C-terminal bombesin sequence requirements for binding and effects on protein synthesis in Swiss 3T3 cells

• Published in: Biochemical journal 1987-10, Vol.247 (2), p.427-432
• Main Authors: Gargosky, S E, Wallace, J C, Upton, F M, Ballard, F J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Bombesin - pharmacology; Cell Line; Electrophoresis, Polyacrylamide Gel; Gastrin-Releasing Peptide; Growth Substances - pharmacology; Ligands; Peptides - pharmacology; Protein Binding - drug effects; Protein Biosynthesis; Thymidine - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2470427

1. Synthetic peptides corresponding to the five, seven, nine and eleven C-terminal amino acids of the tetradecapeptide bombesin as well as bombesin itself and gastrin-releasing peptide have been evaluated in Swiss 3T3 cells in order to define the minimal peptide length needed for biological responsiveness. 2. Gastrin-releasing peptide, bombesin, the undecapeptide and nonapeptide had nearly equipotent abilities to compete for binding of labelled gastrin-releasing peptide to the cell receptors and showed half-maximal competition at 5-10 nM. The heptapeptide and pentapeptide were ineffective. 3. Cross-linking experiments demonstrated specific binding of gastrin-releasing peptide to a 100 kDa receptor subunit. 4. Total cell protein synthesis was stimulated equally by the nonapeptide and longer peptides with a half-maximal effect at 0.5 nM, while a more than 1000-fold higher concentration of the heptapeptide was required to produce a similar response. Comparable results were found when insulin was also present. 5. Neither an inhibition of protein breakdown nor a stimulation of DNA labelling could be demonstrated by bombesin or gastrin-releasing peptide. 6. We conclude that a C-terminal peptide ligand comprising more than seven but no more than nine amino acids is required to achieve high-affinity binding and receptor-mediated responses via the bombesin receptor.