Abstract
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. We made two cell lines stably expressing the human BRS-3 (hBRS-3). hBRS-3 was overexpressed in the human non-small cell lung cancer cells, NCI-H1299, and the other was made in Balb 3T3 cells, which lack endogenous BRS-3. [D-Phe6,beta-Ala11,Phe13, Nle14]Bn-(6-14) (where Nle represents norleucine) was discovered to have high potency for stimulating inositol phosphate formation in both cell lines. [125I-D-Tyr6,beta-Ala11,Phe13, Nle14]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 Kd <1000 nM. Twenty-six synthetic peptides that are high affinity agonists or antagonists at other bombesin receptors had an affinity >1000 nM. Guanosine 5'-(beta,gamma-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.
Highlights
From the ‡Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1804, §National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland 20850, the ¶Division of Neuroscience, Oregon Primate Research Center, Beaverton, Oregon 97006, and ʈPeptide Research Laboratories, Tulane University, New Orleans, Louisiana 70117
We demonstrate for the first time that the human BRS-3 (hBRS-3) possesses a unique pharmacology for mammalian bombesin receptors, that bombesin receptor subtype 3 (BRS-3) is G protein-coupled, and that none of the existing natural occurring bombesin-related peptides are the natural ligand for this receptor
To determine whether any of the bombesin receptors existed in the Balb 3T3 cells or the human non-small cell lung cancer cell line, NCI-H1299, which we planned to use to make stable cell lines containing BRS-3, we initially used Northern blot analysis (Fig. 1) and did not detect hBRS-3 mRNA in either cell line; nor was the hGRP receptor or hNMB receptor mRNA detected (Fig. 1)
Summary
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) and 47% for the neurome-. 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. We demonstrate for the first time that the hBRS-3 possesses a unique pharmacology for mammalian bombesin receptors, that BRS-3 is G protein-coupled, and that none of the existing natural occurring bombesin-related peptides are the natural ligand for this receptor
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