In this study, we analysed the agonist-promoted trafficking of human B2 (B2R) and B1 (B1R) bradykinin (BK) receptors using wild-type and green fluorescent protein (GFP)-tagged receptors in HEK293 cells. B2R was sequestered to a major extent upon exposure to BK, as determined by the loss of cell-surface B2R using radioligand binding and by imaging of B2R–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 B1R was sequestered to a considerably lesser extent upon binding of des-Arg10-kallidin. B2R sequestration was rapid (half-life ∼ 5min) and reached a steady-state level that was significantly lower than that of BK sequestration. B2R 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 β-arrestin respectively. Furthermore, the B2R-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 β2-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 B2R is sequestered to a significantly greater extent than is B1R upon agonist treatment in HEK293 cells. Furthermore, B2R appears to be recycled in the process of sequestering BK, and this process occurs in a dynamin- and β-arrestin-independent manner and, at least in part, involves caveolae.
Agonist-promoted trafficking of human bradykinin receptors: arrestin- and dynamin-independent sequestration of the B2 receptor and bradykinin in HEK293 cells
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Maria E. LAMB, Willem F. C. DE WEERD, L. M. Fredrik LEEB-LUNDBERG; Agonist-promoted trafficking of human bradykinin receptors: arrestin- and dynamin-independent sequestration of the B2 receptor and bradykinin in HEK293 cells. Biochem J 1 May 2001; 355 (3): 741–750. doi: https://doi.org/10.1042/bj3550741
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