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 heptahelical G-protein receptor family and so named because it shares a 50% amino acid homology with receptors for the mammalian bombesin-like peptides neuromedin B (NMB) and gastrin-releasing peptide. In a recent targeted disruption study, in which BRS-3-deficient mice were generated, the mice developed obesity, diabetes, and hypertension. To date, BRS-3's natural ligand remains unknown, its pharmacology unclear, and cellular basis of action undetermined. Furthermore, there are few tissues or cell lines found that express sufficient levels of BRS-3 protein for study. To define the intracellular signaling properties of BRS-3, we examined the ability of [d-Phe⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]Bn-(6–14), a newly discovered peptide with high affinity for BRS-3, and various Bn receptor agonists and antagonists to alter cellular function in hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299 non-small cell lung cancer cells, which natively express very low levels of hBRS-3. This ligand stimulated a 4–9-fold increase in [³H]inositol phosphate formation in both cell lines under conditions where it caused no stimulation in untransfected cells and also stimulated an increase in [³H]IP₁, [³H]IP₂, and ³H]IP₃. The elevation of [³H]IP was concentration-dependent, with an EC₅₀ of 20–35 nm in both cell lines. [d-Phe⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]Bn-(6–14) stimulated a 2–3-fold increase in [Ca²⁺]_i, a 3-fold increase 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 peptides or three synthetic Bn analogues reported to activate hBRS-3 did so with high affinity. No high affinity Bn receptor antagonists had high affinity for the hBRS-3 receptor, although two low affinity antagonists for gastrin-releasing peptide and NMB receptors, [d-Arg¹,d-Trp^7,9,Leu¹¹]substance P and [d-Pro⁴,d-Trp^7,9,10]substance P-(4–11), inhibited hBRS-3 receptor activation. The NMB receptor-specific antagonistd-Nal,Cys,Tyr,d-Trp,Lys,Val, Cys,Nal-NH₂ inhibited hBRS-3 receptor activation in a competitive fashion (K _i = 0.5 μm). Stimulation of p125^FAK tyrosine phosphorylation by hBRS-3 activation was not inhibited by the protein kinase C inhibitor, GF109203X, or thapsigargin, alone or in combination. These results show that hBRS-3 receptor activation increases phospholipase C activity, which causes generation of inositol phosphates and changes in [Ca²⁺]_i and is also coupled to tyrosine kinase activation, but is not coupled to adenylate cyclase activation or inhibition. hBRS-3 receptor activation results in tyrosine phosphorylation of p125^FAK, and it is not dependent on activation of either limb of the phospholipase C cascade. Although the natural ligand is not a known bombesin-related peptide, the availability of [d-Phe⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]Bn-(6–14), which functions as a high affinity agonist in conjunction with hBRS-3-transfected cell lines and the recognition of three classes of receptor antagonists including one with affinity of 0.5 μm, should provide important tools to assist in the identification of its natural ligand, the development of more potent selective receptor antagonists and agonists, and further exploration of the signaling properties of the hBRS-3 receptor.