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-aspart ate (NMDA) receptor, a subtype of the ionotropic glutamate receptor fa mily. The glycine binding site of this hetero-oligomeric ion channel p rotein is formed by two distinct extracellular regions, S1 and S2, of the NR1 subunit, whereas the homologous domains of the NR2 subunit med iate glutamate binding. Here, segments SI and S2 of the NR1 polypeptid e were fused via a linker peptide followed by N-. and C-terminally tag ging with Flag and His, epitopes, respectively. Infection of High Five insect cells with a recombinant baculovirus containing this glycine b inding site construct resulted in efficient secretion of a soluble fus ion protein of about 53 kDa. After affinity purification to near-homog eneity, the fusion protein bound the competitive glycine site antagoni st [H-3]MDL105,519 with high affinity (K-d = 5.22 +/- 0.13 nM) similar to that determined with rat brain membrane fractions. This high affin ity binding could be competed by the glycine site antagonist 7-chlorok ynurenic 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 subu nit are sufficient for the formation of a glycine binding site that di splays pharmacological properties similar to those of the NMDA recepto r in vivo.