Expression and initial characterization of a soluble glycine binding domain of the N-methyl-D-aspartate receptor NR1 subunit.
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 His6 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 [3H]MDL105,519 with high affinity (Kd = 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.
Department of Neurochemistry, Max-Planck-Institute for Brain Research, Deutschordenstrabetae 46, D-60528 Frankfurt/Main, Germany.
A
A
Ivanovic
H
H
Reiländer
B
B
Laube
J
J
Kuhse
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive / Q000502:physiology; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D007736:Kynurenic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics
D018691:Excitatory Amino Acid Antagonists; D007211:Indoles; C101788:MDL 105519; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D012694:Serine; D007736:Kynurenic Acid; C057013:7-chlorokynurenic acid; D005998:Glycine