Phosphoproteomic analysis of protease-activated receptor-1 biased signaling reveals unique modulators of endothelial barrier function

Abstract

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.

A cellular perspective of bias at G protein‐coupled receptors

“…Phosphoproteomics is an alternative approach to understand how GPCR activation changes kinase and phosphatase activity in the cell. One advantage to phosphoproteomics is that it can be used to detect and identify thousands of phosphosites; thus, examining GPCR activity from the “cellular perspective.” For example, GPCR activation is estimated to change 1–10% of all phosphorylation sites in the cell 46–49 . As a comparison, an estimated 16–20% of phosphosites can be regulated upon receptor tyrosine kinase (RTK) stimulation 50,51 …”

“…A more recent study examined how the cellular phosphoproteome was remodeled by activation of the protease‐activated receptor‐1 (PAR1) with thrombin or anticoagulant protease (APC) 49 . Thrombin cleavage of PAR1 causes coupling to Gα12/13, Gαq, and β‐arrestin while APC causes PAR1 to couple to β‐arrestin 58–61 .…”

“…Thrombin cleavage of PAR1 causes coupling to Gα12/13, Gαq, and β‐arrestin while APC causes PAR1 to couple to β‐arrestin 58–61 . Phosphoproteomic comparison of PAR1 signaling in endothelial cell derived EA.hy926 cells revealed broadly different targets of thrombin and APC‐induced activity 49 . Targets of thrombin activated PAR1 signaling were enriched in proteins linked to endothelial barrier function and adherens junctions while APC‐induced signaling modulated phosphorylation of proteins linked to gene transcription 49 .…”

“…Phosphoproteomic comparison of PAR1 signaling in endothelial cell derived EA.hy926 cells revealed broadly different targets of thrombin and APC‐induced activity 49 . Targets of thrombin activated PAR1 signaling were enriched in proteins linked to endothelial barrier function and adherens junctions while APC‐induced signaling modulated phosphorylation of proteins linked to gene transcription 49 . Additionally, APC modulated a much smaller number of phosphosites than the full agonist thrombin 47,49 .…”

“…Targets of thrombin activated PAR1 signaling were enriched in proteins linked to endothelial barrier function and adherens junctions while APC‐induced signaling modulated phosphorylation of proteins linked to gene transcription 49 . Additionally, APC modulated a much smaller number of phosphosites than the full agonist thrombin 47,49 . Thus, certain biased agonist/GPCR pairs can greatly change the cellular phosphoproteome—and consequently cellular function—compared to their unbiased counterparts.…”