Importance of the C‐terminal phenylalanine of gastrin for binding to the human CCK₂receptor

Abstract

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.

The Production and Role of Gastrin-17 and Gastrin-17-Gly in Gastrointestinal Cancers

“…Substitution of Asp 16 with Ala completely destroyed the peptide’s ability to bind and activate CCK 2 -R, confirming Morley’s earlier work [224–225]. Finally, substitution of Phe 17 with Ala causes a 10 4 -10 5 -fold decrease in affinity [169, 224]. …”

“…Sulfated and non-sulfated gastrin and CCK-8 bind with nearly equal (sub-nanomolar) affinity to CCK 2 -R, and CCK-4 binds with micromolar affinity [167, reviewed in 122]. Despite the importance of sulfation to gastrin processing, it has seemingly no role in the biological activity mediated by CCK 2 -R, in contrast to CCK 1 -R. C-terminal amidation is, however, essential to high binding affinity, as the processing intermediate G17-Gly has less affinity for CCK 2 -R than CCK-4 [168–169]. …”

Bioactivity of analogs of the N-terminal region of gastrin-17

“…The growth-promoting effects of G17-Gly have been shown to be mediated by a putative, non-CCK receptor by several groups, through a mechanism not involving the C-terminal tetrapeptide sequence Trp-Met-Asp-Phe-NH 2 of G17, which is essential for binding and activation of the CCK 2 receptor [3, 7,23–24,26, 37, 39–40]. Nanomolar and micromolar affinity receptors for G17-Gly on primary tissues and on cultured cell lines have been detected [17, 19, 25, 33, 36, 38, 41].…”

Two phenylalanines in the C-terminus of Epstein–Barr virus Rta protein reciprocally modulate its DNA binding and transactivation function

“…Next, we examined the role of phenylalanine or histidine residues in the inhibitory region. These amino acids play important roles in certain protein–protein interactions (Ahmed et al, 2001; Clark et al, 1996; Dowd et al, 2002; Ehlers et al, 1996; Lin et al, 2006). The mutant F600A/F605A was markedly enhanced in DNA binding (Fig.…”

“…For example, F43 in CD4 makes a significant contribution to the high affinity interaction between CD4 and HIV env (Dowd et al, 2002). F17 of gastrin was shown to be important for binding to the human CCK2 receptor (Ahmed et al, 2001). The aromatic ring of F30 also plays a major role in binding of the homopentameric B subunit of verotoxin (VT1) to the glycosphingolipid receptor globotriaosylceramide (Gb3) (Clark et al, 1996).…”