Dataset Information

Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through ?-arrestin and dishevelled-2 scaffolds.

ABSTRACT: Protease-activated receptor-1 (PAR1) is a guanine nucleotide-binding (G) protein-coupled receptor that elicits cellular responses to coagulant and anticoagulant proteases. Activation of PAR1 by the coagulant protease thrombin results in Ras homolog gene family member A (RhoA) activation, disassembly of adherens junctions, and disruption of the endothelial barrier. In contrast, activation of PAR1 with the anticoagulant protease activated protein C (APC) results in activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and endothelial barrier protection. We previously showed that APC cytoprotective signaling requires the compartmentalization of PAR1 in caveolar microdomains. However, the mechanism by which APC-activated PAR1 promotes cytoprotective signaling in human endothelial cells remains poorly understood. Here we show that APC-activated PAR1 cytoprotective signaling is mediated by ?-arrestin recruitment and activation of the dishevelled-2 (Dvl-2) scaffold and not by G protein ? inhibiting activity polypeptide 2 (G?(i)) signaling. In human endothelial cells, PAR1 and ?-arrestins form a preassembled complex and cosegregate in caveolin-1-enriched fractions. Remarkably, we found that depletion of ?-arrestin expression by RNA interference resulted in the loss of APC-induced Rac1 activation but not of thrombin-stimulated RhoA signaling. APC also failed to protect against thrombin-induced endothelial barrier permeability in cells deficient in ?-arrestin expression. We further demonstrate that APC activation of PAR1 results in ?-arrestin-dependent recruitment of Dvl-2, which is critical for Rac1 signaling and endothelial barrier protection but not for thrombin-induced RhoA signaling. Our findings identify a role for ?-arrestin and Dvl-2 scaffolds in APC-activated PAR1 cytoprotective signaling in human endothelial cells.

SUBMITTER: Soh UJ

PROVIDER: S-EPMC3250136 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through β-arrestin and dishevelled-2 scaffolds.

Soh Unice J K UJ Trejo JoAnn J

Proceedings of the National Academy of Sciences of the United States of America 20111121 50

Protease-activated receptor-1 (PAR1) is a guanine nucleotide-binding (G) protein-coupled receptor that elicits cellular responses to coagulant and anticoagulant proteases. Activation of PAR1 by the coagulant protease thrombin results in Ras homolog gene family member A (RhoA) activation, disassembly of adherens junctions, and disruption of the endothelial barrier. In contrast, activation of PAR1 with the anticoagulant protease activated protein C (APC) results in activation of Ras-related C3 bot ...[more]

PMID: 22106258

Similar Datasets

Project description:Activated Gq protein-coupled receptors (GqPCRs) can be desensitized by phosphorylation and ?-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein-tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and ?-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of ?-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of ?-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient ?-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein-coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited ?-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by ?-arrestin to explain why knockdown of ?-arrestin led to secondary depletion of PIP2. Indeed, artificial recruitment of PIP5K removed the secondary loss of PIP2 completely. Altogether, our experimental and theoretical approaches demonstrate roles and dynamics of the protein kinases, ?-arrestin, and PIP5K in the desensitization of PAR2.

Dataset Information

Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through ?-arrestin and dishevelled-2 scaffolds.

Publications

Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through β-arrestin and dishevelled-2 scaffolds.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets