title: Agonist-dependent internalization of the angiotensin II type one receptor (AT1): role of C-terminus phosphorylation in recruitment of beta-arrestins.
abstract: Beta-arrestins play a role in AT1 endocytosis by binding the cytoplasmic, C-terminus region T332-S338, the major site of angiotensin II (Ang II)-induced phosphorylation. However, the processes responsible for recruiting beta-arrestin to the activated receptor are poorly defined. In this study, we used CHO-K1 and HEK 293 cells expressing wild-type or mutant AT1 to investigate two possibilities: activated AT1 induces global relocation of beta-arrestins to the plasma membrane or the phosphorylated C-terminus acts as bait to attract beta-arrestins. Results obtained using high osmolarity and dominant-negative beta-arrestin confirmed that internalization of AT1 in both CHO-K1 and HEK 293 cells is predominately via clathrin-mediated endocytosis involving beta-arrestin, and substitution of T332, S335, T336 and S338 with alanine to preclude phosphorylation markedly attenuated AT1 internalization. Confocal microscopy revealed that wild-type AT1 induced a time-dependent translocation of GFP-tagged beta-arrestins 1 and 2 to the cell surface. In contrast, the TSTS/A mutant did not traffic beta-arrestin 1 at all, and only trafficked beta-arrestin 2 weakly. Results of rescue-type experiments were consistent with the idea that both beta-arrestins are able to interact with the non-phosphorylated receptor, albeit with much lower affinity and beta-arrestin 1 less so than beta-arrestin 2. In conclusion, this study shows that the high affinity binding of beta-arrestins to the phosphorylated C-terminus is the predominant mechanism of agonist-induced beta-arrestin recruitment to the cell surface and AT1 receptor.
authors.affiliation: Science Department, Cabrini College, Radnor, PA, USA.
authors.forename: Chris E
authors.initials: CE
authors.lastname: Kule
authors.forename: Vijaya
authors.initials: V
authors.lastname: Karoor
authors.forename: Jonathan N E
authors.initials: JN
authors.lastname: Day
authors.forename: Walter G
authors.initials: WG
authors.lastname: Thomas
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Baker
authors.forename: Diem
authors.initials: D
authors.lastname: Dinh
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: Acker
authors.forename: George W
authors.initials: GW
authors.lastname: Booz
mesh_terms: D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D016466:CHO Cells; D019556:COS Cells; D002966:Clathrin / Q000378:metabolism; D006224:Cricetinae; D004705:Endocytosis; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D044139:Receptor, Angiotensin, Type 2 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; D019390:Arrestins; D002966:Clathrin; D044139:Receptor, Angiotensin, Type 2; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D000804:Angiotensin II; D049452:Green Fluorescent Proteins