Abstract

Neither the native ligand nor the cell biology of the bombesin (Bn)-related orphan receptor subtype 3 (BRS-3) is known. In this study, we used RT-PCR to identify two human lung cancer lines that contain sufficient numbers of native hBRS-3 to allow study: NCI-N417 and NCI-H720. In both cell lines, [dPhe⁶,βAla¹¹,Phe¹³,Nle¹⁴]Bn(6-14) stimulates [³H]inositol phosphate. In NCI-N417 cells, binding of¹²⁵I-[dTyr⁶,βAla¹¹,Phe¹³,Nle¹⁴]Bn(6-14) was saturable and high-affinity. [dPhe⁶,βAla¹¹,Phe¹³,Nle¹⁴]Bn(6-14) stimulated phospholipase D activity and a concentration-dependent release of [³H]inositol phosphate (EC₅₀ = 25 nM) and intracellular calcium (EC₅₀ = 14 nM); the increases in intracellular calcium were primarily from intracellular stores. hBRS-3 activation was not coupled to changes in adenylate cyclase activity, [³H]-thymidine incorporation or cell proliferation. No naturally occurring Bn-related peptides bound or activated the hBRS-3 with high affinity. Four different bombesin receptor antagonists inhibited increases in [³H]inositol phosphate. Using cytosensor microphysiometry, we found that [dPhe⁶,βAla¹¹,Phe¹³, Nle¹⁴]Bn(6-14) caused concentration-dependent acidification. The results show that native hBRS-3 receptors couple to phospholipases C and D but not to adenylate cyclase and that they stimulate mobilization of intracellular calcium and increase metabolism but not growth. The discovery of human cell lines with native, functional BRS-3 receptors, of new leads for a more hBRS-3-specific antagonist and of the validity of microphysiometry as an assay has yielded important tools that can be used for the identification of a native ligand for hBRS-3 and for the characterization of BRS-3-mediated biological responses.