-Arrestin- and Dynamin-Dependent Endocytosis of the AT 1 Angiotensin Receptor ZSUZSANNA G ´ ABORIK, M ´ ARTA SZASZ ´ AK, L ´ ASZL ´ O SZIDONYA, BORB ´ ALA BALLA, S ´ ANDOR PAKU, KEVIN J. CATT, ADRIAN J. L. CLARK, and L ´ ASZL ´ O HUNYADY Department of Physiology, Semmelweis University Medical School, Budapest, Hungary (Z.G., M.S., L.S., B.B., L.H.); Department of Molecular Pathology, Joint Research Organization of the Semmelweis University and the Hungarian Academy of Sciences, Budapest, Hungary (S.P.); Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (K.J.C.); and Departments of Endocrinology, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, West Smithfield, London, United Kingdom (A.J.L.C.) This paper is available online at http://molpharm.aspetjournals.org ABSTRACT The major mechanism of agonist-induced internalization of G protein-coupled receptors (GPCRs) is -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 embry- onic kidney (HEK) 293 cells, are internalized by a -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 unaf- fected by inhibition of endocytosis via caveolae with filipin. In addition, internalized fluorescein-conjugated angiotensin II ap- peared in endosomes, as demonstrated by colocalization with transferrin. Overexpression of -arrestin1(V53D) and -arres- tin1(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 dy- namin-2, and a pleckstrin homology domain-mutant (K535A) dynamin-2 with impaired phosphoinositide binding, also inhib- ited 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 recep- tor 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 -arrestin- and dynamin-dependent endocy- tosis of the AT 1 receptor via clathrin-coated vesicles at physi- ological angiotensin II concentrations. The pressor octapeptide hormone, angiotensin II (Ang II), exerts the majority of its physiological effects on cardiovas- cular regulation and salt-water balance by activating the G q -coupled AT 1 angiotensin receptor (De Gasparo et al., 2000). The AT 1 receptor also activates intracellular signaling pathways that stimulate cell growth including activation of tyrosine kinases and small GTP-binding proteins (Berk, 1999; De Gasparo et al., 2000), and is rapidly internalized after Ang II binding (Thomas, 1999; Hunyady et al., 2000). Agonist-induced endocytosis of G protein-coupled receptors (GPCRs) initiates a process by which desensitized receptors are resensitized and recycled to the plasma membrane (Krupnick and Benovic, 1998). Sequestration of the  2 -adren- ergic receptor has been shown to require the binding of -ar- restin proteins to its cytoplasmic tail after agonist-induced activation and phosphorylation by G protein-coupled recep- tor kinases (Zhang et al., 1996; Ferguson et al., 1997; Krupnick and Benovic, 1998). -arrestins direct the phos- phorylated receptors to clathrin-coated pits and induce the formation of clathrin-coated vesicles (Goodman et al., 1996). The role of -arrestins in receptor internalization has been demonstrated for several GPCRs (Bu ¨ nemann et al., 1999). Although -arrestins translocate to the plasma membrane upon agonist stimulation of many GPCRs (Zhang et al., 1999), it has been reported that the internalization of some of This work was supported in part by a Collaborative Research Initiative grant from the Wellcome Trust (051804/Z/97/Z), an International Research Scholar’s award from the Howard Hughes Medical Institute (HHMI 75195– 541702) and by grants from the Hungarian Ministry of Culture and Education (FKFP-0318/1999), the Hungarian Science Foundation (OTKA T-032179) and the Semmelweis University. Preliminary data of this work were presented at the American Society for Cell Biology Annual Meeting, Washington, DC, December 1999 [Hunyady L, Ga ´ borik Z, Mihalik B, Clark AJL, Catt KJ (1999) Dynamin-dependent mech- anism of internalization of the AT 1A angiotensin receptor (Abstract). Mol Biol Cell 10:316a]. ABBREVIATIONS: Ang II, Angiotensin II; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; PH, pleckstrin homology; CHO, Chinese hamster ovary; HA, influenza hemagglutinin epitope; HRP, horseradish peroxidase; k e , endocytotic rate constant. 0026-895X/01/5902-239 –247$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 2 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 386/879752 Mol Pharmacol 59:239–247, 2001 Printed in U.S.A. 239 at ASPET Journals on July 20, 2018 molpharm.aspetjournals.org Downloaded from