beta-arrestin- and dynamin-dependent endocytosis of the AT(1) 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 em bryonic kidney (HEK) 293 cells, are internalized by a beta -arrestin- and d ynamin-independent mechanism, and possibly via a clathrin-independent pathw ay. In this study, agonist-induced endocytosis of the rat AT(1A) receptor e xpressed in Chinese hamster ovary (CHO) cells was abolished by clathrin dep letion during treatment with hyperosmotic sucrose and was unaffected by inh ibition of endocytosis via caveolae with filipin. In addition, internalized fluorescein-conjugated angiotensin II appeared in endosomes, as demonstrat ed by colocalization with transferrin. Overexpression of beta -arrestin1( V 53D) and beta -arrestin1(1-349) exerted dominant negative inhibitory effect s 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 bin ding, also inhibited the endocytosis of AT(1) receptors in CHO cells. Simil ar results were obtained in COS-7 and HEK 293 cells. Confocal microscopy us ing fluorescein-conjugated angiotensin II showed that overexpression of dyn amin-1( K44A) and dynamin-2( K44A) isoforms likewise inhibited agonist-indu ced AT(1) receptor endocytosis in CHO cells. Studies on the angiotensin II concentration-dependence of AT(1) receptor endocytosis showed that at highe r agonist concentrations its rate constant was reduced and the inhibitory e ffects of dominant negative dynamin constructs were abolished. These data d emonstrate the importance of beta -arrestin- and dynamin-dependent endocyto sis of the AT(1) receptor via clathrin-coated vesicles at physiological ang iotensin II concentrations.