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

Abstract

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 (NR2⌬2) 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 NR2⌬2 truncations are able to form homomers and heteromers on co-expression with NR1-1a. Furthermore, when NR2⌬2 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 NR2B⌬5 (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. N-methyl-D-aspartate (NMDA)3 receptors are heteromeric complexes primarily assembled from two subunit classes: NR1 and NR2. Co-assembly of NR1 and NR2 subunits is essential for formation of a functional channel, presumed to be a tetramer containing two NR1 and two NR2 subunits (1-2). NR1 is a single subunit with eight splicing variants, which have distinct trafficking and functional properties (3). NR2 subunits are coded by four separate genes, NR2A-D, each of which can endow the receptor channel with different properties (4). Thus, the subunit composition of NMDA receptors is a major determinant of NMDA receptor-mediated activity in the central nervous system. Although much is known about the physiological roles that NMDARs play in long term potentiation (LTP), learning and memory (5-7), much remains to be learned about the mechanisms by which these receptors are assembled, sorted, targeted, and anchored to the appropriate location.The C-terminal domains of the receptor subunits contain critical determinants of subcellular receptor localization. Regulation of receptor trafficking by these determinants ensures that only fully assembled multimeric receptors are expressed on the plasma membrane. When expressed alone in heterogeneous cells, the major NR1 isoform (NR1-1) is retained in the ER because of an RR...

Phosphorylation of Tyrosine 1070 at the GluN2B Subunit Is Regulated by Synaptic Activity and Critical for Surface Expression of N-Methyl-d-aspartate (NMDA) Receptors

“…Whole-cell Patch Clamp Recording-The electrophysiological methods have been described previously (24). The extracellular recording solution contained 147 mM NaCl, 3 mM KCl, 10 mM HEPES, 8 mM glucose, 2 mM MgCl 2 (305 mosM, pH adjusted to 7.30 with NaOH).…”

“…Immunocytochemistry-Surface-staining of HEK293 cells and cortical neurons was performed as described previously (24). Cortical neurons were plated on coverslips and transfected with the indicated plasmid on DIV 7.…”

“…Reagents-Wild-type (WT) GluN1 and GluN2B tagged with extracellular amino-terminal GFP (GFP-GluN2B WT ) in the pRK5 vector were described previously (24). The primary antibodies used were: Tyr(P)-1472 GluN2B (1:500; Millipore, Lake Placid, NY), mouse GluN2B (1:2000; Cell Signaling, Danvers, MA), Ser(P)-845-GluA1 (1:1000; Millipore), GluA1 (1:1000; Millipore), rabbit monoclonal Fyn (1:2000; Epitomics, Burlingame, CA), mouse monoclonal PSD-95 (1:1000; Abcam, Cambridge, MA), ␤-actin (1:20000; Sigma), and rabbit monoclonal GFP (1:2000; Epitomics).…”

Transmembrane Region of N-Methyl-d-aspartate Receptor (NMDAR) Subunit Is Required for Receptor Subunit Assembly

“…Surface Immunostaining and Quantitative Analysis-These methods have been described previously (20,21). Cells were examined through a 60 ϫ 1.4 (numerical aperture) oil immersion objective on a FV1000 confocal laser scanning microscope equipped with imaging analysis software (Olympus, Tokyo, Japan).…”

Role of the fourth membrane domain of the NR2B subunit in the assembly of the NMDA receptor

“…Second, using truncated and chimeric NMDA receptor subunits, we showed recently that M3 of both NR1 and NR2B possess ER retention signals and that these domains as well as M4 of NR1 are responsible for masking the ER retention signals in the M3 domains.10 Third, NR1/NR2 complexes can reach the cell surface membrane even when either one or both C-terminal regions are deleted five or six amino acid residues after M4.10 -12 However, full-length NR2B constructs lacking the HLFY motif, located one amino acid residue after the M4 domain, are not delivered to the surface membrane.6 Fourth, a later study showed that replacement of the HLFY motif with alanines in the NR2B subunit, truncated immediately after the mutated HLFY motif, does not affect the formation of functional channels. 13 Since this study also showed that at least three amino acid residues after NR2B M4 are necessary for surface trafficking of the receptors, this indicates that the NR2B M4 domain and surrounding segments are critical for correct assembly and formation of functional receptors. Finally, co-expression of the NR2 subunit truncated before M4 (NR2 preM4) with the full-length NR1 subunit fails to produce functional receptors.…”