An orphan receptor discovered in 1993 was called bombesin receptor subtype 3 (BRS-3) because of 47-51% amino acid identity with bombesin (Bn) receptors. Its pharmacology is unknown, because no naturally occurring tissues have sufficient receptors to allow studies. ]Bn-(6 -14) bound to both cell lines with high affinity. Neither Bn nor 14 other naturally occurring Bn peptides bound to hBRS-3 with a K d <1000 nM. Twenty-six synthetic peptides that are high affinity agonists or antagonists at other bombesin receptors had an affinity >1000 nM. Guanosine 5-(␤,␥-imido)triphosphate inhibited binding to both cells due to a change in receptor affinity. These results demonstrate hBRS-3 has a unique pharmacology. It does not interact with high affinity with any known natural agonist or high affinity antagonist of the Bn receptor family, suggesting the natural ligand is either an undiscovered member of the Bn peptide family or an unrelated peptide. The availability of these cell lines and the hBRS-3 ligand should facilitate identification of the natural ligand for BRS-3, its pharmacology, and cell biology. We made two cell lines stably expressing the human BRS-3 (hBRS-3). hBRS-3 was overexpressed in the huRecently, an orphan receptor that is a member of the heptahelical superfamily of receptors was described in both human small cell lung cancer cells (1) and guinea pig uterus (2). Because this orphan receptor had a high degree of homology to mammalian bombesin receptors (i.e. 51-52% for the gastrinreleasing peptide receptor (GRP-R) 1 and 47% for the neuromedin B receptor (NMB-R) (1, 2)), it was named the BRS-3 for bombesin receptor subtype-3 in one study (1). Studies of the distribution of the receptor mRNA show that BRS-3 has a pattern of expression limited to rat secondary spermatocytes (1), guinea pig brain and pregnant uterus (2), and some tumor cell lines (various human small cell and non-small cell lung cancer cell lines (1), the human ductal breast cancer cell line T47D (3), and the human epidermal cancer cell line A431 (3)). However, the natural ligand that interacts with the BRS-3 is unknown, and its pharmacology is largely unknown because of the lack of a radioligand. In addition, little is known about the cellular basis of action of BRS-3 except that it is coupled to phospholipase C when expressed in Xenopus oocytes (1) or when transfected into Balb 3T3 cells (4). The ability to elucidate the pharmacology of the BRS-3 is not only limited by the lack of a radioligand but also by the lack of a cell containing native BRS-3 receptors in sufficient numbers to allow binding studies to identify a possible radioligand.To deal with this latter issue, in the present study we have used two different strategies to produce cell lines stably expressing the human BRS-3 (hBRS-3) receptor whose pharmacology and coupling will probably closely resemble that of the native hBRS-3. Furthermore, we have discovered a unique ligand that is a synthetic analogue of bombesin-(6 -14), which interacts with high affinity with the hBRS-3. With ...

The orphan receptor, bombesin (Bn) receptor subtype 3 (BRS-3), shares high homology with bombesin receptors (neuromedin B receptor (NMB-R) and gastrin-releasing peptide receptor (GRP-R)). This receptor is widely distributed in the central nervous system and gastrointestinal tract; target disruption leads to obesity, diabetes, and hypertension, however, its role in physiological and pathological processes remain unknown due to lack of selective ligands or identification of its natural ligand. We have recently discovered (Mantey, S. A., Weber, H. C., Sainz, E., Akeson, M., Ryan, R. R. Pradhan, T. K., Searles, R. P., Spindel, E. R., Battey, J. The 399-amino acid orphan receptor, bombesin receptor subtype 3 (BRS-3), 1 shares 51 and 47% amino acid sequence homology with the mammalian bombesin (Bn) receptors (gastrinreleasing peptide receptor (GRP-R) and the neuromedin B receptor (NMB-R), respectively) (1, 2). Studies of the distribution of this orphan receptor show that the BRS-3 receptor is present in the central nervous system and peripheral tissues although the distribution is more limited than the GRP-R and NMB-R (3-6). The BRS-3 receptor has been found on such diverse structures as secondary spermatocytes, pregnant uterus, a number of brain regions, and some human lung, breast, and epidermal cancer cell lines (1, 2)The role of BRS-3 in physiological or pathological processes is unknown even though BRS-3-deficient mice, produced by targeted disruption, develop obesity, diabetes, and hypertension (7). These results (7) suggest that the BRS-3 receptor may be required for the regulation of glucose metabolism, energy balance, and maintenance of blood pressure. This proposition is yet to be confirmed because the natural ligand of the BRS-3 receptor is still unknown. Results from previous studies (8 -10) have demonstrated that the hBRS-3 receptor has a unique pharmacology compared with that of any of the closely related Bn receptor family.

The mammalian bombesin (Bn)-receptor family[gastrin-releasing peptide-receptor(GRPR-receptor), neuromedin B-receptor(NMB-receptor)], their natural ligands,GRP/NMB, as well as the related orphan-receptor,BRS-3, are widely-distributed, and frequently overexpressed by tumors. There is increased interest in agonists for this receptor family to explore their roles in physiological/pathophysiological processes, and for receptor-imaging/cytotoxicity in tumors. However, there is minimal data on human pharmacology of Bn-receptor agonists and most results are based on nonhuman receptor studies, particular rodent-receptors, which with other receptors frequently differ from human-receptors. To address this issue we compared hNMB/GRP-receptor affinities and potencies/efficacies of cell-activation(assessing phospholipase C activity) for 24 putative Bn-agonists(12-natural,12-synthetic) in four different cells with these receptors, containing native-receptors or receptors expressed at physiological densities, and compared the results to native rat-GRP-receptor-containing cells-(AR42J–cells) or rat-NMB-receptor cells(C6-glioblastoma cells). There were close correlations(r=0.92–99,p<0.0001) between their affinities/potencies for the two hGRP- or hNMB-receptor cells. Twelve analogues had high affinities(≤ 1 nM) for hGRP-receptor with 15 selective for it(greatest=GRP,NMC), 8 had high affinity/potencies for hNMB-receptors and 4 were selective for it. Only synthetic Bn-analogues containing β−alanine11 had high affinity for hBRS-3, but t also had high affinities/potencies for all GRP-/hNMB-receptor cells. There was no correlation between affinities for human-GRP-receptors and rat-GRP-receptors(r=0.131,p=0.54), but hNMB-receptor results correlated with rat-NMB-receptor(r=0.71, p<0.0001). These results elucidate the human- and rat-GRP-receptor pharmacophore for agonists differ markedly,whereas they do not for NMB-receptors, therefore potential GRP-receptor agonists for human studies(such as Bn-receptor-imaging/cytotoxicity) must be assessed on human-Bn-receptors. The current study provides affinities/potencies on a large number of potential agonists that might be useful for human studies.