Phosphorylation of the angiotensin II (AT(1A)) receptor carboxyl terminus: A role in receptor endocytosis

The molecular mechanism of angiotensin II type I receptor (AT(1)) endocytosis is obscure, although the identification of an important serine/threonine rich region (Thr(332)Lys(333)Met(334)Ser(335)Thr(336)Leu(337) Ser(338)) within the carboxyl terminus of the AT(1A) receptor subtype suggests that phosphorylation may be involved. In this study, we examined the phosphorylation and internalization of full-length AT(1A) receptors and compared this to receptors with truncations and mutations of the carboxyl terminus. Epitope-tagged full-length AT(1A) 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 AT(1A) receptors was progressively reduced by serial truncation of the carboxyl terminus, and truncation to Lys(325), which removed the last 34 amino acids, almost completely inhibited Ang II-stimulated P-32 incorporation into the AT(1A) receptor. To investigate the correlation between receptor phosphorylation and endocytosis, an epitope-tagged mutant receptor was produced, in which the carboxyl-terminal residues, Thr(332), Ser(335), Thr(336), and Ser(338), 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)Ile(8)-Ang II, which paradoxically causes internalization of wild-type AT(1A) receptors, also promoted their phosphorylation. In an attempt to directly relate phosphorylation of the carboxyl terminus to endocytosis, the internalization kinetics of wild-type AT(1A) receptors and receptors mutated within the Thr(332)-Ser(338) region were compared. The four putative phosphorylation sites (Thr(332), Ser(335), Thr(336), and Ser(338)) 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. Wildtype AT(1A) receptors, expressed in Chinese hamster ovary cells, rapidly internalized after Ang II stimulation [t(1/2) 2.3 min; maximal level of internalization (Y-max) 78.2%], as did mutant receptors carrying single acidic substitutions (T332E, t(1/2) 2.7 min, Y-max 76.3%; S335D, t(1/2) 2.4 min, Y-max 76.7%; T336E, t(1/2) 2.5 min, Y-max 78.2%; S338D, t(1/2) 2.6 min, Y-max 78.4%)

Neurally-mediated increase in calcineurin activity regulates cardiac contractile function in absence of hypertrophy

Objective: The calcineurin pathway has been involved in the development of cardiac hypertrophy, yet it remains unknown whether calcineurin activity can be regulated in myocardium independently from hypertrophy and cardiac load. Methods: To test that hypothesis, we measured calcineurin activity in a rat model of infrarenal aortic constriction (IR), which affects neurohormonal pathways without increasing cardiac afterload. Results: In this model, there was no change in arterial pressure over the 4-week experimental period, and the left ventricle/body weight ratio did not increase. At 2 weeks after IR, calcineurin activity was increased 1.8-fold (