Abstract
The contribution of the N-terminal region of gastrin-17 (G17) to the binding and activation of the putative gastrin processing intermediate receptor(s) on human colorectal cancer cells is investigated. The C-terminal sequence Trp-Met-Asp-Phe-NH2 has long been recognized to be essential to the binding and activation of CCK2-R by G17. However, research in our laboratory has indicated that the N-terminal portion of the peptide likely plays a role in the promotion of proliferation of cancer cells in vitro, binding to low and high affinity sites which may jointly regulate cell proliferation. Consequently, C-terminally truncated G17 analogs were synthesized and their ability both to bind and activate the putative receptor, as well as the conformational features important to bioactivity, were examined.|The pentaglutamyl sequence-containing peptide G17(1-12) and G17(1-3)-NH2, G17(1-4)-NH2, G17(1-5)-NH2, G17(1-6)-NH2, and their non-amidated variants were made using solid phase peptide synthesis. Activity studies were conducted using radioligand binding assays in suspension and cell proliferation assays in monolayer culture with DLD-1 human colorectal cancer cells. Binding of each peptide to DLD-1 cells in competition with [125I-Tyr12]G17(1-12) was assessed. A homologous competition assay with unlabeled G17(1-12) revealed that [125I-Tyr12]G17(1-12) bound to DLD-1 cells in a displaceable fashion, and was displaced by G17(1-12) from both low and high affinity sites, demonstrating that the C-terminus of G17 is not required for binding to either putative receptor.|Further competition assays with [125I-Tyr12]G17(1-12) using G17(1-6)-NH2, G17(1-6), G17(1-5)-NH2, G17(1-5), G17(1-4)-NH2, G17(1-4) and G17(1-3)-NH2 revealed that these short peptides were able to bind to DLD-1 cells and displace a portion of the radioligand. All of these analogs, except for G17(1-3)-NH2, bound the cells and displaced radioligand in a manner consistent with single site binding at very low affinities, decreasing as the analogs were truncated further. Some radioligand was not displaced at concentrations of unlabeled ligand as high as 10-4 M with the G17(1-6) and G17(1-5) analogs, and 10-3 M with G17(1-4)-NH2 (but not G17(1-4)) even after nonspecific binding subtraction, indicating a second receptor from which the analogs do not displace radioligand. Thus, these analogs seem to be able to selectively bind one of the two G17-Gly receptor / binding sites, and the sequence G17(1-4) is the minimum required sequence required for binding.|DLD-1 cell proliferation conducted in the presence of G17(1-12), G17(1-6)-NH2, G17(1-6), G17(1-5)-NH2, G17(1-5), G17(1-4)-NH2, and G17(1-4) was analyzed by visually counting the cells under an optical microscope. G17(1-12) stimulated proliferation of cells in a non-biphasic manner, similar to how the peptide stimulated the proliferation of HT-29 cells in a previous study. G17(1-6)-NH2 also stimulated proliferation, while G17(1-6) and the shorter analogs did not. This would seem to indicate that, in agreement with a previous study, the N-terminus of G17 is capable of selectively binding and activating the G17-Gly receptor, and additionally, that the full pentaglutamyl sequence is not required for this activation, though residues Leu5 and Glu6 and C-terminal amidation are required over the minimum binding sequence. The lack of activation of the receptor by G17(1-4) and G17(1-5) analogs and G17(1-6) marks these peptides as receptor antagonists and more importantly, potential targets for the design of peptide mimetics as higher affinity antagonists.|In order to ascertain the structural features present in these peptides possibly required for binding and activation of the receptor, ECD and VCD/IR spectroscopy and MD simulations were performed in various solvents. These solvents were chosen to determine the structural properties of the analogs in aqueous, membrane-mimicking, and destabilizing environments. CDSSTR analysis of ECD spectroscopic results in the far-UV region revealed that the helix/turn structure propensity of G17(1-12) climbs substantially when the peptide is placed in the membrane-mimicking solvent SDS and in TFE as compared to solvation in aqueous solution. The G17(1-6) amidated and nonamidated forms are similarly affected, but the amidated form folds more completely than the nonamidated form in SDS and TFE, and overall helix/turn formation is less than in G17(1-12). The shorter analogs also show this difference between amidated and nonamidated forms, though the helix/turn formation is even less. These results, taken with the binding and activation results, may indicate that the helix and turn structures represent a conformation close to the bioactive conformation necessary for activation of the G17-Gly proliferation promoting receptor.|VCD/IR spectroscopy, a newer but less analyzed technique resolving greater conformational detail, was also used to analyze the peptide analogs. G17(1-12) was not sufficiently soluble at the concentrations necessary for VCD analysis in solvents other than DMSO-d6, but experiments using the pre-pentaglutamyl G17 analogs revealed the predominant presence of predominant random meander structure and some indications of ß-turn structure in deuterated TFE and SDS and 15 mM Na2HPO4 / 100 mM NaCl in D2O. Notably, VCD/IR analysis provided spectra of peptides in deuterated DMSO, a hydrogen bond-breaking solvent, which showed a partial shifting of the major amide I’ IR peak, indicating an increasing number of free carbonyl groups and thus partial unfolding in all analogs, including G17(1-12). MD simulations of the peptide analogs in water, DMSO, and TFE also were conducted to more precisely resolve the structures present. Standard and REMD simulations revealed predominant random coil (meander) in all peptides, and localized ß-turn structure to residues pGlu1-Trp4, and revealed this ß-turn structure from dihedral angles and the alpha carbon distances of pGlu1 and Trp4 to likely be type IV in nature. This structure is likely to play a large role in binding to the receptor.|The results of this study are threefold: 1) the N-terminus (and particularly, residues 1-4) is sufficient to bind and activate the G17-Gly receptor, 2) the minimum sequence peptide capable of selectively binding said receptor and the potential of developing non peptide antagonists from it, and 3) the structural and residual requirements for binding to the receptor. Additionally, this investigation has demonstrated the efficacy of using several non-overlapping techniques to examine secondary structure in peptides.