Integration of endothelial protease-activated receptor-1 inflammatory signaling by ubiquitin

Abstract

Purpose of review: The maintenance and integrity of the endothelial barrier is essential for vascular homeostasis. Endothelial barrier dysfunction is mediated by various inflammatory factors, many of which act through G protein-coupled receptors including protease-activated receptors (PARs). PARs are expressed in multiple cell types in the vasculature and mediate cellular responses to thrombin, the key effector protease of the coagulation cascade. Thrombin activation of PAR1 induces endothelial barrier permeability through multiple pathways. Here, we discuss the mechanism by which thrombin activation of PAR1 promotes endothelial barrier breakdown and highlight recent advances that have provided new insight into molecular mechanisms that control endothelial barrier integrity.

Recent findings: Although the signal transduction pathways induced by thrombin activation of PAR1 in endothelial cells have been extensively studied, the key regulatory mechanisms remain poorly understood. Posttranslational modifications are integral to the regulation of PAR1 signaling and recent studies suggest a novel function for ubiquitination of PAR1 in regulation of endothelial barrier permeability.

Summary: An understanding of how endothelial barrier permeability is regulated by thrombin activation of PAR1 is important for the discovery of new drug targets that can be manipulated to control endothelial barrier permeability and prevent progression of vascular inflammation.

Fig. 1. Model of thrombin-activated PAR1 induction of endothelial barrier permeability

Activation of PAR1 by α-thrombin (α-Th) promotes rapid coupling to heterotrimeric Gα_q and Gα_12/13 proteins comprised of α and βγ subunits. PAR1 coupling to Gα_q activates phospholipase C beta (PLC-β), which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) generating inositol-1,4,5 triphosphate (IP3) and diacylglycerol (DAG). IP3 triggers release of intracellular Ca²⁺, which activates calcium/calmodulin (CaM) resulting in the activation of myosin light chain kinase (MLCK). MLCK phosphorylates MLC promoting MLC interaction with F-actin, resulting in stress fiber formation and cell contraction, which facilitates endothelial barrier disruption. The activation of protein kinase C delta (PKC-δ) is mediated by DAG and Ca²⁺, which phosphorylates Rho-GDP guanine nucleotide dissociation inhibitor (GDI) and thereby increases the stability of active RhoA bound to GTP. PAR1 coupling to Gα_12/13 activates Rho GTP exchange factors (GEFs) to activate RhoA, which promotes Rho kinase phosphorylation of MLC phosphatase (P’tase) and thereby prevents MLC dephosphorylation and enhances endothelial barrier permeability. Thrombin-stimulation also promotes activation of p38 MAP kinase, which promotes endothelial barrier disruption through a poorly characterized pathway mediated by caldesmon and stress fiber formation that appears to be independent MLC phosphorylation.

Fig. 2. Model of thrombin-activated PAR1 induction of non-canonical p38 MAP kinase signaling

Activation of PAR1 by α-thrombin (α-Th) promotes rapid coupling to heterotrimeric G proteins comprised of α and βγ subunits. Activated PAR1 is rapidly phosphorylated and ubiquitinated, the latter of which is mediated by the NEDD4-2 E3 ubiquitin ligase. Ubiquitination of activated PAR1 induces recruitment of TAB2 on endosomes. TAB2 associates with TAB1, which binds to p38 MAP kinase promoting autophosphorylation and activation following thrombin stimulation. Intriguingly, TAB1 protein is stabilized following recruitment to activated PAR1-TAB2-p38 MAP kinase signaling complex. Importantly, PAR1-stimulated non-canonical 38 MAP kinase activation causes a significant increase in endothelial barrier permeability.