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

被引:62
作者
Paas, Y
DevillersThiery, A
Changeux, JP
Medevielle, F
Teichberg, VI
机构
[1] WEIZMANN INST SCI,DEPT NEUROBIOL,IL-76100 REHOVOT,ISRAEL
[2] INST PASTEUR,CNRS URA D1284,F-75015 PARIS,FRANCE
关键词
glutamate receptors; guanine nucleotide binding site; kainate binding protein; photoaffinity labelling;
D O I
10.1002/j.1460-2075.1996.tb00499.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 [H-3]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-P-32]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 [H-3]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.
引用
收藏
页码:1548 / 1556
页数:9
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