THERE ARE 3 DISTINCT FORMS OF BOMBESIN - IDENTIFICATION OF [LEU(13)]BOMBESIN, [PHE(13)]BOMBESIN, AND [SER(3),ARG(10),PHE(13)]BOMBESIN IN THE FROG BOMBINA-ORIENTALIS

Amphibian bombesin is the prototypic peptide that defines the bombesin -like peptide family. In this paper we show that in the frog Bombina o rientalis, there are actually 3 distinct forms of bombesin, and each o f these peptides is an agonist with differing affinities for the known bombesin receptors. Oligonucleotides complementary to the 5'- and 3'- untranslated regions of the bombesin mRNA were used to amplify bombesi n-related cDNAs from the skin, brain, and gut of B. orientalis. Three classes of cDNAs were found. One class encoded the previously characte rized form of bombesin which has a Leu at position 13 ([Leu(13)]bombes in). The other two classes, respectively, encoded new bombesin-like pe ptides which we have designated as [Phe(13)]bombesin and [Ser(3),Arg(1 0),Phe(13)]bombesin ([SAP]bombesin). The existence of [SAP]bombesin in skin was confirmed by tandem mass spectrometry. Polymerase chain reac tion analysis of genomic DNA showed the mRNAs for [Leu(13)]bombesin, [ Phe(13)]bombesin, and [SAP]bombesin most likely arise from separate ge nes. Polymerase chain reaction analysis showed different patterns of t issue-specific expression for each form. [Leu(13)]Bombesin and [SAP]bo mbesin were predominantly expressed in skin, brain, and gut; [Phe(13)] bombesin was expressed only in brain, and [Leu(13)]bombesin predominat ed in oocytes, [SAP]Bombesin contained a cleavage site between residue s 4 and 5, which if used would yield the peptide [SAP]bombesin(5-14) w hich has the sequence [Gln(3),Arg(6)]neuromedin B. Thus a frog homolog of NMB could derive from the [SAP]bombesin prohormone. [Phe(13)]Bombe sin, [SAP]bombesin, and [SAP]bombesin(5-14) were synthesized and their affinities for the mammalian bombesin-like peptide (GRP and NMB) rece ptors determined. These peptides acted as agonists for the GRP and NMB receptors, with relative potencies for the GRP receptor of [Leu(13)]b ombesin > [Phe(13)]bombesin > [SAP]bombesin(5-14) > [SAP]bombesin and for the NMB receptor of [Phe(13)]bombesin > [SAP]bombesin(5-14) > [Leu (13)]bombesin > [SAP]bombesin. None of these peptides demonstrated hig h affinity binding for the BRS-3 receptor. The different receptor affi nities and tissue distribution of these peptides suggests distinct phy siologic roles and raises the possibility of as yet uncharacterized ma mmalian homologs of these new amphibian peptides.