title: Importance of N- and C-terminal regions of gastrin-Gly for preferential binding to high and low affinity gastrin-Gly receptors.
abstract: G17-Gly has been shown to stimulate the growth of DLD-1 human colon cancer cells in a biphasic manner via high and low affinity receptors. In the current study, the existence of heterogeneous receptor populations for G17-Gly on the HT-29 human colon cancer cell line was investigated. The effect of either N- or C-terminal peptide truncation on receptor binding and cell growth stimulation was also explored. [Leu15]G17-Gly bound to both high (nM) and low (microM) affinity sites on HT-29 cells. The peptide stimulated cell growth in a dose-dependent and biphasic manner with maximal stimulation at 10(-9) M peptide concentration, suggesting that, as in the case of DLD-1 cells, it is the high affinity receptor which is responsible for the growth-promoting effects. In contrast, G17(1-12) stimulated the growth of HT-29 cells in a sigmoidal fashion with an EC50 of 4.6x10(-9) M. Sequential N-terminal truncation of [Leu15]G17-Gly results in decreased binding to the high affinity G17-Gly receptor on DLD-1 cells. [Leu15]G17(11-17)Gly bound to the low affinity G17-Gly receptor with an affinity similar to that of the full sequence peptide but was unable to displace the radioligand from high affinity sites. G17(1-6)-NH2 was unable to displace [3H]G17-Gly from either site. These results suggest that the important residues for binding to the low affinity receptor are in the C-terminal region of the peptide while those required for interaction with the high affinity receptor lie further towards the N-terminus.
authors.affiliation: Department of Biomedical Science, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE, USA.
authors.forename: Shawn
authors.initials: S
authors.lastname: Ahmed
authors.forename: Richard F
authors.initials: RF
authors.lastname: Murphy
authors.forename: Sándor
authors.initials: S
authors.lastname: Lovas
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism*; D005755:Gastrins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D043887:Receptor, Cholecystokinin B / Q000378:metabolism*
chemical_list: D005755:Gastrins; D010455:Peptides; D043887:Receptor, Cholecystokinin B; C086854:glycine-extended gastrin 17