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Identification of an extracellular motif involved in the binding of guanine nucleotides by a glutamate receptor.
Author(s) -
Paas Y.,
DevillersThiéry A.,
Changeux J. P.,
Medevielle F.,
Teichberg V. I.
Publication year - 1996
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1996.tb00499.x
Subject(s) - biology , guanine , nucleotide , extracellular , motif (music) , biochemistry , genetics , computational biology , gene , physics , acoustics
The chick cerebellar kainate (KA) binding protein (KBP), a member of the family of ionotropic glutamate receptors, harbours a glycine‐rich (GxGxxG) motif known to be involved in the binding of ATP and GTP to kinases and G proteins respectively. Here, we report that guanine, but not adenine, nucleotides interact with KBP by inhibiting [3H]KA binding in a competitive‐like manner, displaying IC50 values in the micromolar range. To locate the GTP binding site, KBP was photoaffinity labelled with [alpha‐32P]GTP. The reaction was blocked by KA, glutamate, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and antibodies raised against a peptide containing the glycine‐rich motif. Site‐directed mutagenesis of residues K72 and Y73 within the glycine‐rich motif followed by the expression of the KBP mutants at the surface of HEK 293 cells showed a decrease in GTP binding affinity by factors of 10 and 100 respectively. The binding of [3H]KA to the K72A/T KBP mutants was not affected but binding to the Y73I KBP mutant was decreased by a factor of 10. Accordingly, we propose that the glycine‐rich motif of KBP forms part of a guanine nucleotide binding site. We further suggest that the glycine‐rich motif is the binding site at which guanine nucleotides inhibit the glutamate‐mediated responses of various members of the subfamily of glutamate ionotropic receptors.