Phosphorylation of the angiotensin II (AT1A) receptor carboxyl terminus: a role in receptor endocytosis.

The molecular mechanism of angiotensin II type I receptor (AT1) endocytosis is obscure, although the identification of an important serine/threonine rich region (Thr332Lys333Met334Ser335Thr336Leu337 Ser338) within the carboxyl terminus of the AT1A receptor subtype suggests that phosphorylation may be involved. In this study, we examined the phosphorylation and internalization of full-length AT1A receptors and compared this to receptors with truncations and mutations of the carboxyl terminus. Epitope-tagged full-length AT1A receptors, when transiently transfected in Chinese hamster ovary (CHO)-K1 cells, displayed a basal level of phosphorylation that was significantly enhanced by angiotensin II (Ang II) stimulation. Phosphorylation of AT1A receptors was progressively reduced by serial truncation of the carboxyl terminus, and truncation to Lys325, which removed the last 34 amino acids, almost completely inhibited Ang II-stimulated 32P incorporation into the AT1A receptor. To investigate the correlation between receptor phosphorylation and endocytosis, an epitope-tagged mutant receptor was produced, in which the carboxyl-terminal residues, Thr332, Ser335, Thr336, and Ser338, previously identified as important for receptor internalization, were substituted with alanine. Compared with the wild-type receptor, this mutant displayed a clear reduction in Ang II-stimulated phosphorylation. Such a correlation was further strengthened by the novel observation that the Ang II peptide antagonist, Sar(1)Ile8-Ang II, which paradoxically causes internalization of wild-type AT1A receptors, also promoted their phosphorylation. In an attempt to directly relate phosphorylation of the carboxyl terminus to endocytosis, the internalization kinetics of wild-type AT1A receptors and receptors mutated within the Thr332-Ser338 region were compared. The four putative phosphorylation sites (Thr332, Ser335, Thr336, and Ser338) were substituted with either neutral [alanine (A)] or acidic amino acids [glutamic acid (E) and aspartic acid (D)], the former to prevent phosphorylation and the latter to reproduce the acidic charge created by phosphorylation. Wild-type AT1A receptors, expressed in Chinese hamster ovary cells, rapidly internalized after Ang II stimulation [t1/2 2.3 min; maximal level of internalization (Ymax) 78.2%], as did mutant receptors carrying single acidic substitutions (T332E, t1/2 2.7 min, Ymax 76.3%; S335D, t1/2 2.4 min, Ymax 76.7%; T336E, t1/2 2.5 min, Ymax 78.2%; S338D, t1/2 2.6 min, Ymax 78.4%). While acidic amino acid substitutions may simply be not as structurally disruptive as alanine mutations, we interpret the tolerance of a negative charge in this region as suggestive that phosphorylation may permit maximal internalization. Substitution of all four residues to alanine produced a receptor with markedly reduced internalization kinetics (T332A/S335A/T336A/S338A, t1/2 10.1 min, Ymax 47.9%), while endocytosis was significantly rescued in the corresponding quadruple acidic mutant (T332E/S335D/T336E/S338D, t1/2 6.4 min, Ymax 53.4%). Double mutation of S335 and T336 to alanine also diminished the rate and extent of endocytosis (S335A/T336A, 3.9 min, Ymax 69.3%), while the analogous double acidic mutant displayed wild type-like endocytotic parameters (S335D/T336E, t1/2 2.6 min, Ymax 77.5%). Based on the apparent rescue of internalization by acidic amino acid substitutions in a region that we have identified as a site of Ang II-induced phosphorylation, we conclude that maximal endocytosis of the AT1A receptor requires phosphorylation within this serine/threonine-rich segment of the carboxyl terminus.