CHARACTERIZATION OF THE KAINATE-BINDING DOMAIN OF THE GLUTAMATE-RECEPTOR GLUR-6 SUBUNIT

Recombinant fragments of the kainate-selective glutamate receptor subu nit GluR-6 were expressed in insect cells and analysed for [H-3]kainat e binding activity in order to characterize the structural determinant s responsible for ligand recognition. Deletion of the N-terminal simil ar to 400 amino-acid-residue segment and the C-terminal similar to 90 residues resulted in a membrane-bound core fragment which displayed ph armacologically native-like [H-3]kainate binding properties. Further r eplacement of the membrane-embedded segments M1-M3 by a hydrophilic li nker peptide gave rise to a soluble polypeptide which was accumulated in the culture medium. When bound to chelating Sepharose beads via a C -terminal histidine tag, the soluble fragment showed low-affinity bind ing of [H-3]kainate, which was displaced in a concentration-dependent manner by unlabelled domoic acid, L-glutamate and 6-cyano-7-nitroquino xaline-2,3-dione. Our results indicate that the kainate-binding site i s formed exclusively by the two discontinuous extracellular segments ( S1 and S2) which are homologous to bacterial amino-acid-binding protei ns. Ligand binding characteristics of soluble S1-S2 chimaeras between the GluR-6 and GluR-D subunits showed that, whereas both S1 and S2 seg ments contribute to agonist-selectivity, the N-terminal one-third of t he GluR-D S2 segment is sufficient to confer o-3-hydroxy-5-methyl-4-is oxazolepropionate-binding capacity to the chimaeric ligand-binding dom ain.