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)-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,betaAla11,Phe13, Nle14]Bn(6-14) stimulates [3H]inositol phosphate. In NCI-N417 cells, binding of 125I-[DTyr6,betaAla11,Phe13,Nle14]Bn(6-14) was saturable and high-affinity. [DPhe6,betaAla11,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,betaAla11,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.
Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
R R
RR
Ryan
H C
HC
Weber
S A
SA
Mantey
W
W
Hou
M E
ME
Hilburger
T K
TK
Pradhan
D H
DH
Coy
R T
RT
Jensen
D016475:3T3 Cells; D000818:Animals; D001839:Bombesin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002455:Cell Division; D000242:Cyclic AMP / Q000096:biosynthesis; D004247:DNA / Q000096:biosynthesis; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism*; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism; D018004:Receptors, Bombesin / Q000032:analysis / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D010716:Phosphatidylinositols; D018004:Receptors, Bombesin; C107120:bombesin receptor subtype 3; D004247:DNA; D000242:Cyclic AMP; D001839:Bombesin; D002118:Calcium