JoAnn Trejo

Signal transduction by protease‐activated receptors

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 β‐arrestin binding. Most classic GPCRs are internalized through a clathrin, dynamin and β‐arrestin‐dependent 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 β‐arrestins. Interestingly, PAR1 is basally ubiquitinated and deubiquitinated after activation and traffics from endosomes to lysosomes independent of ubiquitination. In contrast, β‐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.

Arrestin-2 Interacts with the Ubiquitin-Protein Isopeptide Ligase Atrophin-interacting Protein 4 and Mediates Endosomal Sorting of the Chemokine Receptor CXCR4

The chemokine receptor CXCR4 is rapidly targeted for lysosomal degradation by the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4). Although it is known that AIP4 mediates ubiquitination and degradation of CXCR4 and that perturbations in these events contribute to disease, the mechanisms mediating AIP4-dependent regulation of CXCR4 degradation remain poorly understood. Here we show that AIP4 directly interacts with the amino-terminal half of nonvisual arrestin-2 via its WW domains. We show that depletion of arrestin-2 by small interfering RNA blocks agonist-promoted degradation of CXCR4 by preventing CXCR4 trafficking from early endosomes to lysosomes. Surprisingly, CXCR4 internalization and ubiquitination remain intact, suggesting that the interaction between arrestin-2 and AIP4 is not required for ubiquitination of the receptor at the plasma membrane but perhaps for a later post-internalization event. Accordingly, we show that activation of CXCR4 promotes the interaction between AIP4 and arrestin-2 that is consistent with a time when AIP4 co-localizes with arrestin-2 on endocytic vesicles. Taken together, our data suggest that the AIP4·arrestin-2 complex functions on endosomes to regulate sorting of CXCR4 into the degradative pathway.

Protease-Activated Receptor-2 Is Essential for Factor VIIa and Xa–Induced Signaling, Migration, and Invasion of Breast Cancer Cells

Protease-activated receptors (PAR) are G protein-coupled receptors that function as cell-surface sensors for coagulant proteases, as well as other proteases associated with the tumor microenvironment. PAR1 is activated by thrombin whereas the upstream coagulant protease VIIa bound to tissue factor and Xa can activate both PAR1 and PAR2. PAR1 has been implicated in tumor cell growth, migration, and invasion whereas the function of PAR2 in these processes is largely unknown. Towards defining the functional importance of PAR2 in cancer cells, we used small interfering RNAs to deplete highly invasive breast cancer cells of endogenous PAR proteins. Our findings strongly suggest that PAR2 is critical for MDA-MB-231 and BT549 breast cancer cell migration and invasion towards NIH 3T3 fibroblast conditioned medium. To define the relative importance of PAR1 versus PAR2 in mediating factor VIIa and Xa responses, we assessed signaling in cancer cells lacking either endogenous PAR1 or PAR2 proteins. Strikingly, in MDA-MB-231 cells depleted of PAR2, we observed a marked inhibition of VIIa and Xa signaling to phosphoinositide hydrolysis and extracellular signal-regulated kinase 1/2 activation whereas signaling by VIIa and Xa remained intact in PAR1-deficient cells. Factor VIIa and Xa-induced cellular migration was also impaired in MDA-MB-231 cells deficient in PAR2 but not in cells lacking PAR1. Together, these studies reveal the novel findings that PAR2, a second protease-activated G protein-coupled receptor, has a critical role in breast cancer cell migration and invasion and functions as the endogenous receptor for coagulant proteases VIIa and Xa in these cells.

Role of the Thrombin Receptor's Cytoplasmic Tail in Intracellular Trafficking DISTINCT DETERMINANTS FOR AGONIST-TRIGGERED VERSUS TONIC INTERNALIZATION AND INTRACELLULAR LOCALIZATION

The G protein-coupled thrombin receptor is activated by an irreversible proteolytic mechanism and, perhaps as a result, exhibits an unusual trafficking pattern in the cell. Naive receptors tonically cycle between the cell surface and a protected intracellular pool, whereas receptors cleaved and activated at the cell surface internalize and move to lysosomes. Toward understanding how these trafficking events are regulated, we examined a series of receptor mutants. A receptor with alanine substitutions at all potential phosphorylation sites in the cytoplasmic tail failed to display agonist-triggered internalization but, like wild type receptor, displayed robust signaling, tonic cycling, and localization to both the cell surface and an intracellular pool. A truncation mutant that lacked most of the cytoplasmic tail also signaled robustly, lacked phosphorylation, and was defective in agonist-triggered internalization. However, in contrast to the specific phosphorylation site mutant, the truncation mutant did not display tonic cycling and localized exclusively to the cell surface. An analysis of a series of truncation mutants localized residues important for receptor trafficking to a 10-amino acid stretch in its cytoplasmic tail. These data suggest that phosphorylation may trigger internalization of activated thrombin receptors but that a second phosphorylation-independent signal mediates tonic internalization of naive receptors. They further suggest that maintenance of the intracellular pool of naive thrombin receptors requires tonic receptor internalization.

Caveolae are required for protease-selective signaling by protease-activated receptor–1

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 PAR1. Activated protein C (APC), an anti-coagulant protease, also activates PAR1. 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 PAR1-deficient endothelial cells, indicating that PAR1 is the major effector of protease signaling. To delineate the mechanism responsible for protease-selective signaling by PAR1, 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 PAR1 in caveolae is critical for APC selective signaling to Rac1 activation and endothelial barrier protection. We further report that APC induces PAR1 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 PAR1 in caveolae and a novel mechanism of PAR1 signal regulation.

Protease-activated receptor signaling: new roles and regulatory mechanisms

Purpose of reviewProtease-activated receptors are G-protein-coupled receptors that transmit cellular responses to coagulant proteases in a variety of cell types in the vasculature and other tissues. Several other proteases can activate protease-activated receptors in vitro and may affect their function in vivo. While a role for these receptors in hemostasis and thrombosis has been established, their functions in inflammatory and other responses have yet to be fully elucidated. In addition, the mechanisms responsible for protease and cell type-specific signaling mediated by these receptors are largely undefined. Here, we highlight recent advances in understanding the roles and regulation of protease-activated receptor signaling. Recent findingsRecent studies have increased our knowledge of the function of protease-activated receptor signaling in platelets and its contribution to thrombosis. In other cell types, recent work has revealed new connections between these receptors and signaling effectors important for vascular development and inflammatory responses. Other studies have advanced our understanding of protease and cell type-specific responses as well as novel regulatory mechanisms for control of protease-activated receptor signaling. SummaryThus, elucidating the signaling and regulatory mechanisms of protease-activated receptors in various tissues and cell types is important for understanding their biological function as well as for designing therapeutic strategies to control their function.

ALIX binds a YPX3L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/ MVB sorting

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.

Thrombin Promotes Release of ATP from Lung Epithelial Cells through Coordinated Activation of Rho- and Ca2+-dependent Signaling Pathways

Extracellular ATP controls key aspects of lung function via activation of epithelial cell purinergic receptors, but how ATP is released from cells remains poorly understood. To identify mechanistic components upstream of ATP release, we examined the effect of selected G protein coupled-receptor activation on ATP release from lung epithelial cells. The protease-activated receptor (PAR) agonist thrombin elicited a rapid Ca2+-dependent release of ATP from A549 cells. In contrast, the P2Y2 receptor agonist UTP caused negligible ATP release, despite promoting a robust Ca2+ response. Agonist-elicited ATP release was associated with Rho activation and was reduced in cells transfected with dominant negative mutants of p115-Rho GEF or RhoA, and by inhibitors of Rho kinase (ROCK). However, RhoA activation alone did not promote ATP release if temporally separated from Ca2+ mobilization. PAR3 was the only PAR subtype detected in A549 cells by reverse transcription-PCR. Transfection of cells with human PAR3 cDNA increased thrombin-promoted ATP release, inositol phosphate formation, and RhoA activation. Conversely, small interference RNA against PAR3 diminished thrombin-evoked responses. Thrombin-elicited ATP release was accompanied by an enhanced cellular uptake of propidium iodide in a Ca2+- and ROCK-dependent manner and was inhibited by connexin/pannexin hemichannel blockers. Our data suggest that thrombin promotes ATP release from A549 cells via Rho- and Ca2+-dependent activation of connexin/pannexin hemichannels. The relevance of these findings is highlighted by the observation that exposure of primary cultures of well differentiated human bronchial epithelial cells to thrombin resulted in robust ATP release, which was inhibited by ROCK inhibitors and by connexin/pannexin hemichannel blockers.

Clathrin Adaptor AP2 Regulates Thrombin Receptor Constitutive Internalization and Endothelial Cell Resensitization

ABSTRACT Protease-activated receptor 1 (PAR1), a G protein-coupled receptor for the coagulant protease thrombin, is irreversibly activated by proteolysis. Unactivated PAR1 cycles constitutively between the plasma membrane and intracellular stores, thereby providing a protected receptor pool that replenishes the cell surface after thrombin exposure and leads to rapid resensitization to thrombin signaling independent of de novo receptor synthesis. Here, we show that AP2, a clathrin adaptor, binds directly to a tyrosine-based motif in the cytoplasmic tail of PAR1 and is essential for constitutive receptor internalization and cellular recovery of thrombin signaling. Expression of a PAR1 tyrosine mutant or depletion of AP2 by RNA interference leads to significant inhibition of PAR1 constitutive internalization, loss of intracellular uncleaved PAR1, and failure of endothelial cells and other cell types to regain thrombin responsiveness. Our findings establish a novel role for AP2 in direct regulation of PAR1 trafficking, a process critically important to the temporal and spatial aspects of thrombin signaling.

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

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 β-arrestin recruitment and activation of the dishevelled-2 (Dvl-2) scaffold and not by G protein α inhibiting activity polypeptide 2 (Gαi) 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.