Transmembrane region of N-methyl-D-aspartate receptor (NMDAR) subunit is required for receptor subunit assembly

Abstract

N-Methyl-D-aspartate receptors (NMDARs), one of three main classes of ionotropic glutamate receptors, play major roles in synaptic plasticity, synaptogenesis, and excitotoxicity. Unlike non-NMDA receptors, NMDARs are thought to comprise obligatory heterotetrameric complexes mainly composed of GluN1 and GluN2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, most of the NMDAR subunits can neither leave the endoplasmic reticulum (ER) nor be expressed in the cell membrane because of the ER retention signals. Only when NMDARs are heteromerically assembled can the ER retention signals be masked and NMDARs be expressed in the surface membrane. However, the mechanisms underlying NMDAR assembly remain poorly understood. To identify regions in subunits that mediate this assembly, we made a series of truncated or chimeric cDNA constructs. Using FRET measurement in living cells combined with immunostaining and coimmunoprecipitation analysis, we examined the assembly-determining domains of NMDAR subunits. Our results indicate that the transmembrane region of subunits is necessary for the assembly of NMDAR subunits, both for the homodimer and the heteromer.

FIGURE 1.

FRET detection of NT-deleted NMDAR subunits in HEK293 cells. A, schematic topology of the NMDAR subunit. The dark gray line indicates the ATD, the light gray lines indicate S1 and S2, and the black lines indicate the membrane and intracellular domains. B, schematic representation of fluorescent protein-tagged NMDAR or GABA_A α1 constructs. The green box represents CFP or YFP. The hatched box corresponds to the signal peptide; the long vertical bars are the three transmembrane domains M1, M3, and M4; and the short vertical bar corresponds to the reentrant channel loop, M2. The position of each truncation is indicated by the residue number. C, summary data of FR values between NT-deleted subunit homodimers. D, summarized data of FR values between NT-deleted subunit heteromers. **, p < 0.01 versus coexpression of CFP-GluN1 and GABA_A α1-YFP in HEK293 cells. C, CFP; Y, YFP; X, either CFP or YFP.

FIGURE 2.

Deletion of NT abolishes trafficking of the assembled GluN1/GluN2 complex to the cell membrane. A–C, HEK 293 cells expressing CFP-GluN1/YFP-GluN2A or CFP-GluN1/YFP-GluN2B (A), CFP-GluN1_ΔNT/YFP-GluN2A or CFP-GluN1_ΔNT/YFP-GluN2B (B), CFP-GluN1/YFP-GluN2A_ΔNT or CFP-GluN1/YFP-GluN2B_ΔNT (C) were surface-stained with anti-GFP antibodies. CFP was tagged to the GluN1 or NT-deleted GluN1 subunit (left columns), whereas YFP was tagged to the GluN2 or NT-deleted GluN2 subunit (center columns). Red fluorescence (right columns) was from surface staining using anti-GFP antibody and Cy3-conjugated secondary antibody. Scale bar = 10 μm. D, percentages of cells with detectable surface labeling were determined for over 200 YFP-expressing cells for each combination of cDNAs in three experiments. C, CFP; Y, YFP.

FIGURE 3.

Detection of the assembly of GluN1/GluA1 chimeras with wild-type NMDAR subunits. A, schematic representation of GluN1/GluA1 chimeras and NMDAR subunits tagged with XFP. B, summary of the FRs between different NMDAR constructs. The numbers of cells measured were between 15 and 30. **, p < 0.01 versus coexpression of CFP-GluN1 and GABA_A α1-YFP in HEK293 cells. C, interaction of YFP-tagged GluN1 or GluN1_{M1–4(GluA1)} with wild-type GluN1 using coimmunoprecipitation and immunoprecipitation (IP) with anti-GFP antibody and Western blotting with anti-GluN1 C terminus antibody. The arrowheads indicate the band for the wild-type GluN1 subunit. The arrow indicates the nonspecific band of YFP-GluN1_{M1–4(GluA1).} S, supernatant; P, pellet; C, CFP; Y, YFP; X, either CFP or YFP.

FIGURE 4.

M1–3 region is required for GluN1 homodimer assembly. A, schematic representation of GluN1/GluA1 chimeras and NMDAR subunits tagged with XFP. B, summary of the FRs from HEK293 cells coexpressing the NMDAR chimera and wild-type NMDAR. The numbers of cells measured were between 26 and 35. **, p < 0.01 versus coexpression of CFP-GluN1 and GABA_Aα1-YFP in HEK293 cells. C, CFP; Y, YFP; X, either CFP or YFP.

FIGURE 5.

Membrane segments of GluN1 can assemble as homooligomers. A, schematic representation of different GluN1 transmembrane domain constructs tagged with XFP. B, summarized data of FR values from the oligomerization of different GluN1 membrane domain constructs. The numbers of cells measured were between 22 and 28. **, p < 0.01 versus coexpression of CFP-GluN1 and GABA_Aα1-YFP in HEK293 cells. C, CFP; Y, YFP; X, either CFP or YFP.

FIGURE 6.

Schematic model for native, deleted, and chimeric NMDA receptor assembly. A, N and C termini are not necessary for homomeric and heteromeric assembly of NMDAR subunits. B, the transmembrane region of the NMDAR subunit is essential to homomeric and heteromeric assembly of NMDAR subunits. C, the M1–3 domain of GluN1 is responsible for GluN1 homodimer assembly, and the whole M1–4 is involved in GluN1/GluN2 heteromer assembly.