C末端缺失NR2B亚单位表达载体的构建及其在NMDA受体装配研究中的应用

目的：研究NMDA受体NR2B亚单位细胞内C末端，在NR1—1a／NR2B亚型NMDA受体装配和表面表达中的作用。方法：构建C末端不同缺失和GFP标记的NR2B亚单位表达载体，单独转染或与NR1亚单位共转染到HEK293细胞，用抗GFP多克隆抗体和Cy3荧光素交联的二抗作活细胞表面受体染色。结果：成功构建了8个C末端不同缺失的GFP—NR2B表达质粒(GFP—NR2B△1～△8)。将GFP—NR1—1a，GFP—NR2B及GFP—NR2B△1～△8分别单独转染HEK293细胞，不能获得迭细胞膜表面的表达。而将这些质粒分别与NR1—1a共转染293细胞，发现GFP—NR2B及C末端部分缺失的GFP—NR2B△1～△6，均能与NR1—1a一起表达于细胞膜表面，而仅留有PDZ结合基序的GFP—NR2B△7和C末端全长缺失的GFP—NR2B△8，则不能与NR1—1a一起表达于细胞膜表面。结论：NR1—1a与NR2B的共表达和装配，是形成NR1—1a／NR2B亚型NMDA受体并表达于细胞表面的必要条件。NR2B与NR1—1a装配时，NR2B对NR1—1a C1盒中内质网滞留基序的遮蔽和阻滞作用，并不依赖于NR2BC末端某一特定的区域，相反可能仅要求有一定长度的NR2BC末端。

Construction of expression vectors for C-terminally deleted NR2B subunit mutants and their application in the study of assembling of NMDA receptors

OBJECTIVE: To investigate the role of NR2B subunit C-terminus in assembling and surface expression of NMDA receptor subtype composed of NR1-1a/NR2B subunits. METHODS: Eight vectors NR2BDelta1-Delta8) expressing GFP-tagged NR2B subunit mutants with various deletion in the carboxyl-terminal region were generated by conventional molecular cloning techniques. Each of these vectors was transfected alone, or co-transfected with NR1-1a into HEK293 cells. NR1-1a/GFP-NR2B receptors on membrane surface of the living transfected cells were immuno-stained using rabbit antibody against GFP followed by Cy3 conjugated secondary antibody. RESULTS: The eight vectors NR2BDelta1-Delta8 were successfully constructed. No surface labeling of GFP-tagged NMDA receptor was found for those transfected cells with NR1-1a, GFP-NR2B, and GFP-NR2BDelta1-Delta8 alone. GFP-tagged NMDA receptors were immuno-stained by anti-GFP for those cells co-transfected by NR1-1a and GFP-NR2B or GFP-NR2BDelta1-Delta6, which were mutants with partially deleted c-terminus at different region. However, positive stained was not found for those cells co-transfected by NR1-1a and GFP-NR2BDelta 7 (lack of most C-terminus and with PDZ binding motif fused with TM4) or GFP-NR2BDelta8 (lack of whole C-terminus). CONCLUSIONS: The formation of NR1-1a/NR2B sub-type NMDA receptor requires co-expression and assembling of NR1-1a and NR2B subunits. Shield or inhibition of ER retention motif within C1 cassette of NR1-1a subunit by NR2B subunit when assembling is not dependent on any particular region in NR2B C-terminus.