Agonist-promoted trafficking of human bradykinin receptors: arrestin- and dynamin-independent sequestration of the B2 receptor and bradykinin in HEK293 cells.
In this study, we analysed the agonist-promoted trafficking of human B(2) (B(2)R) and B(1) (B(1)R) bradykinin (BK) receptors using wild-type and green fluorescent protein (GFP)-tagged receptors in HEK293 cells. B(2)R was sequestered to a major extent upon exposure to BK, as determined by the loss of cell-surface B(2)R using radioligand binding and by imaging of B(2)R-GFP using laser-scanning confocal fluorescence microscopy. Concurrent BK sequestration was revealed by the appearance of acid-resistant specific BK receptor binding. The same techniques showed that B(1)R was sequestered to a considerably lesser extent upon binding of des-Arg(10)-kallidin. B(2)R sequestration was rapid (half-life approximately 5 min) and reached a steady-state level that was significantly lower than that of BK sequestration. B(2)R sequestration was minimally inhibited by K44A dynamin (22.4+/-3.7%), and was insensitive to arrestin-(319-418), which are dominant-negative mutants of dynamin I and beta-arrestin respectively. Furthermore, the B(2)R-mediated sequestration of BK was completely insensitive to both mutants, as was the association of BK with a caveolae-enriched fraction of the cells. On the other hand, agonist-promoted sequestration of the beta(2)-adrenergic receptor was dramatically inhibited by K44A dynamin (81.2+/-16.3%) and by arrestin-(319-418) (36.9+/-4.4%). Our results show that B(2)R is sequestered to a significantly greater extent than is B(1)R upon agonist treatment in HEK293 cells. Furthermore, B(2)R appears to be recycled in the process of sequestering BK, and this process occurs in a dynamin- and beta-arrestin-independent manner and, at least in part, involves caveolae.
Lamb
M E
ME
Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
De Weerd
W F
WF
Leeb-Lundberg
L M
LM
D019390:Arrestins / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D001920:Bradykinin / Q000378:metabolism*; D021941:Caveolae / Q000378:metabolism; D002478:Cells, Cultured; D034284:Dynamin I; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000819:agonists / Q000378:metabolism*; D000071557:beta-Arrestins
D019390:Arrestins; D008164:Luminescent Proteins; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins; D001920:Bradykinin