Agonist-dependent internalization of the angiotensin II type one receptor (AT1): role of C-terminus phosphorylation in recruitment of β-arrestins (original) (raw)

h-Arrestins play a role in AT 1 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 h-arrestin to the activated receptor are poorly defined. In this study, we used CHO-K1 and HEK 293 cells expressing wild-type or mutant AT 1 to investigate two possibilities: activated AT 1 induces global relocation of h-arrestins to the plasma membrane or the phosphorylated C-terminus acts as bait to attract h-arrestins. Results obtained using high osmolarity and dominant-negative h-arrestin confirmed that internalization of AT 1 in both CHO-K1 and HEK 293 cells is predominately via clathrin-mediated endocytosis involving h-arrestin, and substitution of T332, S335, T336 and S338 with alanine to preclude phosphorylation markedly attenuated AT 1 internalization. Confocal microscopy revealed that wild-type AT 1 induced a timedependent translocation of GFP-tagged h-arrestins 1 and 2 to the cell surface. In contrast, the TSTS/A mutant did not traffic h-arrestin 1 at all, and only trafficked h-arrestin 2 weakly. Results of rescue-type experiments were consistent with the idea that both h-arrestins are able to interact with the non-phosphorylated receptor, albeit with much lower affinity and h-arrestin 1 less so than h-arrestin 2. In conclusion, this study shows that the high affinity binding of h-arrestins to the phosphorylated C-terminus is the predominant mechanism of agonist-induced h-arrestin recruitment to the cell surface and AT 1 receptor.