CLONING OF THE CDNA FOR A BRAIN GLYCINE-BINDING, GLUTAMATE-BINDING AND THIENYLCYCLOHEXYLPIPERIDINE-BINDING PROTEIN

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), glu tamate (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 identi fied. Northern blot analyses performed on poly(A(+)) RNA from brain re vealed hybridization of the labeled cDNA probes to transcripts of 1.9 kb. E. coli transformed with the pGyBP expressed a protein that was re cognized by the anti-43 kDa Ab's and had recognition sites for Gly, Gl u and TCP. The cloned protein has 2 glycosylation sites, 3 hydrophobic domains, 4 cysteine-rich motifs (C-X(2)-C-X(16-20)C-X(5-11)), and 2 r egions homologous to the NR1 receptor protein. (C) 1995 Academic Press , Inc.