High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells.
We hypothesized that the inducible kinin B(1) receptor (B(1)R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B(1)Rs and related B(2) receptors (B(2)Rs) was investigated. Endocytosis of the B(1)R-yellow fluorescent protein (YFP) conjugate was more intense than that of B(2)R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B(1)R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B(2)R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B(1)R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B(2)R-GFP remained constant. Wild-type B(1)Rs were also cleared faster than B(2)Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B(1)Rs are more rapidly eliminated than B(2)Rs (decreased maximal effect of agonist over 2 h). The highly regulated B(1)R is rapidly degraded, relative to the constitutive B(2)R.
Fortin
Jean-Philippe
JP
Centre Hospitalier Universitaire de Québec, Centre de recherche du Pavillon l'Hôtel-Dieu de Québec, 11 Côte-du-Palais, Quebec City, Quebec, Canada G1R 2J6.
Bouthillier
Johanne
J
Marceau
François
F
D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D001011:Aorta / Q000166:cytology; D001920:Bradykinin / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents / Q000378:metabolism; D007601:Jugular Veins / Q000166:cytology; D007609:Kallidin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011817:Rabbits; D011869:Radioligand Assay; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014316:Tritium
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011500:Protein Synthesis Inhibitors; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011994:Recombinant Proteins; D014316:Tritium; D049452:Green Fluorescent Proteins; D007609:Kallidin; D000841:Anisomycin; C052787:kallidin, des-Arg(10)-; D001920:Bradykinin