Gastrin (G17) and N-carboxymethylgastrin (G17-Gly) have been shown to stimulate the growth of colon cancer cells both in vivo and in vitro. The identity of the receptor mediating these e¡ects is controversial. A recent study demonstrated the presence of a low a⁄nity binding site for G17 and G17-Gly on the DLD-1 human colon cancer cell line.

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The importance of the C-terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK2 receptor were assessed using analogs of [Leu15]G(11-17). The following peptides were synthesized, Ac[Leu15]G(11-17), Ac[Leu15]G(11-16)NH2, [Leu15]G(11-17), [Leu15,Ala17]G(11-17), [Leu15,Abu17]G(11-17), [Leu15,Val17]G(11-17), [Leu15,Leu17]G(11-17), [Leu15,Cha17]G(11-17), [Leu15,Trp17]G(11-17), [Leu15,Tic17]G(11-17), [Leu15, d-Phe17]G(11-17) and [Leu15,p-X-Phe17]G(11-17), where X = F, Cl, Br, I, OH, CH3, NH2 and NO2. Competition binding experiments with [3H]CCK-8 were performed using human CCK2 receptors stably expressed in CHO cells. Phe17 was shown to be important for binding. A hydrophobic side-chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side-chain either in the g+ or g- conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d-Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des-Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK2 receptor.

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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)-NH2 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)-NH2, including nonamidated 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.