Mutation of lysine residues of the 78-kDa gastrin-binding protein reduces gastrin binding

The 78-kDa gastrin-binding protein (GBP) is a likely target for the antipro liferative effects of gastrin/cholecystokinin receptor antagonists on color ectal carcinoma cell lines. Both the N- and C-terminal halves of the GBP bi nd gastrin, but the affinity of the N-terminal half for gastrin is 7.2-fold higher than the affinity of the C-terminal half. In order to define the ga strin-binding sites of the GBP in greater detail, we have constructed a tru ncation mutant lacking residues 221-318 of the N-terminal domain and a seri es of point mutants in which the lysine residues in the first 220 residues of the N-terminal domain were mutated to arginine residues. The effect of t hese mutations on both the extent of covalent cross-linking of iodinated ga strin(2,17) and on the affinity for gastrin(17) was investigated. Deletion of residues 221-318 of the GBP decreased the affinity 5.5-fold and reduced, but did not abolish, the extent of covalent cross-linking. Mutation of the 17 lysines in residues 1-220 of the GBP decreased the affinity for gastrin between 1.7- and 3.5-fold and in some cases reduced, but did not abolish, the extent of covalent cross-linking. We conclude that one or more lysine r esidues are involved in binding of gastrin to the GBP, but that no single l ysine residue is the preferred target for covalent cross-linking of iodinat ed gastrin(2.17) to the GBP.