The family of G protein-coupled receptors (GPCRs) constitutes the largest class of signalling receptors in the human genome, controlling vast physiological responses and are the target of many drugs. After activation, GPCRs are rapidly desensitized by phosphorylation and b-arrestin binding. Most classic GPCRs are internalized through a clathrin, dynamin and b-arrestindependent pathway and then recycled back to the cell surface or sorted to lysosomes for degradation. Given the vast number and diversity of GPCRs, different mechanisms are likely to exist to precisely regulate the magnitude, duration and spatial aspects of receptor signalling. The G protein-coupled protease-activated receptors (PARs) provide elegant examples of GPCRs that are regulated by distinct desensitization and endocytic sorting mechanisms, processes that are critically important for the spatial and temporal fidelity of PAR signalling. PARs are irreversibly activated through proteolytic cleavage and transmit cellular responses to extracellular proteases. Activated PAR1 internalizes through a clathrin-and dynamin-dependent pathway independent of b-arrestins. Interestingly, PAR1 is basally ubiquitinated and deubiquitinated after activation and traffics from endosomes to lysosomes independent of ubiquitination. In contrast, b-arrestins mediate activated PAR2 internalization and function as scaffolds that promote signalling from endocytic vesicles. Moreover, activated PAR2 is modified with ubiquitin, which facilitates lysosomal degradation. Activated PARs also adopt distinct active conformations that signal to diverse effectors and are likely regulated by different mechanisms. Thus, the identification of the molecular machinery important for PAR signal regulation will enable the development of new strategies to manipulate receptor signalling and will provide novel targets for the development of drugs.

A novel MVB/lysosomal sorting pathway for signaling receptors bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be broadly applicable to GPCRs containing YPXnL motifs.

Protease-activated receptor-1 (PAR1) is a guanine nucleotidebinding (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 β-arrestin recruitment and activation of the dishevelled-2 (Dvl-2) scaffold and not by G protein α inhibiting activity polypeptide 2 (Gα i ) signaling. In human endothelial cells, PAR1 and β-arrestins form a preassembled complex and cosegregate in caveolin-1-enriched fractions. Remarkably, we found that depletion of β-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 β-arrestin expression. We further demonstrate that APC activation of PAR1 results in β-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 β-arrestin and Dvl-2 scaffolds in APC-activated PAR1 cytoprotective signaling in human endothelial cells.biased agonism | endothelial dysfunction | inflammation | sepsis

Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor uniquely activated by proteolysis. Thrombin, a coagulant protease, induces inflammatory responses and endothelial barrier permeability through the activation of PAR 1. Activated protein C (APC), an anti-coagulant protease, also activates PAR 1. However, unlike thrombin, APC elicits anti-inflammatory responses and protects against endothelial barrier dysfunction induced by thrombin. We found that thrombin and APC signaling were lost in PAR 1-deficient endothelial cells, indicating that PAR 1 is the major effector of protease signaling. To delineate the mechanism responsible for protease-selective signaling by PAR 1, we examined the effect of APC and thrombin on the activation of RhoA and Rac1, small GTPases that differentially regulate endothelial barrier permeability. Thrombin caused robust RhoA signaling but not Rac1 activation, whereas APC stimulated a marked increase in Rac1 activation but not RhoA signaling, consistent with the opposing functions of these proteases on endothelial barrier integrity. Strikingly, APC signaling and endothelial barrier protection effects were abolished in cells lacking caveolin-1, whereas thrombin signaling remained intact. These findings suggest that compartmentalization of PAR 1 in caveolae is critical for APC selective signaling to Rac1 activation and endothelial barrier protection. We further report that APC induces PAR 1 phosphorylation and desensitizes endothelial cells to thrombin signaling but promotes limited receptor cleavage and negligible internalization and degradation even after prolonged APC exposure. Thus, APC selective signaling and endothelial barrier protective effects are mediated through compartmentalization of PAR 1 in caveolae and a novel mechanism of PAR1 signal regulation.

Protease-activated receptors (PARs) are G protein-coupled receptors (GPCRs) that transmit cellular responses begun by the actions of extracellular proteases. The activation of a PAR occurs by a unique mechanism whereby the extracellular N-terminal segment of the inactive receptor undergoes proteolytic cleavage, resulting in irreversible activation--unlike most GPCRs that are reversibly activated. PARs mediate cellular responses to coagulant proteases in various cell types localized within the vasculature. Additionally, PARs are expressed in other cell types and respond to a plethora of proteases. Recent studies have revealed that different proteases elicit distinct responses through the activation of the same PAR. This phenomenon appears to involve stabilization of distinct active PAR conformations that facilitates selectively coupling to different effectors and is localized to caveolae, a subtype of lipid rafts.