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

Abstract

1 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 LVVhemorphin-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 EC 50 values were 45715 mM for VV-H-7 and 183760 mM for LVV-H-7 in CHO cells, and 1976 mM for VV-H-7 and 38718 mM 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 mM (GRP) and of 1 -2 mM (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 (IP 3 )-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 14); CHO-G a16 -hBRS-3, chinese hamster ovary cells transfected with G a16 and hBRS-3; FAK, focal adhesion kinase; FIU, fluorescence intensity units; FLIPR, fluorimetric imaging plate reader; GPCR, G-protein coupled receptor; GRP, gastrin-releasing peptide (neuromedin C); hBRS-3, human bombesin receptor subtype 3; IP 3 , inositol(1,4,5)trisphosphate; IRAP, insulin-regulated aminopeptidase; LVV-H-7, LVV-hemorphin-7; MAPK, mitogen-activated protein kinase; NMB, neuromedin B; PD98059, 2 0 -amino-3 0 -methyoxyflavone (mitogen activated protein kinase kinase (MEK-1) inhibitor); SCLC, human small cell lung carcinoma; VV-H-7, VV-hemorphin-7

Comparative in vitro degradation of the human hemorphin LVV‐H7 in mammalian plasma analysed by capillary zone electrophoresis and mass spectrometry

“…mass) of the m-opioid receptor-binding family and is enzymatically released from the hemoglobin-b, -g, -d or -e chain by cathepsin D [1,2], chymotrypsin-like enzyme or pepsin [3]. Naturally occurring LVV-H7 has been detected in adrenal and pheochromocytoma tissue [4], placenta [5], blood [4], hemofiltrate [6] and serum [7] corroborating its systemic presence and the regulatory potential. This cationic decapeptide is involved in different physiological processes [6,8].…”

Hemoglobin Is Expressed by Alveolar Epithelial Cells

“…These so-called "hemorphins" are ligands for opioid and possibly G-protein-coupled receptors and have roles in processes as diverse as blood pressure regulation, neurotransmission, and neutrophil activity (43)(44)(45)(46)(47)(48). The best characterized hemorphin, LVVH-7 (derived from ␤-globin), is an inhibitor of angiotensin-converting enzyme (a protein found at the highest concentration in the mammalian lung), and can help regulate blood pressure through the renin-angiotensin system (45).…”

Characterization of human circulating TIG2 as a ligand for the orphan receptor ChemR23

“…The amplified fragment was subcloned into the expression vector pCl and the sequence was verified by DNA sequencing as described previously [16]. This ChemR23 expression vector was transfected into a CHO‐Gα(16) expressing cell line (Molecular Devices, Sunnyvale, CA, USA) and cell clones were selected and tested for stable expression of ChemR23 as described [17].…”

“…Cells were seeded in 96‐well black well plates (Costar Corp., Cambridge, USA) at 20 000 cells/well and cultured overnight. After 40 min incubation at 37°C in loading medium (HEPES‐buffered HBSS, pH 7.4, containing 2.5 mM probenecid and 2 μM Fluo‐4 AM; Molecular Probes, Leiden, The Netherlands); agonist evoked intracellular Ca 2+ measurements [Ca 2+ ] i were performed on a Fluorometric Imaging Plate Reader (FLIPR) (see [17]) and calculated as peak fluorescence intensity units (FIU) minus basal FIU. These data were either presented as maximal values or transient elevation.…”