Identification of an extracellular motif involved in the binding of guanine nucleotides by a glutamate receptor.
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.
Paas
Y
Y
Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel.
Devillers-Thiéry
A
A
Changeux
J P
JP
Medevielle
F
F
Teichberg
V I
VI
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002531:Cerebellum / Q000378:metabolism; D002645:Chickens; D005110:Extracellular Space / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010777:Photochemistry; D017470:Receptors, Glutamate / Q000235:genetics / Q000378:metabolism*; D018092:Kainic Acid Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D000345:Affinity Labels; D006150:Guanine Nucleotides; D006160:Guanosine Triphosphate; D010446:Peptide Fragments; D017470:Receptors, Glutamate; D018092:Kainic Acid Receptors