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

Kari Keinänen, Annukka Jouppila, Arja Kuusinen

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34 Citations (Scopus)


Recombinant fragments of the kainate-selective glutamate recep-tor 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 ~ 400 amino-acid-residue segment and the C-terminal ~ 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 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-binding capacity to the chimaeric ligand-binding domain.
Original languageEnglish
Pages (from-to)1461-1467
JournalBiochemical Journal
Issue number3
Publication statusPublished - 1998
MoE publication typeA1 Journal article-refereed


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