Expression and Initial Characterization of a Soluble Glycine Binding Domain of the N-Methyl-d-aspartate Receptor NR1 Subunit

Abstract

Glycine is an essential co-agonist of the excitatory N-methyl-D-aspartate (NMDA) receptor, a subtype of the ionotropic glutamate receptor family. The glycine binding site of this hetero-oligomeric ion channel protein is formed by two distinct extracellular regions, S1 and S2, of the NR1 subunit, whereas the homologous domains of the NR2 subunit mediate glutamate binding. Here, segments S1 and S2 of the NR1 polypeptide were fused via a linker peptide followed by N-and C-terminally tagging with Flag and His 6 epitopes, respectively. Infection of High Five insect cells with a recombinant baculovirus containing this glycine binding site construct resulted in efficient secretion of a soluble fusion protein of about 53 kDa. After affinity purification to near-homogeneity, the fusion protein bound the competitive glycine site antagonist [ 3 H]MDL105,519 with high affinity (K d ‫؍‬ 5.22 ؎ 0.13 nM) similar to that determined with rat brain membrane fractions. This high affinity binding could be competed by the glycine site antagonist 7-chlorokynurenic acid as well as the agonists glycine and D-serine but not by L-glutamate. This indicates that the S1 and S2 domains of the NR1 subunit are sufficient for the formation of a glycine binding site that displays pharmacological properties similar to those of the NMDA receptor in vivo.Excitatory neurotransmission in the mammalian brain is predominantly mediated by members of the glutamate receptor family of ligand-gated ion channels, which have been grouped into three distinct subfamilies, the ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) 1 receptors, the kainate receptors, and the N-methyl-D-aspartate (NMDA) receptors (1). Among the glutamate receptors, the NMDA receptor has gained particular interest because of a number of unique properties that classify this ligand gated ion channel as a coincidence detecting molecule in synaptic plasticity and memory formation (2). NMDA receptors are highly permeable to Ca 2ϩ (3), they are regulated by a voltage-dependent Mg 2ϩ block (4), and simultaneous binding of both glutamate and the co-agonist glycine is required for efficient activation of NMDA receptors (5, 6). Prolonged stimulation resulting in high Ca 2ϩ influx causes neuronal cell death, which has been implicated in anoxic-ischemic injury and possibly other neurodegenerative disorders (7). Therefore, the binding site for the co-agonist glycine is a target for site-specific NMDA receptor antagonists, which are proved to be neuroprotective in animal stroke models (8).Structural analysis of the NMDA receptor was initiated by expression cloning of the NMDA receptor subunit NMDAR1 (NR1 or ). Subsequently, additional cDNAs including eight NR1 splice variants (NR1a to NR1h) and different isoforms of a second type of NMDA receptor subunits (NR2A-D, ⑀1-4) were identified (reviewed in Ref. 9). At the N terminus of both, the NR1 and NR2 subunits, a cleavable leader sequence is followed by a long extracellular domain and, in the second half of the polypeptides, four hydrophobic m...

Apolipoprotein E4 Domain Interaction Occurs in Living Neuronal Cells as Determined by Fluorescence Resonance Energy Transfer

“…After centrifugation at 13,000 rpm for 15 min in an Eppendorf centrifuge (Westbury, NY), apoE labeled with both YFP and CFP was detected in the supernatant by Western blotting with M2 monoclonal antibodies against the FLAG tag (Sigma) (75).…”

Expression and characterization of a glycine-binding fragment of the N-methyl-D-aspartate receptor subunit NR1

“…Recently, Ivanovic et al [25] reported similar results that a polypeptide fragment containing only the S1 and the S2 regions of the NR1 subunit is able to form a glycine-binding site. In comparison with our soluble receptor, their fragment was devoid of the N-terminal LIVBP-like region preceding the S1 region and the N-terminal portion (seven residues) of the extracellular loop region between M3 and M4.…”

“…In comparison with rat brain membrane preparations, the recombinant NR1-F1 and NR1-L1 show a lower affinity for the agonists glycine and -serine but not for antagonists. The S1-S2 fusion protein did not show a high affinity for the agonists either [25]. It is reported that antagonist binding is less affected by amino acid mutagenesis in the S2 region, whereas agonist binding is affected by amino acid mutation both in S1 and S2 [14,15].…”

“…Thus the participation of both S1 and S2 regions possibly provided by a closed form seems to be predictable for agonist binding. Because our NR1 subunits show a decreased affinity for agonists, the homomeric NR1 proteins might exist in an open form, namely an antagonistpreferring form as postulated [25,26]. Co-expression with NR2 subunits is expected to improve the affinity for agonist.…”

Cysteine Mutagenesis and Homology Modeling of the Ligand-binding Site of a Kainate-binding Protein

“…8, 10, and 16) consists of a portion of the N terminus of the protein before the first transmembrane segment (M1) and a large portion of the protein between the third and fourth membrane associated segments (M3 and M4). The S1S2 domain has been modeled previously based on the relatively low homology with bacterial amino acid-binding proteins (16 -22) and can be expressed independently in bacteria (23,24) and insect cells (25,26) with an apparently native fold. From homology modeling and site-directed mutagenesis, six regions (R1-R6; Fig.…”