Zg. Wo et al., Cysteine mutagenesis and homology modeling of the ligand-binding site of akainate-binding protein, J BIOL CHEM, 274(52), 1999, pp. 37210-37218
Glutamate receptors comprise the most abundant group of neurotransmitter re
ceptors in the vertebrate central nervous system. Cysteine mutagenesis in c
ombination with homology modeling has been used to study the determinants o
f kainate binding in a glutamate receptor subtype, a low molecular weight g
oldfish kainate-binding protein, GFKAR beta. A construct of GFKAR beta with
no cysteines in the extracellular domain was produced, and single cysteine
residues were introduced at selected positions. N Ethylmaleimide or deriva
tized methanethiosulfonate reagents (neutral or charged) were used to modif
y the introduced cysteines covalently, and the effect on [H-3]kainate bindi
ng was determined. In addition, cysteine mutants of GFKAR beta transiently
expressed in HEK293 cells were labeled with a membrane-impermeable biotinyl
ating reagent followed by precipitation with streptavidin beads and specifi
c detection of GFKAR beta by Western blot analysis. The results are consist
ent with the proposal that the energy driving kainate binding is contribute
d both from residues within the binding site and from interactions between
two regions (i.e, two lobes) of the protein that are brought into contact u
pon ligand binding in a manner analogous to that seen in bacterial amino ac
id-binding proteins.