Export from the Endoplasmic Reticulum of Assembled N-Methyl-D-aspartic Acid Receptors Is Controlled by a Motif in the C Terminus of the NR2 Subunit

Abstract

Functional N-methyl-D-aspartic acid (NMDA) receptors are formed from the assembly of NR1 and NR2 subunits. When expressed alone, the major NR1 splice variant and the NR2 subunits are retained in the endoplasmic reticulum (ER), reflecting a quality control mechanism found in many complex multisubunit proteins to ensure that only fully assembled and properly folded complexes reach the cell surface. Recent studies have identified an RRR motif in the C terminus of the NR1 subunit, which controls the ER retention of the unassembled subunit. Here we investigated the mechanisms controlling the ER retention of the NR2 subunit and the export of the assembled complex from the ER. We found that Tac chimeras of the C terminus of the NR2B subunit show that an ER retention signal is also present in the NR2B subunit. In assembled complexes, ER retention signals on the individual subunits must be overcome to allow the complex to leave the ER. One common mechanism involves mutual masking of the signals on the individual subunits. Our data do not support such a mechanism for regulating the release of assembled NMDA receptors from the ER. We found that the motif, HLFY, immediately following transmembrane domain 4 of the NR2 subunit, is required for the assembled complex to exit from the ER. Mutation of this motif allowed the assembly of NR1 and NR2 subunits into a complex that was functional, based on MK-801 binding, but it is retained in the ER. These results are consistent with HLFY functioning as a signal that is necessary for the release of the assembled functional NMDA receptor complex from the ER. N-Methyl-D-aspartic acid (NMDA)1 receptors are multisubunit complexes assembled from three subunit classes, NR1, NR2, and NR3. Co-assembly of NR1 and NR2 subunits is required to form a functional channel, generally considered to be a tetramer containing two NR1 and two NR2 subunits (1).NR3 subunits can assemble with NR1 alone to form a glycine receptor (2), and they also can have a modulatory role on NR1/NR2 complexes by associating in a yet undefined fashion (3). NR1 is a single subunit that can be expressed in eight splice variants, which have distinct trafficking and functional properties. There are four NR2 subunits, NR2A-D, each of which differs in its distribution and developmental expressions. The type of NR2 subunit present in the complex has a major impact on the functional properties of the channel. For example, NR1/ NR2A channels display rapidly desensitizing currents and close association with the synapse compared with NR1/NR2B channels (4, 5). Therefore, the subunit composition of NMDA receptors is a major determinant of NMDA receptor-mediated activity and localization in the central nervous system. Several splice variants of NR1 and different subunits of NR2 are present in the same neurons, leading to multiple receptors with different functional and trafficking properties (6). For example, a neuron that expresses NR2A and NR2B subunits can contain three functionally distinct receptors: NR1/NR2A, NR1/NR2B, and NR1/NR2A...

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

“…Therefore, both of our findings can be interpreted similarly that the three amino acid segment following TM4, EHL, is critical for forming a functional receptor on the cell surface. However, we noted that mutations of four amino acids (HLFY) in the study of Hawkins et al (10) were made in the fulllength NR2B subunit, whereas our NR2B truncations were made by removal of all but a few amino acids after TM4. We asked the question whether the mutation of EHLFY in our NR2B truncates (with removal of all the rest of the C-terminus but the five amino acids) would also lead a complete loss of functional surface NMDARs.…”

“…ER retention is a common feature of the quality control mechanism for complex proteins, ensuring that unassembled or otherwise defective proteins are not exported from the ER. Previous studies indicate the presence of ER retention motifs on the C terminus of both NR1 and NR2B subunits, but the mechanism by which the assembled complex overrides ER retention to reach the cell surface is largely unknown (10).…”

“…The presence of NR2 subunits can overcome the ER retention mediated by the RRR motif in NR1-1 and deliver the NR1-1/ NR2 receptor complexes to the membrane surface. The NR2 subunit is also retained in the ER when expressed alone in heterogeneous cells (10) and in neurons lacking the NR1 subunit (11). That the intracellular C terminus of the NR2B subunit plays a role in its ER retention is suggested by a study using Tac chimeras of its C terminus (10), but to date there is no particular domain or motif identified as being responsible for the ER retention.…”

“…The NR2 subunit is also retained in the ER when expressed alone in heterogeneous cells (10) and in neurons lacking the NR1 subunit (11). That the intracellular C terminus of the NR2B subunit plays a role in its ER retention is suggested by a study using Tac chimeras of its C terminus (10), but to date there is no particular domain or motif identified as being responsible for the ER retention. Partial truncation of the C terminus of NR2 subunits does not abolish the assembly and surface expression of func-tional NMDA receptor channels in neurons from genetically modified mice, although the synaptic localization of the receptors is impaired as a result of loss of the PDZ binding domain (12)(13)(14)(15)(16).…”

“…Partial truncation of the C terminus of NR2 subunits does not abolish the assembly and surface expression of func-tional NMDA receptor channels in neurons from genetically modified mice, although the synaptic localization of the receptors is impaired as a result of loss of the PDZ binding domain (12)(13)(14)(15)(16). Mutation of the four amino acids (HLFY) following the TM4 region of the NR2B subunit markedly reduces the level of surface expression of NR1/NR2B complexes in heterologous cells (10). Taken together, these studies strongly suggest that the several amino acids following the TM4 domain of the NR2 subunit play a crucial role in releasing the assembled functional NMDA receptor complex from the ER.…”