Phosphoproteomics for studying signaling pathways evoked by hormones of the renin-angiotensin system: A source of untapped potential

Abstract

The Renin-Angiotensin System (RAS) is a complex neuroendocrine system consisting of a single precursor protein, angiotensinogen (AGT), which is processed into various peptide hormones, including the angiotensins [Ang I, Ang II, Ang III, Ang IV, Ang-(1-9), Ang-(1-7), Ang-(1-5), etc] and Alamandine-related peptides [Ang A, Alamandine, Ala-(1-5)], through intricate enzymatic pathways. Functionally, the RAS is divided into two axes with opposing effects: the classical axis, primarily consisting of Ang II acting through the AT₁ receptor (AT₁R), and in contrast the protective axis, which includes the receptors Mas, AT₂R and MrgD and their respective ligands. A key area of RAS research is to gain a better understanding how signaling cascades elicited by these receptors lead to either "classical" or "protective" effects, as imbalances between the two axes can contribute to disease. On the other hand, therapeutic benefits can be achieved by selectively activating protective receptors and their associated signaling pathways. Traditionally, robust "hypothesis-driven" methods like Western blotting have built a solid knowledge foundation on RAS signaling. In this review, we introduce untargeted mass spectrometry-based phosphoproteomics, a "hypothesis-generating approach", to explore RAS signaling pathways. This technology enables the unbiased discovery of phosphorylation events, offering insights into previously unknown signaling mechanisms. We review the existing studies which used phosphoproteomics to study RAS signaling and discuss potential future applications of phosphoproteomics in RAS research including advantages and limitations. Ultimately, phosphoproteomics represents a so far underused tool for deepening our understanding of RAS signaling and unveiling novel therapeutic targets.

Keywords: cellular signaling; phosphoproteome; phosphoproteomics; renin‐angiotensin system.

FIGURE 1

An overview of the Renin‐Angiotensin System. (A) RAS peptide hormones are formed by the limited proteolysis of the protein precursor angiotensinogen. The amino acid sequence (one letter code) of each peptide is represented below its name. (B) RAS receptors and their ligands. Downstream effects associated with receptor activation is also shown. 1TM, single‐pass transmembrane protein; 7TM, seven transmembrane protein; ACE, angiotensin‐converting enzyme; ACE2, angiotensin‐converting enzyme type 2; Ang, angiotensin; APA, aminopeptidase A; APN, aminopeptidase N; AT₁R, angiotensin AT₁ receptor; AT₂R, angiotensin AT₂ receptor DAP, dipeptidyl aminopeptidases; DC, decarboxylase; IRAP, insulin‐regulated aminopeptidase; MrgD, Mas‐related G‐protein‐coupled receptor member D; NEP, neprilysin; PEP, prolyl endopeptidase; PRCP, prolyl carboxypeptidase; PRR, prorenin receptor; RAS, Renin‐Angiotensin System; THOP, thimet oligopeptidase.

FIGURE 2

Typical proteomics and phosphoproteomics workflow to study cell signaling. Cells are treated with a suitable agonist to trigger the signaling cascades of interest. Cells are then lysed, and proteins and phosphoproteins extracted. Proteins' thiol groups are reduced (e.g., with dithiothreitol) and alkylated (e.g., with iodoacetamide). Subsequently, proteins are digested with specific proteases (e.g., trypsin) followed by an enrichment step to increase the phosphopeptide content in the sample. At this point, the protocol can be continued either as untargeted (hypothesis‐generating approach) or targeted (hypothesis‐driven approach) proteomics/phosphoproteomics. In the former approach (right panel), peptides/phosphopeptides are analyzed by LC–MS to obtain a global map of the cell's proteome/phosphoproteome. After bioinformatic analysis, lists of regulated proteins and phosphorylation sites are used to infer signaling cascades activated by the respective agonist (hypothesis‐generation). In the latter approach (left panel), potential effectors of a given signaling cascade are selected (pre‐defined target list) and specifically analyzed by MS for changes in protein abundances and phosphorylation status (hypothesis‐driven).

FIGURE 3

AT₁R signaling. Summary of the knowledge acquired by MS‐based phosphoproteomics regarding AT₁R signaling using unbiased (Ang II) or β‐arrestin‐biased (TRV023, TRV027) agonists. Novel components of β‐arrestin‐biased pathway (depicted in yellow) were identified by several research groups, providing further insights into AT₁R signaling. LCP1, PKC, and PKD (depicted in gray) were observed to be activated by Ang II treatment and, while it is tempting to assume these proteins are related to G‐Protein‐biased pathways, confirmation with G‐Protein‐biased agonists (such as TRV055 or TRV056) is much warranted.

FIGURE 4

Ang‐(1–7)/MasR signaling. Comparison of the classical Western blot‐based hypothesis‐driven approach (A) and MS‐based hypothesis‐generating approach (B) in the study of Ang‐(1–7)/MasR signaling. A solid knowledge about the Ang‐(1–7)/MasR signaling was built using Western blotting resulting for example, in the identification of PI3K‐Akt pathway activation by Ang‐(1–7) to induce NO generation in endothelial cells. The use of a MS‐based shotgun phosphoproteomics method (hypothesis‐generating approach) allowed the identification of 79 potential new downstream effectors of Ang‐(1–7)/MasR signaling (some represented in the figure), including the validated new effector FOXO‐1.

FIGURE 5

Common signaling pathways. Non‐exhaustive list of signaling effectors and phosphorylation events shared by receptors of the protective axis of the RAS as determined by phosphoproteomics. Akt, serine/threonine protein kinase (protein kinase B); AKT1S1, proline‐rich AKT1 substrate 1; AMPK, AMP‐activated protein kinase; C21, Compound 21; ERK1/2, extracellular signal‐regulated kinase 1/2; FOXO‐1, forkhead box protein O1; HDAC1, histone deacetylase 1; MAPK1, mitogen‐activated protein kinase 1; p53, tumor protein p53 (tumor suppressor protein).