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

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• The 78-kDa gastrin-binding protein (GBP) is implicated in the antiproliferative effects of gastrin/cholecystokinin receptor antagonists on colorectal carcinoma.
• Both N- and C-terminal domains of GBP bind gastrin, with the N-terminal domain exhibiting a 7.2-fold higher affinity.

Purpose of the Study:

• To precisely define the gastrin-binding sites within the N-terminal domain of GBP.
• To investigate the role of specific lysine residues in gastrin binding and covalent cross-linking.

Main Methods:

• Construction of a truncation mutant of GBP (lacking residues 221-318).
• Generation of point mutants by converting lysine residues (1-220) to arginine.
• Assessment of the impact of these mutations on gastrin17 affinity and covalent cross-linking with iodinated gastrin2,17.

Main Results:

• Deletion of residues 221-318 reduced gastrin affinity by 5.5-fold and decreased, but did not eliminate, covalent cross-linking.
• Mutation of 17 lysine residues in the N-terminal domain decreased gastrin affinity by 1.7- to 3.5-fold.
• Mutations reduced, but did not abolish, covalent cross-linking, indicating multiple lysines are involved.

Conclusions:

• One or more lysine residues within the N-terminal domain of GBP are essential for gastrin binding.
• No single lysine residue serves as the primary site for covalent cross-linking of iodinated gastrin2,17 to GBP.