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

Christian Madry,Ingo Bartholomäus,Ivana Mesic,Bodo Laube,Thomas Schüler

doi:10.1074/jbc.m703539200

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(DeltaNTD)) abrogating NR1 homo-oligomerization did not affect NR1/NR3A receptor stoichiometry or function. Hence, homo-oligomerization of the NR1 subunit is not essential for proper NR1/NR3 receptor assembly. Because identical results were obtained for NR1(DeltaNTD)/NR2 NMDA receptors (Madry, C., Mesic, I., Betz, H., and Laube, B. (2007) Mol. Pharmacol., 72, 1535-1544) 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.

Highlights

Excitatory neurotransmission in the mammalian brain is mainly mediated by ionotropic glutamate receptors.4 Based on pharmacological studies, iGluRs have been grouped into three distinct subfamilies: (RS)-2-amino-3-(3-hydroxy-5methyl-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]
A biophysical and biochemical approach based on fluorescence resonance energy transfer (FRET) and BN-PAGE was used to investigate the role of the NR1, NR2, and NR3 subunits in NMDA receptor oligomerization

Summary

Introduction

Excitatory neurotransmission in the mammalian brain is mainly mediated by ionotropic glutamate receptors (iGluRs). Based on pharmacological studies, iGluRs have been grouped into three distinct subfamilies: (RS)-2-amino-3-(3-hydroxy-5methyl-4-isoxazolyl)propionic acid (AMPA) receptors, kainate receptors, and N-methyl-D-aspartic acid (NMDA) receptors [1]. 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]. The so-called “conventional” type of NMDA receptors is a tetrameric membrane protein composed of two NR1 and NR2 subunits, each [3], that requires both glutamate and the coagonist glycine for channel activation [4]. Triheteromeric receptors containing NR1, NR2, and NR3 subunits reconstitute glycine- and glutamate-gated channels, which strikingly differ in channel properties from the respective NR1/ NR2 and NR1/NR3 di-heteromers [7, 8]. Our results support the idea that NR1/NR2 or NR1/NR3 heterodimers are intermediate states of NMDA receptor assembly and that subunit composition of tetrameric NMDA receptors is defined by a dimer of heterodimers

Results

Discussion

Conclusion