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Characterization of the kainate-binding domain of the glutamate receptor GluR-6 subunit

Recombinant fragments of the kainate-selective glutamate recepto subunit GluR-6 were expressed in insect cells and analysed for [3H]kainate binding activity in order to characterize the structural determinants responsible for ligand recognition. Deletion of the N-terminal approximately 400 amino-aci...

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Bibliographic Details
Published in:Biochemical journal 1998-03, Vol.330 ( Pt 3) (3), p.1461-1467
Main Authors: Keinänen, K, Jouppila, A, Kuusinen, A
Format: Article
Language:English
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Summary:Recombinant fragments of the kainate-selective glutamate recepto subunit GluR-6 were expressed in insect cells and analysed for [3H]kainate binding activity in order to characterize the structural determinants responsible for ligand recognition. Deletion of the N-terminal approximately 400 amino-acid-residue segment and the C-terminal approximately 90 residues resulted in a membrane-bound core fragment which displayed pharmacologically native-like [3H]kainate binding properties. Further replacement of the membrane-embedded segments M1-M3 by a hydrophilic linker 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 binding of [3H]kainate, which was displaced in a concentration-dependent manner by unlabelled domoic acid, L-glutamate and 6-cyano-7-nitroquinoxaline-2,3-dione. Our results indicate that the kainate-binding site is formed exclusively by the two discontinuous extracellular segments (S1 and S2) which are homologous to bacterial amino-acid-binding proteins. Ligand binding characteristics of soluble S1-S2 chimaeras between the GluR-6 and GluR-D subunits showed that, whereas both S1 and S2 segments contribute to agonist-selectivity, the N-terminal one-third of the GluR-D S2 segment is sufficient to confer alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-binding capacity to the chimaeric ligand-binding domain.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj3301461