Phorbol-induced surface expression of NR2A subunit homologues in HEK293 cells.
OBJECTIVE
N-methyl-D-aspartate receptors (NMDAR) are heteromeric complexes primarily assembled from NR1 and NR2 subunits. In normal conditions, NR2 subunits assemble into homodimers in the endoplasmic reticulum (ER). These homodimers remain in the ER until they coassemble with NR1 dimers and are trafficked to the cell surface. However, it still remains unclear whether functional homomeric NMDAR exist in physiological or pathological conditions.


METHODS
We transfected GFP-NR2A alone into HEK293 cells, treated the cells with PKC activator 12-myristate-13 acetate (PMA), and then detected surface NR2A subunits with a live cell immunostaining method. We also used a series of NR2A mutants with a partial deletion of its C-terminus to identify the regions that are involved in the PMA-mediated surface expression of NR2A subunits.


RESULTS
NR2A subunits were expressed on the cell membrane after incubation with PMA (200 nmol/L, 30 min), although no functional NMDA channels were detected after PMA-induced membrane trafficking. Immunostaining with an ER marker also revealed that NR2A subunits were exported from the ER after PMA treatment. Furthermore, the deletion of amino acids between 1149-1347 or 1354-1464 of NR2A inhibited PMA-induced surface expression of NR2A subunits.


CONCLUSIONS
First, our data suggests that PMA treatment can induce the surface expression of homomeric NR2A subunits. Furthermore, this process is probably mediated by the NR2A C-terminal region between positions 1149 and 1464.
Zheng
Chan-ying
CY
Department of Neurobiology, Zhejiang University School of Medicine, Hangzhou 310031, China.
Yang
Xiu-juan
XJ
Fu
Zhan-yan
ZY
Luo
Jian-hong
JH
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D009154:Mutation; D010957:Plasmids; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology; D014162:Transfection
C473649:NR1A NMDA receptor, rat; C120997:NR2A NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D049452:Green Fluorescent Proteins; C016658:phorbolol myristate acetate; D013755:Tetradecanoylphorbol Acetate