Identification and functional characterization of hemorphins VVH-7 and LVV-H-7 as low-affinity agonists for the orphan bombesin receptor subtype 3

1 The human orphan G-protein coupled receptor bombesin receptor subtype 3 (hBRS-3) was screened for peptide ligands by a Ca2+ mobilization assay resulting in the purification and identification of two specific ligands, the naturally occurring VV-hemorphin-7 (VV-H-7) and LVV-hemorphin-7 (LVV-H-7), from human placental tissue. These peptides were functionally characterized as full agonists with unique specificity albeit low affinity for hBRS-3 compared to other bombesin receptors. 2 VV-H-7 and LVV-H-7 induced a dose-dependent response in hBRS-3 overexpressing CHO cells, as well as in NCI-N417 cells expressing the hBRS-3 endogenously. The affinity of VV-H-7 was higher in NCI-N417 cells compared to overexpressing CHO cells. In detail, the EC50 values were 45 +/- 15 muM for VV-H-7 and 183 +/- 60 muM for LVV-H-7 in CHO cells, and 19 +/- 6 muM for VV-H-7 and 38 +/- 18 muM for LVV-H-7 in NCI-N417 cells. Other hemorphins had no effect. Gastrin-releasing peptide (GRP) and neuromedin B (NMB) showed similar EC50 values of 13 - 20 muM (GRP) and of 1 - 2 muM (NMB) on both cell lines. 3 Structure-function analysis revealed that both the N-terminal valine and the C-terminal phenylalanine residues of VV-H-7 are critical for the ligand-receptor interaction. 4 Endogenous hBRS-3 in NCI-N417 activated by VV-H-7 couples to phospholipase C resulting in changes of intracellular calcium, which is initially released from an inositol trisphosphate (IP3)-sensitive store followed by a capacitive calcium entry from extracellular space. 5 VV-H-7-induced hBRS-3 activation led to phosphorylation of p42/p44-MAP kinase in NCI-N417 cells, but did not stimulate cell proliferation. In contrast, phosphorylation of focal adhesion kinase (p125(FAK)) was not observed.