Cloning of the cDNA for a brain glycine-, glutamate- and thienylcyclohexylpiperidine-binding protein.

K N Kumar, K K Babcock, P S Johnson, X Chen, M Ahmad, E K Michaelis

Department of Pharmacology and Toxicology, University of Kansas, Lawrence 66045-2505, USA.

Biochemical and biophysical research communications 1995 Nov 2

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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.

Citation

K N Kumar, K K Babcock, P S Johnson, X Chen, M Ahmad, E K Michaelis. Cloning of the cDNA for a brain glycine-, glutamate- and thienylcyclohexylpiperidine-binding protein. Biochemical and biophysical research communications. 1995 Nov 2;216(1):390-8