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

Recently, a fourth member of the bombesin (Bn) receptor family (fBB(4)-R) w as isolated from a cDNA library from the brain of the frog, Bombina orienta lis. Its pharmacology and cell biology are largely unknown, and no known na tural cell lines or tissues possess sufficient numbers of fBB(4)-R's to all ow either of these to be determined. To address these issues, we have used three different strategies. fBB(4)-R expression in cells widely used for ot her Bn receptor subtypes was unsuccessful as was expression in two frog cel l lines. However, stable fBB(4)-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. [DPhe (6),beta Ala(11),Phe(13),Nle1(4)]Bn(6-14) was found to have high affinity ( K-i = 0.4 nM) for the fBB(4)-R receptor and I-125-[DTyr(6),beta ala(11),Phe (13),Nle(14)]Bn(6-14) to be an excellent ligand for this receptor. The fBB( 4)-R had a unique pharmacology for naturally occurring Bn-related agonists, with the presence of a penultimate phenylalanine being critical for high-a ffinity interaction. It also had a unique profile for six classes of Bn ant agonists. The fBB(4)-R was coupled to phospholipase C with activation incre asing [H-3]inositol phosphates and mobilizing Ca2+ almost entirely from cel lular sources. There was a close correlation between agonist the receptor o ccupation and the receptor activation. Three of the five classes of Bn rece ptor antagonists that interacted with higher affinity with the fBB(4)-R fun ctioned as fBB(4)-R antagonists and two as partial agonists, fBB(4)-R activ ation stimulated increases in phospholipase D (PLD) over the same range of concentrations at which it activated phospholipase C. These results demonst rate that the fBB(4) receptor has a unique pharmacology for agonists and an tagonists and is coupled to phospholipase C and D. The availability of thes e cell lines, this novel ligand, and the identification of three classes of antagonists that can be used as lead compounds should facilitate the furth er investigation of the pharmacology and cell biology of the BB4 receptor.