Abstract
Amidated and nonamidated gastrins elicit different biological effects via distinct receptors in different tissues. Amidated gastrin 17 stimulates gastric acid secretion and the development of gastric carcinoids, whereas glycine-extended gastrin 17 stimulates proliferation of the colonic mucosa and the development of colorectal cancers. Because glycine-extended gastrin 17 binds two ferric ions with high affinity (Baldwin, G. S., Curtain, C. C., and Sawyer, W. H. (2001) Biochemistry 40, 10741-10746), we have investigated the identity of the iron ligands and the role of ferric ions in biological activity. Here we report the solution structure of glycine-extended gastrin 17, determined by NMR spectroscopy. The spectral changes observed upon the addition of ferric ions revealed that Glu(7) acted as a ligand at the first ferric binding site, and that Glu(8) and Glu(9) acted as ligands at the second ferric ion binding site. Fluorescence quenching experiments confirmed that a GglyE7A mutant bound only one ferric ion. The inability of this mutant to stimulate proliferation or migration in the IMGE-5 cell line and the observation that the iron chelator desferrioxamine selectively blocked the effects of glycine-extended gastrin 17 indicated that binding of a ferric ion to Glu(7) was essential for biological activity. This is the first report of an essential role for a metal ion in the action of a hormone.
Highlights
Metal ions are critically important in many biological processes [1]
In aqueous solution high affinity binding of divalent metal ions to gastrin was not detected by NMR spectroscopy [7]
Structure of Ggly—The structure of Ggly in aqueous solution was determined by 1H NMR spectroscopy
Summary
Metal ions are critically important in many biological processes [1]. For example, the redox properties of iron are utilized in mitochondrial oxidation, calcium is a necessary stabilizing element for numerous proteins, and zinc plays an essential catalytic role in many enzyme reactions. Binding of zinc is important in storage of insulin in pancreatic granules [2], but the possible involvement of metal ions in the biological activity of other hormones is a neglected area. In aqueous solution high affinity binding of divalent metal ions to gastrin was not detected by NMR spectroscopy [7]. We recently demonstrated by fluorescence quenching that gastrin binds two trivalent ferric ions with high affinity in aqueous solution [8]. Amidated gastrin (Gamide) was first characterized as a stimulant of gastric acid secretion [9], it is involved in several other biological processes. The aims of this work were to identify the amino acids in the Ggly sequence important for ferric ion binding and to elucidate the role of this interaction in biological activity. The complete lack of activity in both assays of a Ggly mutant in which Glu was replaced by Ala (GglyE7A) and the inhibition of Ggly activity by the iron chelator desferrioxamine (DFO) are consistent with the hypothesis that ferric ion binding is essential for biological activity
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