Differential inhibition of receptor activation by two mouse monoclonal antibodies specific for the human leukotriene B4 receptor, BLT1.
The inflammatory mediator leukotriene B(4) (LTB(4)) binds to and activates a G-protein-coupled receptor named BLT(1). We have previously produced two monoclonal antibodies, named 7B1 and 14F11, that bind specifically to this receptor. Using a HeLa cell line expressing human BLT(1), we find that both antibodies inhibit LTB(4)-induced calcium release, and activation of a MAP-kinase-sensitive luciferase reporter system. The normal chemotactic movement of polymorphonuclear cells towards higher LTB(4) concentrations was also strongly inhibited by both antibodies. Neither antibody was found to activate BLT(1), and experiments using cyclic peptide fragments of the BLT(1) n-terminal and extracellular loops showed that these antibodies bind only to complex epitopes in the tertiary, membrane bound, conformation of the receptor protein. In ligand binding experiments, 7B1 was found to be a competitive antagonist, while 14F11 was a noncompetitive antagonist that inhibited receptor activation, but not agonist (LTB(4)) binding. 14F11 will be a useful tool for studying the mechanisms of receptor activation.
Sabirsh
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Molecular Neurobiology, Lund University, BMC A12 Tornavägen 10, 224 81 Lund, Sweden. Alan.Sabrish@mphy.lu.se
Pettersson
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Boketoft
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Kotarsky
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Owman
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005454:Fluorescence Polarization; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D020024:Leukotriene Antagonists; D007975:Leukotriene B4 / Q000096:biosynthesis / Q000276:immunology; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D018102:Receptors, Leukotriene B4 / Q000096:biosynthesis / Q000276:immunology* / Q000502:physiology
D000911:Antibodies, Monoclonal; D020024:Leukotriene Antagonists; D043562:Receptors, G-Protein-Coupled; D018102:Receptors, Leukotriene B4; D007975:Leukotriene B4; D008156:Luciferases; D002118:Calcium