Bradykinin B(2) receptor endocytosis, recycling, and down-regulation assessed using green fluorescent protein conjugates.
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 DeltaS/T (with previously identified Ser/Thr phosphorylation sites in the C-terminal tail mutated to Ala), exhibited specific and saturable binding (K(D) 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 DeltaS/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 B(2)R 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.
Centre Hospitalier Universitaire de Québec, Centre de recherche, Pavillon l'Hôtel-Dieu de Québec, 11 Côte-du-Palais, Québec, Canada G1R 2J6.
D R
DR
Bachvarov
S
S
Houle
M
M
Bachvarova
J
J
Bouthillier
A
A
Adam
F
F
Marceau
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D001920:Bradykinin / Q000378:metabolism / Q000494:pharmacology*; D019556:COS Cells; D015536:Down-Regulation; D004705:Endocytosis; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism*; D011817:Rabbits; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D000806:Angiotensin-Converting Enzyme Inhibitors; D008164:Luminescent Proteins; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001920:Bradykinin