells and cultured hippocampal neurons. The results showed that when the C-terminal of NR2A subunit was truncated, the downstream of TM4 remained 59,5 and 3 amino acid residues (2A 螖 C59C5 and 2A 螖 C3), which could form a functional receptor channel with NR1-1a and express on the surface of cell membrane. However, compared with the untruncated NR2A, the expression of EHLFY on the surface of TM4 of 2A 螖 C5 was decreased, and when the only 5 amino acid EHLFY of TM4 of 2A 螖 C5 mutated to EAAAA (2A 螖 C5 _ (EAAAA), it could form receptor complex with NR1-1a and express on the surface of cell membrane. If the C-terminal of NR2A was truncated with only one amino acid (2A 螖 C1) of TM4, the expression of the receptor complex on the cell membrane and the glutamate induced current could not be recorded when the mutant was co-transfected with NR1-1a. Interestingly, when NR1-4a was co-transfected with 2A 螖 C1, the NR1-4a / 2A 螖 1 receptor complex could still be expressed on the surface of HEK293 cells and the channel function could be detected. Here, unlike NR1-1a, NR1-4a does not contain endoplasmic reticulum retention motifs. These results suggest that the three amino acids of NR2A subunit after TM4 are necessary for the surface expression of NR1-1a/NR2A receptor complex. However, the C-terminal of NR2A subunit is not necessary for the assembly and formation of functional channels between NR2A and NR1. But it can help NR1-1a overcome endoplasmic reticulum retention and make NMDA receptor complex express on the surface of cell membrane. In the second part of this study, the membrane surface expression was studied when NR2A subunits were transfected alone. Previous research
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R33
本文编号：2207339
[Abstract]:NMDA receptor is an heteropolymer complex composed of NR1 and NR2 subunits. The assembly between subunits is very important for the formation and surface expression of NMDA receptor channels. Previous studies suggest that the C-terminal region of NR2 subunit may be involved in regulating the surface expression and synaptic localization of NMDA receptor. In the first part of this study, a series of C-terminal truncated NR2A subunit mutants were constructed and the surface expression and function of NMDA receptor were detected. The role of C-terminal of NR2A subunit in the assembly and surface expression of NMDA receptor was studied in HEK293 cells and cultured hippocampal neurons. The results showed that when the C-terminal of NR2A subunit was truncated, the downstream of TM4 remained 59,5 and 3 amino acid residues (2A 螖 C59C5 and 2A 螖 C3), which could form a functional receptor channel with NR1-1a and express on the surface of cell membrane. However, compared with the untruncated NR2A, the expression of EHLFY on the surface of TM4 of 2A 螖 C5 was decreased, and when the only 5 amino acid EHLFY of TM4 of 2A 螖 C5 mutated to EAAAA (2A 螖 C5 _ (EAAAA), it could form receptor complex with NR1-1a and express on the surface of cell membrane. If the C-terminal of NR2A was truncated with only one amino acid (2A 螖 C1) of TM4, the expression of the receptor complex on the cell membrane and the glutamate induced current could not be recorded when the mutant was co-transfected with NR1-1a. Interestingly, when NR1-4a was co-transfected with 2A 螖 C1, the NR1-4a / 2A 螖 1 receptor complex could still be expressed on the surface of HEK293 cells and the channel function could be detected. Here, unlike NR1-1a, NR1-4a does not contain endoplasmic reticulum retention motifs. These results suggest that the three amino acids of NR2A subunit after TM4 are necessary for the surface expression of NR1-1a/NR2A receptor complex. However, the C-terminal of NR2A subunit is not necessary for the assembly and formation of functional channels between NR2A and NR1. But it can help NR1-1a overcome endoplasmic reticulum retention and make NMDA receptor complex express on the surface of cell membrane. In the second part of this study, the membrane surface expression was studied when NR2A subunits were transfected alone. Previous research
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R33
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