A three amino acid tail following the TM4 region of the N-methyl-D-aspartate receptor (NR) 2 subunits is sufficient to overcome endoplasmic reticulum retention of NR1-1a subunit.
The cytoplasmic C-terminal domains of NR2 subunits have been proposed to modulate the assembly and trafficking of NMDA receptors. However, questions remain concerning which domains in the C terminus of NR2 subunits control the assembly of receptor complexes and how the assembled complexes are selectively trafficked through the various cellular compartments such as endoplasmic reticulum (ER) to the cell surface. In the present study, we found that the three amino acid tail after the TM4 region of NR2 subunits is necessary for surface expression of functional NMDA receptors, while truncations with only two amino acids following the TM4 region (NR2Delta2) completely eliminated surface expression of the NMDA receptor on co-expression with NR1-1a in HEK293 cells. FRET (fluorescence resonance energy transfer) analysis showed that these NR2Delta2 truncations are able to form homomers and heteromers on co-expression with NR1-1a. Furthermore, when NR2Delta2 subunits were cotransfected with either the NR1-4a or NR1-1a(AAA) mutant, lacking the ER retention motif (RRR), functional NMDA receptors were detected in the transfected HEK293 cells. Unexpectedly, we found that the replacement of five residues after TM4 with alanines gave results indistinguishable from those of NR2BDelta5 (EHLFY), demonstrating the short tail following the TM4 of NR2 subunits is not sequence-specific-dependent. Taken together, our results show that the C terminus of the NR2 subunits is not necessary for the assembly of NMDA receptor complexes, whereas a three amino acid long cytoplasmic tail following the TM4 of NR2 subunits is sufficient to overcome the ER retention existing in the C terminus of NR1, allowing the assembled NMDA receptors to reach the cell surface.
Yang
Wei
W
Department of Neurobiology, Institute for Neuroscience, Zhejiang University School of Medicine, Hangzhou 3100058, China.
Zheng
Chanying
C
Song
Qilin
Q
Yang
Xiujuan
X
Qiu
Shuang
S
Liu
Chunqing
C
Chen
Zhong
Z
Duan
Shumin
S
Luo
Jianhong
J
D000818:Animals; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry*
C409466:NR1 NMDA receptor; C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate