Identification of a 70-kDa gastrin-binding protein on DLD-1 human colorectal carcinoma cells

Gastrin(17)gly acts as a growth factor for the colonic mucosa. Studies of t he receptor involved have generally been restricted to its binding properti es, and no investigation of the structure of gastrin(17)gly receptors on hu man colorectal carcinoma cell lines has yet been reported. The aim of this study was to optimise the conditions for binding of gastrin(17)gly to the h uman colorectal carcinoma cell line DLD-1, and to investigate the structure of the receptor responsible. Binding of I-125[Me-15]gastrin(17)gly to DLD- 1 cells was measured in competition experiments with increasing concentrati ons of either gastrin(17)gly or gastrin(17), or with single concentrations of gastrin receptor antagonists. The molecular weights of the gastrin(17)gl y binding proteins were determined by gel electrophoresis and autoradiograp hy after covalent cross-linking of I-125[Nle(15)]gastrin(2,17)gly to cells or membranes with disuccinimidyl suberate. The IC50 value for binding of ga strin(17)gly to DLD-1 cells was 2.1 +/- 0.4 muM. Binding was inhibited by t he non-selective gastrin/cholecystokinin receptor antagonists proglumide an d benzotript, but not by the cholecystokinin-A receptor antagonist L364,718 , or the gastrin/cholecystokinin-B receptor antagonist L365,260. The molecu lar weight of the major gastrin binding protein on DLD-1 cells or membranes was 70,000. We conclude that the major gastrin(17)gly binding site on the human colorectal carcinoma cell line DLD-1 is clearly distinct from the cho lecystokinin-A and gastrin/cholecystokinin-B receptors, but is similar in s ome respects to the gastrin/cholecystokinin-C receptor. (C) 2001 Elsevier S cience Ltd. All rights reserved.