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Antibodies raised against a 43 kDa component of a complex of synaptic membrane proteins with ligand binding sites characteristic of glutamate/N-methyl-D-aspartate (NMDA) receptors, were used previously to clone a cDNA for a glycine-, glutamate-, and thienylcyclohexylpirperidine (TCP)-binding protein, pGlyBP (Kumar et al., Biochem. Biophys. Res. Commun. 216, 390-398, 1995). In the present studies, the antibodies were shown to label a 60 kDa protein, in synaptic membranes, that was relatively hydrophilic as demonstrated by its predominant separation in the detergent-depleted phase of proteins solubilized with Triton X-114. A 55-60 kDa protein was purified from rat brain synaptic membranes by chromatographic separation through matrices derivatized with 5,7-di-chlorokynurenic acid (5,7-DCK) followed by chromatography on a matrix derivatized with 8-hydroxyquinoline (8-OHQ). The isolated fractions were highly enriched in strychnine-insensitive [3H]glycine, NMDA- and glutamate-sensitive L-[3H]glutamate, and MK-801-sensitive [3H]TCP binding sites. The purified protein bound [3H]glycine with a stoichiometry of 1.1-1.2 mol glycine per mol protein and exhibited both high (KD = 280 nM) and low affinity (KD = 30 microM) glycine binding sites. Glycine binding was inhibited by D-serine and R-(+)-3-amino-1-hydroxypyrrolidin-2-one(R-(+)-HA-966). The KD values for high and low affinity sites of glycine binding as well as those for the inhibition by R-(+)-HA-966 were very similar to the KDs for glycine binding to the expressed pGlyBP. Both L-glutamate and glycine activated [3H]TCP binding to the isolated proteins, but with relatively low affinity. The anti-43 kDa antibodies reacted strongly with the 55-60 kDa protein. Based on these results, it appears that the 60 kDa glycoprotein in brain synaptic membranes described in the present study is the same protein as the cloned pGlyBP.

The functional reconstitution of glutamate receptor proteins purified from mammalian brain has been difficult to accomplish. However, channels activated by L-glutamate (L-Glu) and N-methyl-D-aspartate (NMDA) were detected in planar lipid bilayer membranes (PLMs) following the reconstitution of a complex of proteins with binding sites for NMDA receptor (NMDAR) ligands. The presence of glycine was necessary for optimal activation. A linear current-voltage relationship was observed with the reversal potential being zero. Channels activated by L-Glu had conductances of 23, 47 and 65 pS, and were suppressed partially by competitive and fully by noncompetitive inhibitors of NMDARs. Magnesium had little effect on the reconstituted channels.

Polyclonal antibodies (Ab's) were raised against a 43-kDa component of a protein complex that has ligand recognition sites similar to those of brain N-methyl-D-aspartate (NMDA) receptors. The Ab's were used to immunopurify from brain synaptic membranes a 60-kDa glycine (Gly), glutamate (Glu) and thienylcyclohexylpiperidine (TCP)-binding protein and to screen a rat hippocampal cDNA expression library. A 1.85-kb clone, pGlyBP, coding for a protein of 470 amino acids (52.7 kDa) was identified. Northern blot analyses performed on poly(A+) RNA from brain revealed hybridization of the labeled cDNA probes to transcripts of 1.9 kb. E. coli transformed with the pGyBP expressed a protein that was recognized by the anti-43 kDa Ab's and had recognition sites for Gly, Glu and TCP. The cloned protein has 2 glycosylation sites, 3 hydrophobic domains, 4 cysteine-rich motifs (C-X2-C-X16-20-C-X5-11), and 2 regions homologous to the NR1 receptor protein.

A method was developed for the solubilization of approximately 50% of proteins in synaptic membranes that have ligand-binding characteristics of N-methyl-D-aspartate (NMDA) receptors. Affinity chromatographic separation of the solubilized proteins through L-glutamate-derivatized matrices and subsequent elution by NMDA-containing buffers led to the purification of four predominant proteins with estimated sizes of 67-70, 53-62, 41-43, and 28-36 kDa. The co-purification of NMDA-sensitive L-glutamate binding, dizocilpine-sensitive thienylcyclohexyl piperidine (TCP)-binding, and strychnine-insensitive glycine-binding proteins was achieved by this affinity chromatographic procedure. Glutamate, glycine, and the polyamine spermidine increased both the "on" rate and the equilibrium level of [3H]TCP binding to the isolated proteins. The group of proteins eluted by NMDA from the glutamate-derivatized matrices could be further purified through size exclusion chromatography of the NMDA-sensitive glutamate-binding from the dizocilpine-sensitive TCP-binding proteins. Polyclonal and monoclonal antibodies to the cloned NMDA receptor protein NMDAR1 did not react with any proteins in the solubilized membrane proteins or the purified fractions. However, immunoreaction of antibodies raised against a glutamate-binding protein and a phosphonoaminocarboxylic acid-binding protein indicated that these are two of the major proteins in the purified fractions. These studies indicate that these two proteins might be components of a complex that has some of the characteristics of NMDA receptors and that neuronal membranes may contain varieties of NMDA-like receptors composed of protein subunits that differ from the NMDAR1 and NMDAR2 receptor proteins.