Association of beta-arrestin 1 with the type 1A angiotensin II receptor involves phosphorylation of the receptor carboxyl terminus correlates with receptor internalization

Arrestins bind to phosphorylated G protein-coupled receptors and participat e in receptor desensitization and endocytosis. Although arrestins traffic w ith activated type 1 (AT(1A)) angiotensin II (AngII) receptors, the contrib ution of arrestins to AT1A receptor internalization is controversial, and t he physical association of arrestins with the AT1A receptor has not been es tablished. In this study, by coimmunoprecipitating AT(1A) receptors and bet a -arrestin 1, we provide direct evidence for an association between arrest ins and the AT1A receptor that was agonist- and time-dependent and continge nt upon the level of beta -arrestin I expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of ig-arrestin 1 assoc iation, which correlated with decreases in receptor phosphorylation. Trunca tion of the AT1A receptor to lysine(325) prevented Angll-induced phosphoryl ation and beta -arrestin I association as well as markedly inhibiting recep tor internalization, indicating a close correlation between these receptor parameters. Angll-induced association was also dramatically reduced in a ph osphorylation- and internalization-impaired receptor mutant in which four s erine and threonine residues in the central portion of the AT(1A) receptor carboxyl terminus (Thr(332), Ser(335), Thr(338), Ser(338)) were substituted with alanine. In contrast, substitutions in another serine/threonine-rich region (Ser(346), Ser(347), Ser(348)) and at three PKC phosphorylation site s (Ser(331), Ser(338), Ser(348)) had no effect on Angll-induced beta -arres tin I association or receptor internalization. While AT(1A) receptor intern alization could be inhibited by a dominant-negative beta -arrestin 1 mutant (beta arr1(319-418)), treatment with hyperosmotic sucrose to inhibit inter nalization did not abrogate the differences in arrestin association observe d between the wild-type and mutant receptors, indicating that arrestin bind ing precedes, and is not dependent upon, receptor internalization. Interest ingly, a substituted analog of Angll, [Sar(1)Ile(4)Ile(8)]-Angll, which pro motes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong beta -arrestin 1 association with the full-le ngth AT(1A) receptor. These results identify the central portion of the AT( 1A) receptor carboxyl terminus as the important determinant for beta -arres tin I binding and internalization and indicate that AT(1A) receptor phospho rylation is crucial for beta -arrestin docking.