Roles Of The Intracellular C-Terminus Of NR2A Subunit In Assembling, Trafficking And Surface Expression Of NMDA Receptors

N-methyl-D-aspartate (NMDA) receptors are heteromeric complexes primarily assembled from two subunit classes: NR1 and NR2 subunits. Co-assembly of NR1 and NR2 subunits is essential for the formation of a functional channel, presumed to be a tetramer containing two NR1 and two NR2 subunits. The cytoplasmic C-terminal domains of NR2A subunits have been proposed to modulate the surface expression and synaptic localization of NMDA receptors. In this study, we further investigated the role of the intracellular C- terminus of NR2A subunit in assembling and surface expression of NMDA receptor channels in both HEK293 cells and cultured hippocampal neurons using a series of C- terminally truncated NR2A mutants. The results showed that the mutants 2AAC59,2Aâ–³C5, and 2AAC3, which had 59, 5, and 3 amino acid remind following the TM4 region respectively, were able to form functional NMDA receptors with NRl-la and expressed on membrane surface in co-transfeceted HEK293 cells, although the surface expression level was decreased. Even 2AAC5eaaaa did so, in which the remained residues EHLFY had been replaced to EAAAA. Removal of all but one amino acid of NR2A C-terminus completely eliminated surface expression of recombinant NMDA receptors composed by NR1 -1a and 2Aâ–³C1. Interestingly, when contransfected with NR1 -4a in HEK293 cells, 2Aâ–³C1 was able to reach the cell surface and form functional NMDA receptor channels. Unlike NRl-la, NRl-4a isoform lacks ER retention motif RXR in the C1 cassette. Thus, our data indicate that 1) a three amino acid tail (EHL) following the TM4 of NR2A subunit is required for surface expression of NMDA recetors;2) C- terminus of NR2A subunit is not necessary for assembling between NR1 and NR2 subunits and formation of a functional channel;and 3) a certain length of C terminal tail following the TM4 region of NR2A subunit may behelpful in inhibiting the effect of ER retention motit during the process of assembly between NR1 -1 a and NR2A.Furthmore, we invetigated the surface expression of NR2A subunit expressed alone in HEK293 cells. As indicated by previous studies, NR2A subunit could not traffick to membrane surface when transfected in HEK293 cells alone, and Protein kinase C (PKC) were able to enhance surface expression of NMDA receptors via phosphoration sites in NR1 C terminus. In our experiments, we transfected GFP-NR2A alone into HEK293 cells, and treated the cells with 200 nM PMA, a activator of PKC, for 15 min. Interestingly, it was found that 4.1 + 1.5% GFP-NR2A expressing cells were labled with anti-GFP antibody in living cell immunochemistry, indicating some of transfected cells could deliver NR2A subunit to cell surface. Using NR2A mutants with partial deletion of C terminus, the segment between 1149D and 1464V was identified to involve this function. However, a glutamate-mediated current was not recorded in the cells with NR2A subunit surface expression, and the stoichiometry of such surface NR2A remains unknown.We also examined influence on NR2A memrane trafficking by the scafold protein PSD95. Some particles were found to have high density of both NR2A and PSD95 existing in the plasma of HEK293 cells co-transfected with NR2A and PSD95, and these particles was also positively stained by Golgi tracker and early endosome antibody, indicating that PSD95 may promote release of NR2A from ER. Moreover, an application of 1 mM PMA could increase the percentage of the ells with such particles by three times, whereas the phenomena was not observed in the cells co-transfected with PSD95 and the NR2A mutant with C terminual truncation. It is suggested that interaction between NR2A and PSD95 and phosphoration at early stage may involved in membran tranfficking of NR2A-containing NMDA receptors.