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

Mol Endocrinol. 2001 Oct;15(10):1706-19. doi: 10.1210/mend.15.10.0714.

Abstract

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 beta-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 beta-arrestin 1 expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of beta-arrestin 1 association, which correlated with decreases in receptor phosphorylation. Truncation of the AT(1A) receptor to lysine(325) prevented AngII-induced phosphorylation and beta-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 (Thr(332), Ser(335), Thr(336), 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 sites (Ser(331), Ser(338), Ser(348)) had no effect on AngII-induced beta-arrestin 1 association or receptor internalization. While AT(1A) receptor internalization could be inhibited by a dominant-negative beta-arrestin 1 mutant (beta arr1(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, [Sar(1)Ile(4)Ile(8)]-AngII, which promotes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong beta-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 beta-arrestin 1 binding and internalization and indicate that AT(1A) receptor phosphorylation is crucial for beta-arrestin docking.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Angiotensin II / pharmacology
  • Animals
  • Arrestins / genetics
  • Arrestins / metabolism*
  • CHO Cells
  • Cricetinae
  • Endocytosis
  • Immunosorbent Techniques
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Receptor, Angiotensin, Type 1
  • Receptors, Angiotensin / chemistry
  • Receptors, Angiotensin / genetics
  • Receptors, Angiotensin / metabolism*
  • Transfection
  • beta-Arrestins

Substances

  • Arrestins
  • Receptor, Angiotensin, Type 1
  • Receptors, Angiotensin
  • beta-Arrestins
  • Angiotensin II