Time-Resolved Quantitative Phosphoproteomics: New Insights into Angiotensin-(1–7) Signaling Networks in Human Endothelial Cells

Abstract

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous ligand of the Mas receptor and induces vasodilation, positive regulation of insulin, and antiproliferative and antitumorigenic activities. However, little is known about the molecular mechanisms behind these biological properties. Aiming to identify proteins involved in the Ang-(1-7) signaling, we performed a mass spectrometry-based time-resolved quantitative phosphoproteome study of human aortic endothelial cells (HAEC) treated with Ang-(1-7). We identified 1288 unique phosphosites on 699 different proteins with 99% certainty of correct peptide identification and phosphorylation site localization. Of these, 121 sites on 79 proteins had their phosphorylation levels significantly changed by Ang-(1-7). Our data suggest that the antiproliferative activity of Ang-(1-7) is due to the activation or inactivation of several target phosphoproteins, such as forkhead box protein O1 (FOXO1), mitogen-activated protein kinase 1 (MAPK), proline-rich AKT1 substrate 1 (AKT1S1), among others. In addition, the antitumorigenic activity of Ang-(1-7) is at least partially due to FOXO1 activation, since we show that this transcriptional factor is activated and accumulated in the nucleus of A549 lung adenocarcinoma cells treated with Ang-(1-7). Moreover, Ang-(1-7) triggered changes in the phosphorylation status of several known downstream effectors of the insulin signaling, indicating an important role of Ang-(1-7) in glucose homeostasis. In summary, this study provides new concepts and new understanding of the Ang-(1-7) signal transduction, shedding light on the mechanisms underlying Mas activation.

International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli

“…Ligand treatment with Ang(1-7) or AVE0991 in MAS-transfected cells couples calcium-independent activation of nitric oxide synthase to the phosphatidylinositol 3-kinase/protein kinase B, AKT pathway (Sampaio Verrilli et al, 2012;Savergnini et al, 2013;Than et al, 2013), or activation of phospholipase A2 (Santos et al, 2003b). Ang(1-7) altered the phosphorylation of MAP kinases (Sampaio et al, 2007a;Zimpelmann and Burns, 2009;Liu et al, 2012;Verano-Braga et al, 2012). Thus, Ang(1-7) action through MAS is proposed to be production of arachidonic acid and activation of nitric oxide synthase, which may not involve cAMP, IP3, and calcium signaling.…”

G-Protein–Coupled Receptor MrgD Is a Receptor for Angiotensin-(1–7) Involving Adenylyl Cyclase, cAMP, and Phosphokinase A

“…The need of such second messenger is also illustrated by the fact that 2 publications during the past years intensively investigated phosphorylation of intracellular molecules 28 and quantities of intracellular proteins, 29 respectively, in response to Ang-(1-7). Although aimed to identify useful molecules for receptor pharmacology, the data published failed to add useful tools to this approach.…”

Mas and Its Related G Protein–Coupled Receptors, Mrgprs

“…4). Our recent phosphoproteomic study identified several further possible signaling pathways of cells induced by Ang-(1-7) stimulation, such as the Mas-Related Genes forkhead box protein O1 (Verano-Braga et al, 2012). It is not yet clear how these early signaling events induced by Ang-(1-7) are linked to the NO and phospholipase A 2 pathway.…”