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Gastrin-17 (G17) processing intermediates bind to non-CCK receptors which mediate growth of the colonic mucosa but also the formation and development of colonic cancers. In previous studies, we removed the C-terminal region of G17 to form G17(1-12) and considerably shorter C-terminally amidated and non-amidated analogs. Peptides as short as G17(1-4) continued to bind to a single site on DLD-1 human colonic carcinoma cells, while only the G17(1-6)-NH(2) and G17(1-12) peptides retained the ability to activate the receptor and stimulate cell proliferation in vitro. In this report, we studied the structure of these analogs, using a combination of ECD and VCD spectroscopy and replica exchange molecular dynamics (REMD) simulations in water, TFE, and membrane-mimicking environments, in order to determine preferred conformations that may have importance in promoting the biological activities. Mostly random meander structures, punctuated by a beta-turn at residues 1-4, were found in most peptides by REMD simulations. G17(1-3)-NH(2), which cannot form a beta-turn, failed to bind the non-CCK receptor, suggesting the importance of this feature for binding. Additionally, the beta-turn appeared more frequently in longer sequences, possibly explaining the higher affinity of the non-CCK receptor for these peptides seen previously. Finally, C-terminally amidated peptides generally showed greater formation of turn structure than their non-amidated counterparts as shown by ECD spectra, suggesting the importance of peptide length in stabilizing turn structure in N-terminal sequences, and perhaps explaining the ability of G17(1-6)-NH(2) to activate the non-CCK receptor where as the non-amidated G17(1-6) and shorter peptides do not.

Gastrin-17-Gly (G17-Gly) has been shown to bind to non-CCK nanomolar and micromolar affinity sites on DLD-1 and HT-29 human colonic carcinoma cells and to stimulate cellular proliferation. However, in previous studies, we showed that C-terminal truncation of the gastrin-17 (G17) to the G17 analog G17(1-12) and then to G17(1-6)-NH(2) did not remove the ability to bind to DLD-1 cells or to activate proliferation. This implies that residues and/or structural motifs required for bioactivity at these receptors rest in the N-terminal region of G17. In this work, radioligand binding studies conducted with further C-terminally truncated analogs revealed that sequences as short as G17(1-4) still bind to a single receptor with micromolar affinity. Additionally, cell proliferation assays showed that G17(1-12) stimulates proliferation of DLD-1 cells, as of HT-29 cells, but the sequences shorter than G17(1-6)-NH(2), including non-amidated G17(1-6), were incapable of stimulating proliferation. These observations indicate that the tetrapeptide pGlu-Gly-Pro-Trp is the minimum N-terminal sequence for binding to the probable growth-promoting site on DLD-1 cells. Since analogs shorter than G17(1-6) are able to bind the receptor, these peptides may be of use for developing selective antagonists.

Our previous studies have shown that stimulation of proliferation of DLD-1 and HT29 human colonic cancer cells by nanomolar gastrin (G17) and carboxymethyl gastrin (G17Gly) and reversal of growth by micromolar G17 and G17Gly involves binding sites which can neither be CCK1 nor CCK2 receptors; the N terminal fragment, G17(1-12), is sufficient to increase the number of HT-29 cells by binding the higher affinity binding site but is without a suppressing effect through the lower affinity site. In this study with DLD-1 cells, competitive binding using 125I-G17(1-12) showed that G17(1-12) binds both high and low affinity sites, as do G17 and G17Gly. G17(1-6)-NH2, even without the central-to-C-terminal portion of G17, was still able to bind a single site and to promote a dose-dependent increase in cell number at nanomolar concentrations. The results indicate the presence of a non-CCK receptor on human colonic cancer cells which could mediate the tumor-promoting activity of the N-terminal-to-central portion of G17Gly which, unlike G17, is produced by such cells.