Subunit assembly of N-methyl-d-aspartate receptors analyzed by fluorescence resonance energy transfer.
N-methyl-d-aspartate (NMDA) receptors play major roles in synaptic transmission and plasticity, as well as excitotoxicity. NMDA receptors are thought to be tetrameric complexes mainly composed of NMDA receptor (NR)1 and NR2 subunits. The NR1 subunits are required for the formation of functional NMDA receptor channels, whereas the NR2 subunits modify channel properties. Biochemical and functional studies indicate that subunits making up NMDA receptors are organized into a dimer of dimers, and the N termini of the subunits are major determinants for receptor assembling. Here we used a biophysical approach, fluorescence resonance energy transfer, to analyze the assembly of intact, functional NMDA receptors in living cells. The results showed that NR1, NR2A, and NR2B subunits could form homodimers when they were expressed alone in HEK293 cells. Subunit homodimers were also found existing in heteromeric NMDA receptors formed between NR1 and NR2 subunits. These findings are consistent with functional NMDA receptors being arranged as a dimer of dimers. In addition, our data indicated that the conformation of NR1 subunit homodimers was affected by the partner NR2 subunits during the formation of heteromeric receptor complexes, which might underlie the mechanism by which NR2 subunits modify NMDA receptor function.
Qiu
Shuang
S
Department of Neurobiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310031, China.
Hua
Yu-Lin
YL
Yang
Fan
F
Chen
Yi-Zhang
YZ
Luo
Jian-Hong
JH
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D009474:Neurons / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014162:Transfection
D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; C485182:Cyan Fluorescent Protein; C054562:yellow fluorescent protein, Bacteria