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 antiproliferative effects of gastrin/cholecystokinin receptor antagonists on colorectal carcinoma cell lines. Both the N- and C-terminal halves of the GBP bind 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 gastrin-binding sites of the GBP in greater detail, we have constructed a truncation mutant lacking residues 221-318 of the N-terminal domain and a series 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 these mutations on both the extent of covalent cross-linking of iodinated gastrin2,17 and on the affinity for gastrin17 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 residues are involved in binding of gastrin to the GBP, but that no single lysine residue is the preferred target for covalent cross-linking of iodinated gastrin2,17 to the GBP.
Rorison
K A
KA
University Department of Surgery, A&RMC, Melbourne, Victoria, Australia.
Lee
D J
DJ
Baldwin
G S
GS
D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003432:Cross-Linking Reagents; D005755:Gastrins / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000235:genetics; D063991:Mitochondrial Trifunctional Protein; D008958:Models, Molecular; D009097:Multienzyme Complexes; D017354:Point Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D005755:Gastrins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D063991:Mitochondrial Trifunctional Protein; D008239:Lysine