Study of Receptor Binding and Growth Effect of Gastrin Gene Products
Loading...
Authors
Wibowo, Felicitas I.
Issue Date
2002
Volume
Issue
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
Colorectal cancer is the third leading cancer in US, accounted for 160,000 cases and 57,000 deaths per year. Gastrin (G17) has been postulated as a growth factor for colorectal cancer development. Gastrin, a heptadecapeptide, is a gut hormone produced by the G cells in the gastric antrum and functions primarily in stimulating acid secretion from the parietal cells. Besides its role as an acid secretagogue in the stomach, gastrin has also been reported to act as growth factor for both normal and malignant cells. Various reports have shown the trophic effect of gastrin gene products on carcinoma cells, especially colonic cancer cells. The C-terminal tetrapeptide of gastrin, shared with Cholecystokinin (CCK), is the minimal sequence required for its biological effect through the CCK2 receptor. Structure-functional studies of gastrin and the CCK2 receptor revealed the importance of Phe in residue 17 of gastrin in receptor recognition. Removal of Phe" abolishes the binding of gastrin to the human CCK2 receptor but leads to an antagonist for the rat CCK2 receptor-mediated acid secretagogue effect of gastrin. Hence, interspecies differences bring some concerns on the application of animal models in studying the interaction of peptides with the human receptor. Gastrin heptapeptide, G(ll-17), which has been shown to have a high affinity for the human CCK2 receptor with similar affinity to G17, is used as the basic peptide for the study. To determine what characteristic of gastrin residues are important for the binding to the CCK2 receptor, several gastrin analogs were tested, which include: (i) para-substituted analogs, [Leu15,p-X-Phe17]G(ll-17), where X=C1, CH3, and NH2, (ii) analogs in which Phe" was substituted with hydrophobic amino acids of increasing size, Ala, Abu, Val, Leu and Cha and (iii) analogs in which Phe" was replaced by another aromatic amino acid, Trp.
Using [^H-propionyl]CCK8, the affinity of gastrin analogs for the human CCK2 receptor was analyzed by competitive binding studies with membrane preparations from CHO cells expressing the human CCK2 receptor. All para-substituted analogs bound to the human CCK2 receptor with affinities similar to the basic peptide. Replacement of Phe with Trp and Cha was well tolerated by the human CCK2 receptor, but replacement of Phe with Ala, Abu, Val and Leu abolished the binding of the peptides to the receptor. The results indicate that aromaticity is not a strict requirement and a side-chain of hydrophobic amino acid larger than Leu is required for the interaction with the receptor. Further, the size and electron density of aromatic ring of Phe do not significantly affect the binding property of gastrin to the human CCK2 receptor.
N-carboxymethyl gastrin, gastrin with C-terminal glycine, is the post tranlational precursor of gastrin. TV-carboxymethyl gastrin 18 (G17Gly) is produced and secreted into the circulation along with gastrin from antral G cells and found in roughly equal concentration to gastrin in the plasma. G17Gly potentiates gastrin-stimulated acid secretion in the rat stomach. G17Gly is the predominant gastrin gene product in human colonic cancers and has been shown to have mitogenic effect on these cancers. However, the role of G17Gly on the growth of human colonic tumors is still disputed since the presence of CCK.2 receptor on human colon tumors is still controversial. Therefore, the interaction of G17Gly with cell membranes prepared from human colonic cancer cell lines, DLD-1 and HT-29, by comparison with CHO cells expressing human CCK2 receptor, was investigated by competitive binding studies using [3H]G17Gly, [3H]G(11- 17)Gly and [3^H-propionyl]CCK8. Cell proliferation assays, thymidine incorporation and SRB assays, were then performed to determine whether or not G17Gly stimulates cell growth through a receptor. CCK8, G17, G17Gly and G(ll-17)Gly bound to human CCK2 receptor; the K, value of G(1 l-17)Gly was one order of magnitude higher than of G17Gly. G17Gly and G(ll-17)Gly bound specifically to the HT-29 and DLD-1 cell membranes, albeit with low affinity, but CCK8 and G17 did not. These result indicate the existence of a specific binding site in human colonic cancer cell lines, which recognize G17Gly and its C-terminal partial sequence but which is distinct from the CCKi and CCK2 receptors which would bind CCK8 and G17. However, the results from thymidine incorporation and SRB assays showed that G17Gly and G (ll-17)Gly do not significantly stimulate colonic cancer cell proliferation. Further studies are needed to establish the significance of the specific binding to N-carboxymethyl gastrin to the colonic cancer cell membranes.
Description
Citation
Publisher
Creighton University
License
Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.
Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.
Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.