Identification and functional characterization of hemorphins VV-H-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 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. 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 45+/-15 microM for VV-H-7 and 183+/-60 microM for LVV-H-7 in CHO cells, and 19+/-6 microM for VV-H-7 and 38+/-18 microM 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 microM (GRP) and of 1-2 microM (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(FAK)) was not observed.
IPF PharmaCeuticals GmbH, Feodor-Lynen-Strasse 31, 30625 Hannover, Germany.
Hans-Peter
HP
Lammerich
Annette
A
Busmann
Christian
C
Kutzleb
Martin
M
Wendland
Petra
P
Seiler
Claudia
C
Berger
Peter
P
Eickelmann
Markus
M
Meyer
Wolf-Georg
WG
Forssmann
Erik
E
Maronde
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D006454:Hemoglobins / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D018004:Receptors, Bombesin / Q000819:agonists* / Q000096:biosynthesis / Q000235:genetics
D006454:Hemoglobins; D010446:Peptide Fragments; D018004:Receptors, Bombesin; C107120:bombesin receptor subtype 3; C074309:LVV-hemorphin-7; C094394:VV-hemorphin-7