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 t he family of ionotropic glutamate receptors, harbours a glycine-rich ( GxGxxG) motif known to be involved in the binding of ATP and GTP to ki nases and G proteins respectively, Here, we report that guanine, but n ot adenine, nucleotides interact with KBP by inhibiting [H-3]KA bindin g in a competitive-like manner, displaying IC50 values in the micromol ar range, To locate the GTP binding site, KBP was photoaffinity labell ed with [alpha-P-32]GTP. The reaction was blocked by KA, glutamate, 6- cyano-7-nitroquinoxaline-2,3-dione and antibodies raised against a pep tide containing the glycine-rich motif, Site-directed mutagenesis of r esidues K72 and Y73 within the glycine-rich motif followed by the expr ession of the KBP mutants at the surface of HEK 293 cells showed a dec rease in GTP binding affinity by factors of 10 and 100 respectively, T he binding of [H-3]KA to the K72A/T KBP mutants was not affected but b inding to the Y73I KBP mutant was decreased by a factor of 10, Accordi ngly, we propose that the glycine-rich motif of KBP forms part of a gu anine nucleotide binding site, We further suggest that the glycine-ric h motif is the binding site at which guanine nucleotides inhibit the g lutamate-mediated responses of various members of the subfamily of glu tamate ionotropic receptors.