Tyrosine modification increases the affinity of gastrin for ferric ions

The peptide hormone gastrin(17), which occurs naturally in both tyrosine sulphated and unsulphated forms, binds two ferric ions with pM affinities. The aim of this study was to investigate the hypothesis that sulphation or phosphorylation of gastrin(17) altered ferric ion binding, and/or affinity for the CCK1 or CCK2 receptor. To investigate the effect of tyrosine modification on ferric ion binding, the changes in absorbance of gastrin(17), gastrin(17)SO(4) and gastrin(17)PO(4) on addition of Fe(3+) ions were monitored. Binding of gastrin(17), gastrin(17)SO(4) and gastrin(17)PO(4) to the human CCK1 and CCK2 receptors was assessed by competition with [(125)I]-Bolton and Hunter-labelled cholecystokinin(8) in transiently transfected COS cells. Tyrosine sulphation or phosphorylation increased the affinity of gastrin(17) for the first ferric ion bound from 267 to 83 pM and 14 pM, respectively, but had no effect on the stoichiometry of ferric ion binding. In contrast the affinity of gastrin(17) for the second ferric ion bound was reduced from 94 pM to 7.32 µM and 671 nM, respectively. While sulphation of gastrin(17) increased its affinity for the CCK2 receptor approximately 50 fold, phosphorylation had no effect on receptor binding. These results demonstrate that tyrosine modification may have profound effects on the interaction of gastrins with ferric ions and with the CCK2 receptor.