TY - JOUR T1 - Bradykinin B<sub>2</sub> Receptor Endocytosis, Recycling, and Down-Regulation Assessed Using Green Fluorescent Protein Conjugates JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 19 LP - 26 VL - 297 IS - 1 AU - Dimcho R. Bachvarov AU - Steeve Houle AU - Magdalena Bachvarova AU - Johanne Bouthillier AU - Albert Adam AU - François Marceau Y1 - 2001/04/01 UR - http://jpet.aspetjournals.org/content/297/1/19.abstract N2 - Agonist-induced endocytosis and/or down-regulation have been evaluated using green fluorescent protein (GFP) conjugates of the rabbit bradykinin (BK) B2 receptor (B2R). COS-1 cells transiently transfected with vectors coding for either of two rabbit B2R fluorescent variants, B2R-GFP and B2R-GFP ΔS/T (with previously identified Ser/Thr phosphorylation sites in the C-terminal tail mutated to Ala), exhibited specific and saturable binding (KD in the lower nM range). The acute addition of BK (10–100 nM) to HEK 293 cells stably expressing B2R-GFP in the presence of cycloheximide was rapidly followed by translocation of the surface receptors into the cells, with essentially complete recycling of the surface receptors in 1 to 3 h (confocal microscopy, cell fractionation). Adding captopril to inhibit angiotensin I-converting enzyme activity increased the half-life of BK in the culture medium (enzyme immunoassay) and, accordingly, promoted B2R-GFP internalization for at least 3 h. However, agonist-induced down-regulation was not observed under conditions optimal for endocytosis (microscopy, immunoblot using anti-GFP antibodies). In contrast, B2R-GFP was partially degraded following a short treatment of cells with trypsin. B2R-GFP internalized following agonist treatment was colocalized with fluorescent transferrin, supporting translocation of the receptor to recycling endosomes. B2R-GFP ΔS/T failed to translocate into the cells following treatment with BK, but exhibited at baseline an altered subcellular distribution relative to B2R-GFP. The agonist BK promotes B2R receptor endocytosis followed by recycling to the cell surface, but does not promote receptor down-regulation in the heterologous system that we used here. Digestion initiated by extracellular proteases may be involved in pathological B2R down-regulation, as suggested by the simulation involving trypsin. The American Society for Pharmacology and Experimental Therapeutics ER -