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 usin g green fluorescent protein (GFP) conjugates of the rabbit bradykinin (BK) B-2 receptor (B2R). COS-1 cells transiently transfected with vectors coding for either of two rabbit B2R fluorescent variants, B2R-GFP and B2R-GFP Del taS/T (with previously identified Ser/Thr phosphorylation sites in the C-te rminal 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 cel ls stably expressing B2R-GFP in the presence of cycloheximide was rapidly f ollowed by translocation of the surface receptors into the cells, with esse ntially complete recycling of the surface receptors in 1 to 3 h (confocal m icroscopy, cell fractionation). Adding captopril to inhibit angiotensin I-c onverting enzyme activity increased the half-life of BK in the culture medi um (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 ant i-GFP antibodies). In contrast, B2R-GFP was partially degraded following a short treatment of cells with trypsin. B2R-GFP internalized following agoni st treatment was colocalized with fluorescent transferrin, supporting trans location of the receptor to recycling endosomes. B2R-GFP DeltaS/T failed to translocate into the cells following treatment with BK, but exhibited at b aseline an altered subcellular distribution relative to B2R-GFP. The agonis t BK promotes B2R receptor endocytosis followed by recycling to the cell su rface, but does not promote receptor down-regulation in the heterologous sy stem that we used here. Digestion initiated by extracellular proteases may be involved in pathological B2R down-regulation, as suggested by the simula tion involving trypsin.