Vol. 287, Issue 1, 366-380, October 1998

Pharmacology and Intracellular Signaling Mechanisms of the Native Human Orphan Receptor BRS-3 in Lung Cancer Cells

Richard R. Ryan, H. Christian Weber, Samuel A. Mantey, Wei Hou, Mary E. Hilburger, Tapas K. Pradhan, David H. Coy and Robert T. Jensen

Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (R.R.R., H.C.W., S.A.M., W.H., T.K.P., R.T.J.), National Institutes of Health, Bethesda, Maryland, Laboratory of Tumor Immunology and Biology, National Cancer Institute (M.E.H.), National Institutes of Health, Bethesda, Maryland and Peptide Research Laboratories (D.H.C.), Tulane University, New Orleans, Louisiana

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,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) stimulates [³H]inositol phosphate. In NCI-N417 cells, binding of ¹²⁵I-[DTyr⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn(6-14) was saturable and high-affinity. [DPhe⁶,Ala¹¹,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,Ala¹¹,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.