PHARMACOLOGY AND INTRACELLULAR SIGNALING MECHANISMS OF THE NATIVE HUMAN ORPHAN RECEPTOR BRS-3 IN LUNG-CANCER CELLS

Neither the native ligand nor the cell biology of the bombesin (Bn)-re lated orphan receptor subtype 3 (BRS-3) is known. In this study, we us ed RT-PCR to identify two human lung cancer lines that contain suffici ent numbers of native hBRS-3 to allow study: NCI-N417 and NCI-H720. In both cell lines, [DPhe(6),beta Ala(11),Phe(13),Nle(14)]Bn(6-14) stimu lates [H-3]inositol phosphate. In NCI-N417 cells, binding of I-125-[DT yr(6),beta Ala(11),Phe(13),Nle(14)]Bn(6-14) was saturable and high-aff inity. [DPhe(6),beta Ala(11),Phe(13),Nle(14)]Bn(6-14) stimulated phosp holipase D activity and a concentration-dependent release of [H-3]inos itol phosphate (EC50 = 25 nM) and intracellular calcium (EC50 = 14 nM) ; the increases in intracellular calcium were primarily from intracell ular stores, hBRS-3 activation was not coupled to changes in adenylate cyclase activity, [H-3]-thymidine incorporation or cell proliferation . No naturally occurring Bn-related peptides bound or activated the hB RS-3 with high affinity. Four different bombesin receptor antagonists inhibited increases in [H-3]inositol phosphate. Using cytosensor micro physiometry, we found that [DPhe(6),beta Ala(11),Phe(13), Nle(14)]Bn(6 -14) caused concentration-dependent acidification. The results show th at native hBRS-3 receptors couple to phospholipases C and D but not to adenylate cyclase and that they stimulate mobilization of intracellul ar calcium and increase metabolism but not growth. The discovery of hu man cell lines with native, functional BRS-3 receptors, of new leads f or a more hBRS-3-specific antagonist and of the validity of microphysi ometry as an assay has yielded important tools that can be used for th e identification of a native ligand for hBRS-3 and for the characteriz ation of BRS-3-mediated biological responses.