Expression and characterization of a glycine-binding fragment of the N-methyl-D-aspartate receptor subunit NR1.
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.
Miyazaki
J
J
Department of Molecular Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
Nakanishi
S
S
Jingami
H
H
D000581:Amidohydrolases / Q000378:metabolism; D000634:Aminoquinolines / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics; D005998:Glycine / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006031:Glycosylation; D007211:Indoles / Q000037:antagonists & inhibitors / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007313:Insecta / Q000166:cytology / Q000235:genetics; D008024:Ligands; D008970:Molecular Weight; D010446:Peptide Fragments / Q000819:agonists / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D012995:Solubility
D000634:Aminoquinolines; D007211:Indoles; D008024:Ligands; C101788:MDL 105519; D010446:Peptide Fragments; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; C074617:4-trans-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D005998:Glycine