The interaction of
gastrin with the
cholecystokinin 2 (CCK2)/
gastrin receptor has been studied extensively in relation to gastric acid secretion. However, not much is known about the contribution of individual
amino acids of
gastrin interacting with the
CCK2 receptor, when
gastrin is acting as a
tumor growth factor. The purpose of the present study was to determine the significance of each individual
amino acid residue of human
gastrin-17 with respect to
CCK2 receptor-mediated cell proliferation. Activation of this receptor was assessed using an in vitro bioassay based on
gastrin-induced expression of a c-fos-
luciferase reporter, transfected in AR42JB13 and Colo 320 cells, a rat pancreatic and human colorectal cell line respectively.
Gastrin-17 dose dependently increased c-fos induction in both
cancer cell lines. L365,260, a known
CCK2 receptor antagonist, completely blocked the
gastrin signal, demonstrating the specificity of this assay. We demonstrated for the first time that four carboxy-terminal
amino acids of
gastrin-17 are essential for activation of the
CCK2 receptor with respect to c-fos induction. Also other residues of
gastrin-17, notably glycine-2 for the rat
CCK2 receptor and
glutamic acid 8-10 and tyrosine-12 for the human receptor, were found to be important, although to a lesser extent.
Alanine-substitution variants of each of the four carboxy-terminal
amino acids of
gastrin-17 showed strongly reduced receptor activation but did not act as competitive inhibitors of
gastrin-17. Identification of the essential role of the carboxy-terminal tetrapeptide of
gastrin-17 in
CCK2 receptor-mediated c-fos induction indicates that
gastrin inhibitory therapeutic strategies should mainly be targeted toward this region of
gastrin.