Pharmacology and cell biology of the bombesin receptor subtype 4 (BB₄- R)

Recently, a fourth member of the bombesin (Bn) receptor family (fBB₄- R) was isolated from a cDNA library from the brain of the frog, Bombina orientalis. Its pharmacology and cell biology are largely unknown, and no known natural cell lines or tissues possess sufficient numbers of fBB₄-R's to allow either of these to be determined. To address these issues, we have used three different strategies. fBB₄-R expression in cells widely used for other Bn receptor subtypes was unsuccessful as was expression in two frog cell lines. However, stable fBB₄-R cell lines were obtained in CHO-K1 cells which were shown to faithfully demonstrate the correct pharmacology of the related Bn receptor, the GRP receptor, when expressed in these cells. [DPhe⁶,βAla¹¹,Phe¹³,Nle¹⁴]Bn(6-14) was found to have high affinity (K(i) = 0.4 nM) for the fBB4 receptor and ¹²⁵I- [DTyr⁶,βala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) to be an excellent ligand for this receptor. The fBB₄-R had a unique pharmacology for naturally occurring Bn- related agonists, with the presence of a penultimate phenylalanine being critical for high-affinity interaction. It also had a unique profile for six classes of Bn antagonists. The fBB₄-R was coupled to phospholipase C with activation increasing [³H]inositol phosphates and mobilizing Ca²⁺ almost entirely from cellular sources. There was a close correlation between agonist the receptor occupation and the receptor activation. Three of the five classes of Bn receptor antagonists that interacted with higher affinity with the fBB₄-R functioned as fBB₄-R antagonists and two as partial agonists, fBB₄-R activation stimulated increases in phospholipase D (PLD) over the same range of concentrations at which it activated phospholipase C. These results demonstrate that the fBB₄ receptor has a unique pharmacology for agonists and antagonists and is coupled to phospholipase C and D. The availability of these cell lines, this novel ligand, and the identification of three classes of antagonists that can be used as lead compounds should facilitate the further investigation of the pharmacology and cell biology of the BB₄ receptor.