β-arrestin- and dynamin-dependent endocytosis of the AT1 angiotensin receptor

The major mechanism of agonist-induced internalization of G protein-coupled receptors (GPCRs) is beta -arrestin- and dynamin-dependent endocytosis via clathrin-coated vesicles. However, recent reports have suggested that some GPCRs, exemplified by the AT(1) angiotensin receptor expressed in human embryonic kidney (HEK) 293 cells, are internalized by a beta -arrestin- and dynamin-independent mechanism, and possibly via a clathrin-independent pathway. In this study, agonist-induced endocytosis of the rat AT(1A) receptor expressed in Chinese hamster ovary (CHO) cells was abolished by clathrin depletion during treatment with hyperosmotic sucrose and was unaffected by inhibition of endocytosis via caveolae with filipin. In addition, internalized fluorescein-conjugated angiotensin II appeared in endosomes, as demonstrated by colocalization with transferrin. Overexpression of beta -arrestin1( V53D) and beta -arrestin1(1-349) exerted dominant negative inhibitory effects on the endocytosis of radioiodinated angiotensin II in CHO cells. GTPase-deficient (K44A) mutant forms of dynamin-1 and dynamin-2, and a pleckstrin homology domain-mutant (K535A) dynamin-2 with impaired phosphoinositide binding, also inhibited the endocytosis of AT(1) receptors in CHO cells. Similar results were obtained in COS-7 and HEK 293 cells. Confocal microscopy using fluorescein-conjugated angiotensin II showed that overexpression of dynamin-1( K44A) and dynamin-2( K44A) isoforms likewise inhibited agonist-induced AT(1) receptor endocytosis in CHO cells. Studies on the angiotensin II concentration-dependence of AT(1) receptor endocytosis showed that at higher agonist concentrations its rate constant was reduced and the inhibitory effects of dominant negative dynamin constructs were abolished. These data demonstrate the importance of beta -arrestin- and dynamin-dependent endocytosis of the AT(1) receptor via clathrin-coated vesicles at physiological angiotensin II concentrations.