title: Agonist-induced translocation of the kinin B(1) receptor to caveolae-related rafts.
abstract: The kallikrein-kinin system, activated during inflammatory conditions and the regulation of specific cardiovascular and renal functions, includes two G protein-coupled receptors for bradykinin (BK)-related peptides. The B(1) receptor (B(1)R) subtype is not believed to undergo agonist-induced phosphorylation and endocytosis. A conjugate made of the rabbit B(1)R fused with the yellow variant of green fluorescent protein (YFP) was expressed in mammalian cells. In COS-1 or human embryonic kidney (HEK) 293 cells, the construction exhibited a nanomolar affinity for the agonist radioligand [(3)H]Lys-des-Arg(9)-BK or the antagonist ligand [(3)H]Lys-[Leu(8)]des-Arg(9)-BK and a pharmacological profile virtually identical to that of wild-type B(1)R. Lys-des-Arg(9)-BK stimulation of HEK 293 cells stably expressing B(1)R-YFP but not stimulation of untransfected cells released [(3)H]arachidonate in a phospholipase A(2) assay. B(1)R-YFP was visualized as a continuous labeling of the plasma membranes in stably transfected HEK 293 cells (confocal microscopy). Addition of Lys-des-Arg(9)-BK (1-100 nM) rapidly concentrated the receptor-associated fluorescence into multiple aggregates that remained associated with the plasma membrane (no significant internalization) and colocalized with caveolin-1. This reaction was slowly reversible upon agonist washing at 37 degrees C and prevented pretreatment with a B(1)R antagonist. beta-Cyclodextrin treatment, which extracts cholesterol from membranes and disrupts caveolae-related rafts, prevented agonist-induced redistribution of B(1)R-YFP but not the PLA(2) activation mediated by this receptor. The agonist radioligand copurified with caveolin-1 to a greater extent than the tritiated antagonist in buoyant fractions of HEK 293 cells treated with the ligands. Agonist-induced cellular translocation of the kinin B(1)R to caveolae-related rafts without endocytosis is a novel variation on the theme of G protein-coupled receptor adaptation.
authors.affiliation: Centre Hospitalier Universitaire de Québec, Centre de Recherche du Pavillon l'Hôtel-Dieu de Québec, Québec, Canada.
authors.forename: Thierry
authors.initials: T
authors.lastname: Sabourin
authors.forename: Luc
authors.initials: L
authors.lastname: Bastien
authors.forename: Dimcho R
authors.initials: DR
authors.lastname: Bachvarov
authors.forename: François
authors.initials: F
authors.lastname: Marceau
mesh_terms: D000818:Animals; D016824:Antigens, Human Platelet / Q000032:analysis; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D001920:Bradykinin / Q000378:metabolism; D019556:COS Cells; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002458:Cell Fractionation; D002478:Cells, Cultured; D003505:Cyclodextrins / Q000494:pharmacology; D004705:Endocytosis / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D007609:Kallidin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000378:metabolism; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D011817:Rabbits; D043783:Receptor, Bradykinin B1; D018002:Receptors, Bradykinin / Q000819:agonists / Q000378:metabolism*; D013347:Subcellular Fractions; D014162:Transfection; D014316:Tritium; D047392:beta-Cyclodextrins
chemical_list: D016824:Antigens, Human Platelet; D001426:Bacterial Proteins; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D003505:Cyclodextrins; D008164:Luminescent Proteins; D043783:Receptor, Bradykinin B1; D018002:Receptors, Bradykinin; D047392:beta-Cyclodextrins; C083451:human platelet antigen 1b; C054562:yellow fluorescent protein, Bacteria; D014316:Tritium; D049452:Green Fluorescent Proteins; D007609:Kallidin; C052787:kallidin, des-Arg(10)-; C031215:betadex; D001920:Bradykinin