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-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.
Keinänen
K
K
VTT Biotechnology and Food Research, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland. kari.keinanen@helsinki.fi
Jouppila
A
A
Kuusinen
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011869:Radioligand Assay; D051381:Rats; D017470:Receptors, Glutamate / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D010446:Peptide Fragments; D017470:Receptors, Glutamate; D011994:Recombinant Proteins; C109433:glutamate receptor type D