ABILITY OF VARIOUS BOMBESIN RECEPTOR AGONISTS AND ANTAGONISTS TO ALTER INTRACELLULAR SIGNALING OF THE HUMAN ORPHAN RECEPTOR BRS-3

Bombesin (Bn) receptor subtype 3 (BRS-3) is an orphan receptor that is a predicted member of the hepta-helical G-protein receptor family and so named because it shares a 50% amino acid homology with receptors f or the mammalian bombesin-like peptides neuromedin B (NMB) and gastrin -releasing peptide. In a recent targeted disruption study, in which BR S-3-deficient mice were generated, the mice developed obesity, diabete s, and hypertension. To date, BRS-3's natural ligand remains unknown, its pharmacology unclear, and cellular basis of action undetermined. F urthermore, there are few tissues or cell lines found that express suf ficient levels of BRS-3 protein for study. To define the intracellular signaling properties of BRS-3, we examined the ability of [D-Phe(6),b eta-Ala(11),Phe(13),Nle(14)]Bn-(6-14), a newly discovered peptide with high affinity for BRS-3, and various Bn receptor agonists and antagon ists to alter cellular function in hBRS-3-transfected BALB 3T3 cells a nd hBRS-3-transfected NCI-H1299 non-small cell lung cancer cells, whic h natively express very low levels of hBRS-3. This ligand stimulated a 4-9-fold increase in [H-3]inositol phosphate formation in both cell l ines under conditions where it caused no stimulation in untransfected cells and also stimulated an increase in [H-3]IP1, [H-3]IP2, and H-3]I P3. The elevation of [H-3]IP was concentration dependent, with an EC50 of 20-35 nM in both cell lines. [D-Phe(6),beta-Ala(11),Phe(13),Nle(14 )]Bn-(6-14) stimulated a 2-3-fold increase in [Ca2+](i), a 3-fold incr ease in tyrosine phosphorylation of p125(FAK) with an EC50 of 0.2-0.7 nm, but failed to either stimulate increases in cyclic AMP or inhibit forskolin-stimulated increases. None of nine naturally occurring Bn pe ptides or three synthetic Bn analogues reported to activate hBRS-3 did so with high affinity. No high affinity Bn receptor antagonists had h igh affinity for the hBRS-3 receptor, although two low affinity antago nists for gastrin-releasing peptide and NMB receptors, [D-Arg(1),n-Trp (7,9),Leu(11)]substance P and [D.Pro(4),D-Trp(7,9,10)]substance P-(4-1 1), inhibited hBRS-3 receptor activation. The NMB receptor-specific an tagonist D-Nal,Cys,Tyr,D-Trp,Lys,Val, Cys,Nal-NH2 inhibited hBRS-3 rec eptor activation in a competitive fashion (K-i = 0.5 mu M) Stimulation of p125(FAK) tyrosine phosphorylation by hBRS-3 activation was not in hibited by the protein kinase C inhibitor, GF109203X, or thapsigargin, alone or in combination. These results show that hBRS-3 receptor acti vation increases phospholipase C activity, which causes generation of inositol phosphates and changes in [Ca2+](i) and is also coupled to ty rosine kinase activation, but is not coupled to adenylate cyclase acti vation or inhibition. hBRS-3 receptor activation results in tyrosine p hosphorylation of p125(FAK) and it is not dependent on activation of e ither limb of the phospholipase C cascade. Although the natural ligand is not a known bombesin-related peptide, the availability of [D-Phe(6 ),beta-Ala(11),Phe(13),Nle(14)]Bn-(6-14), which functions as a high af finity agonist in conjunction with hBRS-3-transfected cell lines and t he recognition of three classes of receptor antagonists including one with affinity of 0. 5 mu M, should provide important tools to assist i n the identification of its natural ligand, the development of more po tent selective receptor antagonists and agonists, and further explorat ion of the signaling properties of the hBRS-3 receptor.