Formation of NR1/NR2 and NR1/NR3 Heterodimers Constitutes the Initial Step in N-Methyl-D-aspartate Receptor Assembly

Abstract

N-Methyl-D-aspartate (NMDA) receptors are tetrameric protein complexes composed of the glycine-binding NR1 subunit with a glutamate-binding NR2 and/or glycine-binding NR3 subunit. Tri-heteromeric receptors containing NR1, NR2, and NR3 subunits reconstitute channels, which differ strikingly in many properties from the respective glycine-and glutamate-gated NR1/NR2 complexes and the NR1/NR3 receptors gated by glycine alone. Therefore, an accurate oligomerization process of the different subunits has to assure proper NMDA receptor assembly, which has been assumed to occur via the oligomerization of homodimers. Indeed, using fluorescence resonance energy transfer analysis of differentially fluorescence-tagged subunits and blue native polyacrylamide gel electrophoresis after metabolic labeling and affinity purification revealed that the NR1 subunit is capable of forming homo-oligomeric aggregates. In contrast, both the NR2 and the NR3 subunits formed homo-and hetero-oligomers only in the presence of the NR1 subunit indicating differential roles of the subunits in NMDA receptor assembly. However, co-expression of the NR3A subunit with an N-terminal domain-deleted NR1 subunit (NR1 ⌬NTD ) abrogating NR1 and NR1-containing hetero-oligomers are readily formed, we assume that heterodimerization of the NR1 with an NR3 or NR2 subunit, which is followed by the subsequent association of two heterodimers, is the key step in determining proper NMDA receptor subunit assembly and stoichiometry.Excitatory neurotransmission in the mammalian brain is mainly mediated by ionotropic glutamate receptors (iGluRs). 4Based on pharmacological studies, iGluRs have been grouped into three distinct subfamilies: (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, kainate receptors, and N-methyl-D-aspartic acid (NMDA) receptors (1). All iGluR subunits share a common modular design of four distinct regions: (i) an extracellular N-terminal domain (NTD) of about 400 amino acids sharing homology with the bacterial leucine-, isoleucine-, and valine-binding protein, implicated to play a role in iGluR oligomerization and modulation, (ii) an extracellular S1S2 ligand binding domain sharing homology with the bacterial glutamine-binding protein that binds agonists in a Venus-flytrap like mechanism, (iii) a membrane-associated domain composed of four membrane segments forming the ion channel, and (iv) an intracellular C-terminal domain involved in linking the receptor to the membrane scaffold and signal transduction proteins (2).The NMDA subtype of iGluRs is an obligatory hetero-oligomeric membrane protein composed of homologous NR1, NR2, and/or NR3 subunits and plays a key role in brain development, synaptic plasticity, and memory formation (1). Cloning of NMDA receptor subunits revealed that the glycine-binding NR1 subunit is a single gene product occurring in eight splice variants (a-h), whereas the glutamate-binding NR2 and the glycine-binding NR3 subunits are encoded by four (NR2A-NR2D) and two (NR3A and -B) different genes...

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

“…Weak interactions can be preserved and reliably detected. Another group using FRET measurement found that the full-length GluN1 subunit assembles in a dimeric form, whereas neither GluN2A nor GluN2B show defined aggregation (18). Their results are inconsistent with our result showing that the GluN2 subunit can also form homodimers when expressed alone (Fig.…”

“…Studies using biochemical methods and immunostaining indicate that the C terminus and/or M4 are not involved in the heteromer formation between GluN1 and GluN2 subunits (9,15), whereas the ATD or even the whole N terminus is necessary for NMDAR subunit assembly (15,16). However, still other research indicates that the ATD mainly plays modulatory roles in NMDAR trafficking and function and is not directly involved in receptor assembly (10,17,18).…”

Supralinear potentiation of NR1/NR3A excitatory glycine receptors by Zn 2+ and NR1 antagonist

“…Because these NR1/ NR2/NR3A receptors are tetrameric complexes composed of NR1/ NR2 and NR1/NR3A heterodimers (11,33), they may share modulatory mechanisms with NR1/NR3A receptors. Hence, compounds targeting the potentiating mechanisms described here might provide novel leads for therapeutic intervention in diseases involving NR3A containing NMDA receptors, such as stroke, vascular dementia, or schizophrenia (19-21, 34, 35).…”

“…Coexpression of the glycinebinding NR3A or NR3B subunit with NR1 and NR2 subunits generates heteromeric receptors composed of NR1, NR2, and NR3 subunits, which also require glutamate and glycine for gating but exhibit altered biophysical properties (7)(8)(9)(10). In contrast, recombinant NMDA receptors assembled from two NR1 and two NR3 subunits (11) do not respond to glutamate but are exclusively activated by glycine (''excitatory glycine receptors''; ref. 12).…”

Psychiatric phenomena as initial manifestation of encephalitis by anti‐NMDAR antibodies

“…The NMDAR1 subunit (NR1) cDNA fused in frame to YFP was a kind gift from B. Laube (17). HEK293 cells (ATCC #CRL1537) were grown on glass coverslips in DMEM and Ham's F10 (Gibco, Breda, The Netherlands) supplemented with 10% FBS (Gibco) and 1% penicillin and streptomycin (Gibco).…”