Differential activation of human gastrin-releasing peptide receptor-mediated responses by bombesin analogs

To enable the detailed pharmacological characterization of five bombesin (BN) analogs with respect to the human gastrin-releasing peptide (GRP) receptor, we ectopically expressed the receptor in BALB/3T3 cells. In such cells (termed GR1 cells), GRP stimulated DNA synthesis and Ca2+ mobilization. Two of these analogs, D-Phe6-BN(6-13) methyl ester (Ki = 1.38 +/- 0.07 nM) and 4-pyridyl-CO-His7-D-Ala11-Lys12-COCH2CH2-phenyl- BN(7-13) methyl amide (Ki = 2.17 +/- 0.05 nM), were pure antagonists of GRP-stimulated DNA synthesis in GR1 cells (IC50 = 14 +/- 8.5 nM and 5.1 +/- 2.0 nM, respectively), whereas three analogs, Leu13-psi-Leu14-BN (Ki = 21.6 +/- 2.2 nM), D-Phe6-BN(6-13) ethyl amide (Ki = 5.17 +/- 0.64 nM), and D-Phe6-BN(6-13) propyl amide (Ki = 0.68 +/- 0.01 nM), displayed significant partial agonistic activity. Although three analogs promoted mitogenesis in GR1 cells, none of the analogs stimulated calcium mobilization in GR1 cells. This dichotomy was not limited to transfected cells, because the same result was obtained for D-Phe6-BN(6-13) propyl amide using human fetal lung cells, which naturally express the GRP receptor. We also assessed the effect of BN analogs on calcium mobilization in transfected GR9 cells expressing about 30 times higher levels of the GRP receptor, compared with GR1 cells. D-Phe6-BN(6-13) ethyl amide, D-Phe6-BN(6-13) propyl amide, and Leu13-psi-Leu14-BN were partial agonists of the intracellular Ca2+ mobilization response of GR9 cells. One conclusion consistent with our data is that GRP-stimulated DNA synthesis requires the activation of far fewer receptors than does GRP-stimulated calcium mobilization. Thus, analogs with a small amount of agonist activity can trigger a mitogenic response but not an intracellular Ca2+ mobilization response, unless cells express a high level of receptors. These studies also provide evidence that the promotion of DNA synthesis in quiescent GR1 or human fetal lung cells via the GRP receptor does not require mobilization of intracellular Ca2+.