Inhibition of the preferential binding of actin to the N-terminal hydratase domain of the 78-kDa gastrin-binding protein by nonsteroidal anti-inflammatory drugs and gastrin receptor antagonists

The 78 kDa gastrin-binding protein (GBP) is a likely target for the antiproliferative effects of gastrin receptor antagonists and non-steroidal anti-inflammatory drugs (NSAIDs) on colorectal carcinoma cells (Baldwin GS, Murphy VJ, Yang Z, and Hashimoto T. J Pharmacol Exp Ther 1998;286:1110-14). This study tested the hypotheses that the GBP bound actin, and that the interaction could be disrupted by gastrin receptor antagonists and NSAIDs. Binding of actin to the GBP was assessed by competition with I-125-[Nle(15)] gastrin(2.17) in a covalent cross-linking assay, and by comparison of I-125-actin binding to the N- and C-terminal GBP domains, which had been expressed independently in E. coli as glutathione-S-transferase (GST) fusion proteins. The ability of gastrin receptor antagonists and NSAIDs to interfere with the actin-GBP interaction was measured by release of I-125-actin from preformed complexes with the N- and C-terminal domain-GST fusion proteins. Actin purified from skeletal muscle or from gastric mucosal cytosol competed with I-125-[NIe(15)]-gastrin(2.17) for binding to the GBP with Ic,, values of 2.6 +/- 0.7 muM, and 2.1 +/- 0.7 muM, respectively. The amount of I-125-actin from either source bound to the N-terminal GBP domain was 8.2 times greater than the amount bound to the C-terminal domain. Binding of actin to both domains was inhibited by the gastrin receptor antagonists proglumide and benzotript, and by NSAIDs, We conclude that the GBP may associate with the cytoskeleton via an interaction between its N-terminal domain and actin, and that the association may be disrupted either by gastrin receptor antagonists or by NSAIDs. (C) 2001 Elsevier Science Inc. All rights reserved.