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Association of ß-Arrestin 1 with the Type 1A Angiotensin II Receptor Involves Phosphorylation of the Receptor Carboxyl Terminus and Correlates with Receptor Internalization

Molecular Endocrinology Laboratory, Baker Medical Research Institute, Melbourne 8008, Australia

Address all correspondence and requests for reprints to: Dr. Walter G. Thomas, Molecular Endocrinology Laboratory, Baker Medical Research Institute, PO Box 6492, St. Kilda Road Central, Melbourne Victoria 8008, Australia. E-mail: walter.thomas{at}baker.edu.au

Arrestins bind to phosphorylated G protein-coupled receptors and participate in receptor desensitization and endocytosis. Although arrestins traffic with activated type 1 (AT_1A) angiotensin II (AngII) receptors, the contribution of arrestins to AT_1A receptor internalization is controversial, and the physical association of arrestins with the AT_1A receptor has not been established. In this study, by coimmunoprecipitating AT_1A receptors and ß-arrestin 1, we provide direct evidence for an association between arrestins and the AT_1A receptor that was agonist- and time-dependent and contingent upon the level of ß-arrestin 1 expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of ß-arrestin 1 association, which correlated with decreases in receptor phosphorylation. Truncation of the AT_1A receptor to lysine³²⁵ prevented AngII-induced phosphorylation and ß-arrestin 1 association as well as markedly inhibiting receptor internalization, indicating a close correlation between these receptor parameters. AngII-induced association was also dramatically reduced in a phosphorylation- and internalization-impaired receptor mutant in which four serine and threonine residues in the central portion of the AT_1A receptor carboxyl terminus (Thr³³², Ser³³⁵, Thr³³⁶, Ser³³⁸) were substituted with alanine. In contrast, substitutions in another serine/threonine-rich region (Ser³⁴⁶, Ser³⁴⁷, Ser³⁴⁸) and at three PKC phosphorylation sites (Ser³³¹, Ser³³⁸, Ser³⁴⁸) had no effect on AngII-induced ß-arrestin 1 association or receptor internalization. While AT_1A receptor internalization could be inhibited by a dominant-negative ß-arrestin 1 mutant (ßarr1^319–418), treatment with hyperosmotic sucrose to inhibit internalization did not abrogate the differences in arrestin association observed between the wild-type and mutant receptors, indicating that arrestin binding precedes, and is not dependent upon, receptor internalization. Interestingly, a substituted analog of AngII, [Sar¹Ile⁴Ile⁸]-AngII, which promotes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong ß-arrestin 1 association with the full-length AT_1A receptor. These results identify the central portion of the AT_1A receptor carboxyl terminus as the important determinant for ß-arrestin 1 binding and internalization and indicate that AT_1A receptor phosphorylation is crucial for ß-arrestin docking.

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