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Expression and initial characterization of a soluble glycine binding domain of the N-methyl-D-aspartate receptor NR1 subunit.

Ivanovic, A. and Reiländer, H. and Laube, Bodo and Kuhse, J. (1998):
Expression and initial characterization of a soluble glycine binding domain of the N-methyl-D-aspartate receptor NR1 subunit.
In: The Journal of biological chemistry, pp. 19933-7, 273, (32), ISSN 0021-9258, [Article]

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 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.

Item Type: Article
Erschienen: 1998
Creators: Ivanovic, A. and Reiländer, H. and Laube, Bodo and Kuhse, J.
Title: Expression and initial characterization of a soluble glycine binding domain of the N-methyl-D-aspartate receptor NR1 subunit.
Language: English
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 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.

Journal or Publication Title: The Journal of biological chemistry
Volume: 273
Number: 32
Divisions: 10 Department of Biology
10 Department of Biology > Neurophysiology and Neurosensory Systems
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Date Deposited: 11 Apr 2011 13:40
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