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Vol. 297, Issue 1, 19-26, April 2001
Centre Hospitalier Universitaire de Québec, Centre de
recherche du Pavillon l'Hôtel-Dieu de Québec, Québec
(Québec), Canada (D.R.B., S.H., M.B., J.B., F.M.); and
Faculté de Pharmacie, Université de Montréal,
Montréal (Québec), Canada (A.A.)
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 
S/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 S/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.
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