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

The human orphan G‐protein coupled receptor bombesin receptor subtype 3 (hBRS‐3) was screened for peptide ligands by a Ca 2+ 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. 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 EC 50 values were 45±15 μ M for VV‐H‐7 and 183±60 μ M for LVV‐H‐7 in CHO cells, and 19±6 μ M for VV‐H‐7 and 38±18 μ M for LVV‐H‐7 in NCI‐N417 cells. Other hemorphins had no effect. Gastrin‐releasing peptide (GRP) and neuromedin B (NMB) showed similar EC 50 values of 13–20 μ M (GRP) and of 1–2 μ M (NMB) on both cell lines. 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. 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 (IP 3 )‐sensitive store followed by a capacitive calcium entry from extracellular space. 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.British Journal of Pharmacology (2003) 138 , 1431–1440. doi: 10.1038/sj.bjp.0705177