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 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. 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 μM for VV-H-7 and 183±60 μM for LVV-H-7 in CHO cells, and 19±6 μM for VV-H-7 and 38±18 μM 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 μM (GRP) and of 1–2 μM (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 (IP3)-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 (p125FAK) was not observed. Keywords: Orphan receptor, hBRS-3, hemorphin, GPCR, signal transduction Introduction The human bombesin receptor subtype 3 (hBRS-3) exhibits about 50% amino-acid sequence homology with human neuromedin B (hNMB-R) and gastrin-releasing peptide (hGRP-R) receptors, and together they form the bombesin-like receptor group (Gorbulev et al., 1992). Gastrin-releasing peptide (GRP) and neuromedin B (NMB) are high-affinity ligands for hGRP-R and hNMB-R, respectively, while both peptides bind only with low affinity to hBRS-3. Recently, a synthetic peptide [DPhe6,β-Ala11,Phe13,Nle14]-bombesin (6-14) (B9P) was described to have high affinity for hBRS-3 (Mantey et al., 1997), but so far a natural high-affinity ligand for hBRS-3 has not been identified. The expression of hBRS-3 is limited to a few brain regions (Ohki-Hamazaki et al., 1997a), placenta (Whitley et al., 1996), pancreatic islets (Fleischmann et al., 2000), secondary spermatocytes and certain tumor cell lines (Fathi et al., 1993; Gorbulev et al., 1994). Targeted disruption of the hBRS-3 gene in mice leads to mild obesity, impaired glucose metabolism, and hypertension (Ohki-Hamazaki et al., 1997b). Therefore, hBRS-3 has been implicated in the regulation of neuroendocrine function and energy metabolism. Several studies described a role of bombesin-like peptides (NMB, GRP) in growth regulation of, for example, small cell lung carcinoma (SCLC) (Cuttitta et al., 1985), human breast cancer (Nelson et al., 1991), and gastrointestinal tissue (Lehy et al., 1983). They are also involved in development of the lung (Johnson et al., 1982) and induce the contraction of smooth muscle (Minamino et al., 1983). Bombesin-like peptides were postulated as mitogens for bronchial epithelial cells and SCLC, and lead to increased fetal lung growth and maturation in utero as well as in organ cultures (Sunday et al., 1998). Furthermore, it has been reported that bombesin-like peptides induce the phosphorylation of focal adhesion kinase (FAK) resulting in proliferation and invasion of cancer cells (Leyton et al., 2001). Since the native specific ligand of hBRS-3 is still unknown, the receptor is classified as orphan receptor and the pharmacology and physiology of the receptor remain speculative. Here, we describe the isolation of two naturally derived ligands VV-H-7 and LVV-H-7 for the hBRS-3 from human placenta tissue. Originally, LVV-H-7 was isolated from pig hypothalami (Chang et al., 1980) characterized as nonclassical opioid peptides (Brantl et al., 1986; Piot et al., 1992) and binding to the AT4 receptor with affinity in the nanomolar range (Moeller et al., 1997; Garreau et al., 1998). In the present study, we characterized the structure–function relation between hemorphins and hBRS-3, and compare the biological activity with GRP and NMB. Additionally, we demonstrate the signal transduction mechanism of the interaction between hemorphin VV-H-7 and the hBRS-3 expressed in NCI-N417 lung cancer cells. We show that endogenous hBRS-3 receptor couples to phospholipase C resulting in changes of intracellular calcium which is initially released from an IP3-sensitive store followed by a capacitive calcium entry from extracellular space. We carried out a physiological characterization of the VV-H-7/hBRS-3 interaction regarding the involvement of kinases in proliferation and adhesion.