Phospho‐proteomic Analysis of Protease‐activated Receptor‐1 Biased Signaling Reveals Novel Modulators of Endothelial Barrier Function

Abstract

Dysregulation of the vascular endothelium is a hallmark of inflammation and promotes barrier disruption, vascular leakage and tissue edema. Vascular endothelial dysfunction is mediated by vasoactive agents and inflammatory mediators, many of which signal through G protein‐coupled receptors (GPCRs). A key GPCR inflammatory mediator is thrombin, a procoagulant protease generated in response to vascular injury and inflammation. Thrombin activates protease‐activated receptor‐1 (PAR1) in endothelial cells to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC) is an anticoagulant protease that activates PAR1 through a distinct mechanism and promotes endothelial barrier stabilization. The ability of distinct tethered ligands formed through cleavage of PAR1 by different proteases results in stabilization of unique receptor active conformational states that promote opposite signaling effects is termed biased signaling. Despite progress in understanding PAR1 bias signaling in endothelial cells, the proteins and pathways by which thrombin and APC regulate the endothelial barrier integrity are largely unknown. Here, we report the global phosphoproteome of thrombin and APC signaling using tandem‐mass‐tag (TMT) mass spectrometry and computational analyses. Our results reveal unique temporal dynamics profiles of thrombin and APC signaling and the identification of multiple enriched phosphoproteome associated biological functions including microtubules, adherens junctions, actin and signaling, all key modulators of endothelial barrier permeability. We selected candidates that showed statistically significant changes in phosphorylation following thrombin or APC stimulation, and validated the function of four proteins including: afadin (AFDN), CDC42 binding protein kinase alpha (BPA), adducin 1 (ADD1), and GPCR interacting protein 1 (GIT1) using an siRNA‐targeted approach. Using target‐specific siRNAs to deplete candidate proteins in endothelial cells, we found that depletion of afadin and adducin had statistically significant effects on endothelial barrier permeability. Afadin depletion led to an increase in thrombin‐induced permeability, while adducin depletion virtually ablated thrombin‐stimulated permeability. Immunofluorescence microscopy imaging of morphological changes in F‐actin stress fiber formation supported these results and revealed corresponding changes in thrombin‐induced gap formation consistent with the observed changes in permeability. In addition, depletion of candidate proteins also modulated thrombin‐stimulated p38 pro‐inflammatory signaling and APC‐induced AKT cytoprotective signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 bias signaling in endothelial cells.Support or Funding InformationThis work was supported by NIH/NIGMS R01 GM116597 and R35 GM127121 (J.T.), UC Tobacco‐related Disease Research Program Predoctoral Fellowship (C.C.R), and NIH/NIGMS K12 GM068524 (O.M.I., J.D.L.)