Identification of an extracellular motif involved in the binding of guanine nucleotides by a glutamate receptor

Yoav Paas, Anne Devillers-Thiéry, Jean Pierre Changeux, Francois Medevielle, Vivian I. Teichberg

Research output: Contribution to journalArticlepeer-review

59 Scopus citations

Abstract

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 [α-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.

Original languageEnglish
Pages (from-to)1548-1556
Number of pages9
JournalEMBO Journal
Volume15
Issue number7
DOIs
StatePublished - 1 Apr 1996
Externally publishedYes

Keywords

  • Glutamate receptors
  • Guanine nucleotide binding site
  • Kainate binding protein
  • Photoaffinity labelling

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