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Expression and characterization of a glycine-binding fragment of the N-methyl-D-aspartate receptor subunit NR1.
Biochem J. 1999 Jun 15; 340 (Pt 3):687-92.BJ

Abstract

N-Methyl-D-aspartate receptor channels are composed of an NR1 subunit and at least one of the NR2 subunits (NR2A-D). Activation of the N-methyl-d-aspartate receptor requires the co-agonists glycine and glutamate. It has been proposed that the NR1 subunit possesses a glycine-binding site. We have expressed a soluble form of the NR1 subunit, which was produced by connecting the N-terminal extracellular region with the extracellular loop between the third and fourth membrane segments, by a baculovirus system along with full-length and truncated membrane-bound forms. The soluble NR1 receptor was efficiently secreted into the culture medium and showed a high affinity for ligands. The Kd of a glycine-site antagonist, [3H]MDL 105,519 [(E)-3-(2-phenyl-2-carboxyethenyl)-4, 6-dichloro-1H-indole-2-carboxylic acid], for the soluble receptor was 3.89+/-0.97 nM, which was comparable to the Kd of 4.47+/-1.39 nM for the membrane-bound full-length form. These values were close to the values reported previously with the use of rat brain membranes and Chinese hamster ovary cells expressing the full-length form of the NR1 subunit. The Ki values of other glycine-site antagonists, L-689,560 (trans-2-carboxy-5,7-dichloro - 4 - phenylaminocarbonylamino - 1,2,3,4 - tetrahydroquinoline), 5, 7-dichlorokynurenate and 5,7-dinitroquinoxaline-2,3-dione, for the soluble receptor were also similar to those for the full-length form of NR1. [3H]MDL 105,519 binding was also inhibited by the agonists glycine and d-serine. Thus the affinity and selectivity of ligand-binding characteristics of the NR1 subunit is conferred on the soluble form of the NR1 subunit. This soluble receptor provides a good experimental tool for initiating a biophysical analysis of the N-methyl-d-aspartate receptor channel protein.

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Authors+Show Affiliations

Miyazaki J
Department of Molecular Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
Nakanishi S
No affiliation info available
Jingami H
No affiliation info available

MeSH

AmidohydrolasesAminoquinolinesAnimalsBaculoviridaeBinding SitesBlotting, WesternCell LineGenetic VectorsGlycineGlycosylationIndolesInhibitory Concentration 50InsectaLigandsMolecular WeightPeptide FragmentsPeptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine AmidaseReceptors, N-Methyl-D-AspartateRecombinant Fusion ProteinsSolubility

Pub Type(s)

Journal Article

Language

eng

PubMed ID

10359652

Citation

Miyazaki, J, et al. "Expression and Characterization of a Glycine-binding Fragment of the N-methyl-D-aspartate Receptor Subunit NR1." The Biochemical Journal, vol. 340 (Pt 3), 1999, pp. 687-92.
Miyazaki J, Nakanishi S, Jingami H. Expression and characterization of a glycine-binding fragment of the N-methyl-D-aspartate receptor subunit NR1. Biochem J. 1999;340 (Pt 3):687-92.
Miyazaki, J., Nakanishi, S., & Jingami, H. (1999). Expression and characterization of a glycine-binding fragment of the N-methyl-D-aspartate receptor subunit NR1. The Biochemical Journal, 340 (Pt 3), 687-92.
Miyazaki J, Nakanishi S, Jingami H. Expression and Characterization of a Glycine-binding Fragment of the N-methyl-D-aspartate Receptor Subunit NR1. Biochem J. 1999 Jun 15;340 (Pt 3):687-92. PubMed PMID: 10359652.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Expression and characterization of a glycine-binding fragment of the N-methyl-D-aspartate receptor subunit NR1. AU - Miyazaki,J, AU - Nakanishi,S, AU - Jingami,H, PY - 1999/6/9/pubmed PY - 1999/6/9/medline PY - 1999/6/9/entrez SP - 687 EP - 92 JF - The Biochemical journal JO - Biochem J VL - 340 (Pt 3) N2 - N-Methyl-D-aspartate receptor channels are composed of an NR1 subunit and at least one of the NR2 subunits (NR2A-D). Activation of the N-methyl-d-aspartate receptor requires the co-agonists glycine and glutamate. It has been proposed that the NR1 subunit possesses a glycine-binding site. We have expressed a soluble form of the NR1 subunit, which was produced by connecting the N-terminal extracellular region with the extracellular loop between the third and fourth membrane segments, by a baculovirus system along with full-length and truncated membrane-bound forms. The soluble NR1 receptor was efficiently secreted into the culture medium and showed a high affinity for ligands. The Kd of a glycine-site antagonist, [3H]MDL 105,519 [(E)-3-(2-phenyl-2-carboxyethenyl)-4, 6-dichloro-1H-indole-2-carboxylic acid], for the soluble receptor was 3.89+/-0.97 nM, which was comparable to the Kd of 4.47+/-1.39 nM for the membrane-bound full-length form. These values were close to the values reported previously with the use of rat brain membranes and Chinese hamster ovary cells expressing the full-length form of the NR1 subunit. The Ki values of other glycine-site antagonists, L-689,560 (trans-2-carboxy-5,7-dichloro - 4 - phenylaminocarbonylamino - 1,2,3,4 - tetrahydroquinoline), 5, 7-dichlorokynurenate and 5,7-dinitroquinoxaline-2,3-dione, for the soluble receptor were also similar to those for the full-length form of NR1. [3H]MDL 105,519 binding was also inhibited by the agonists glycine and d-serine. Thus the affinity and selectivity of ligand-binding characteristics of the NR1 subunit is conferred on the soluble form of the NR1 subunit. This soluble receptor provides a good experimental tool for initiating a biophysical analysis of the N-methyl-d-aspartate receptor channel protein. SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/10359652/Expression_and_characterization_of_a_glycine_binding_fragment_of_the_N_methyl_D_aspartate_receptor_subunit_NR1_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/10359652/ DB - PRIME DP - Unbound Medicine ER -