Agonist-induced translocation of the kinin B(1) receptor to caveolae-related rafts

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.