[Construction of expression vectors for C-terminally deleted NR2B subunit mutants and their application in the study of assembling of NMDA receptors].
To investigate the role of NR2B subunit C-terminus in assembling and surface expression of NMDA receptor subtype composed of NR1-1a/NR2B subunits.



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.



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).



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.
Yang
Wei
W
Department of Neurobiology, College of Medicine, Zhejiang University, Hangzhou 310006, China.
Luo
Jian-hong
JH
Gao
Ying
Y
Huang
Man
M
Zheng
Chan-ying
CY
D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D009154:Mutation; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics*; D014162:Transfection
C409466:NR1 NMDA receptor; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate