Abstract
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.
Footnotes
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Send reprint requests to: François Marceau, M.D., Ph.D., Centre Hospitalier Universitaire de Québec, Centre de recherche, Pavillon l'Hôtel-Dieu de Québec, 11 Côte-du-Palais, Québec (Québec), Canada G1R 2J6. E-mail: francois.marceau{at}crhdq.ulaval.ca
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This study was supported by the Canadian Institutes of Health Research (Grant MOP-14077). D.R.B. is a recipient of the E. J. B. Tomlinson Scholarship Award from the Kidney Foundation of Canada and of a FRSQ Scholarship. S.H. is a recipient of a Studentship from the Canadian Institutes of Health Research.
- Abbreviations:
- B2R
- B2 receptor
- BK
- bradykinin
- GFP
- green fluorescent protein
- LDL
- low-density lipoprotein
- ACE
- angiotensin I-converting enzyme
- B1R
- B1 receptor
- Received August 9, 2000.
- Accepted December 5, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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