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 assemblydetermining 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.

Key Amino Acid Residues within the Third Membrane Domains of NR1 and NR2 Subunits Contribute to the Regulation of the Surface Delivery of N-methyl-d-aspartate Receptors

“…A recent study showed that the transmembrane regions of the NMDA receptor subunits are necessary for the assembly of NMDA receptors (31). Our electrophysiological data show that the replacements of key amino acid residues within both the NR1 and NR2B M3 domains do not interfere with the formation of the functional NMDA receptors.…”

A Eukaryotic Specific Transmembrane Segment is Required for Tetramerization in AMPA Receptors

“…The deletion of the GluN1 M4 segment either blocked (Horak et al, 2008) or permitted (Meddows et al, 2001) surface expression of NMDARs. However, recent experiments have suggested that for NMDAR subunits, the ion channel core (M1–M3) is required for homodimer formation, whereas both the ion channel core and the M4 segment (as well as S2) are necessary for heterotetrameric assembly (Cao et al, 2011). Part of the uncertainty concerning the role of the M4 segment in NMDAR assembly may reflect that it is not required for dimerization but only tetramerization.…”

Agonist binding to the NMDA receptor drives movement of its cytoplasmic domain without ion flow

“…Such conformational changes induced by agonist binding to GPCRs have been measured directly using spectroscopic methods, including FRET (14,15). For the NMDAR, a tetrameric receptor composed of two GluN1 and two GluN2 subunits in the hippocampus (7,16), intrareceptor FRET signals from fluorescent molecules connected to the extracellular domain of subunits have been measured and used to monitor the assembly of the receptors (17). However, detection of transmembrane transduction that couples agonist binding to conformational changes at the cytoplasmic domain of the NMDAR has not been attempted.…”