The cloning and characterization of the gene for the fourth subunit of a glutamate-binding protein complex in rat brain synaptic membranes are described. The cloned rat brain cDNA contained two open reading frames (ORFs) encoding 8.9-(PRO1) and 9.5-kDa (PRO2) proteins. The cDNA sequence matched contiguous genomic DNA sequences in rat chromosome 17. Both ORFs were expressed within the structure of a single brain mRNA and antibodies against unique sequences in PRO1-and PRO2-labeled brain neurons in situ, indicative of bicistronic gene expression. Dicistronic vectors in which ORF1 and ORF2 were substituted by either two different fluorescent proteins or two luciferases indicated concurrent, yet independent translation of the two ORFs. Transfection with noncapped mRNA led to cap-independent translation of only ORF2 through an internal ribosome entry sequence preceding ORF2. In vitro or cell expression of the cloned cDNA led to the formation of multimeric protein complexes containing both PRO1 and PRO2. These complexes had low affinity (؉)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801)-sensitive phencyclidine-binding sites. Overexpression of PRO1 and PRO2 in CHO cells, but not neuroblastoma cells, caused cell death within 24 -48 h. The cytotoxicity was blocked by concurrent treatment with MK-801 or by two tetrahydroisoquinolines that bind to phencyclidine sites in neuronal membranes. Co-expression of two of the other subunits of the protein complex together with PRO1/PRO2 abrogated the cytotoxic effect without altering PRO1/PRO2 protein levels. Thus, this rare mammalian bicistronic gene coded for two tightly interacting brain proteins forming a low affinity phencyclidine-binding entity in a synaptic membrane complex.A complex of four proteins purified from brain synaptic membranes was shown to have recognition sites for L-glutamate, N-methyl-D-aspartate (NMDA), 4 and other ligands characteristic of NMDA receptors in brain, including binding sites for the co-agonist glycine, the modulator spermine, the competitive antagonist (ϩ)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), and the ion channel inhibitors thienylcyclohexylpiperidine (TCP) and (ϩ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) (1, 2). Reconstitution of the purified complex into planar lipid bilayer membranes leads to the formation of channels with four ion conductance levels upon activation by glutamate or NMDA in the presence of glycine (3). These conductances differ from either the predominant NMDA-activated receptor-ion channels of brain neurons or those formed by reconstitution of the NMDA receptor subunits (4), but are similar to those described for ion channels in rat spinal cord motor neurons (5).The genes for three of the proteins in this complex have been cloned and expressed in heterologous cells (6 -10). The gene GRINA for the glutamate-binding protein (GBP) subunit was identified as part of a "learning and memory" module of genes expressed in the entorhinal cortex of the mammalian b...