RESUMO
Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via ß-arrestin-2 (ß-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. aPC/PAR1 cytoprotective responses also require coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a ß-arr2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 coexist in caveolin-1 (Cav1)-rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates ß-arr2-dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced, extracellular signal-regulated kinase 1/2 (ERK1/2) activation is also dependent on ß-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-ß-arr2-mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete, ß-arr2-driven signaling pathways in caveolae.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteína C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor PAR-1/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , beta-Arrestina 2/metabolismo , Anilidas/farmacologia , Apoptose/fisiologia , Células Endoteliais/fisiologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Lactonas/farmacologia , Metanol/farmacologia , Organofosfonatos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Inibidores da Agregação Plaquetária/farmacologia , Proteína C/genética , Proteínas Proto-Oncogênicas c-akt/genética , Piridinas/farmacologia , Pirrolidinas/farmacologia , Receptor PAR-1/genética , Receptores de Esfingosina-1-Fosfato/genética , Sulfonas/farmacologia , beta-Arrestina 2/genéticaRESUMO
Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of ß-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of ß-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation.
Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Megacariócitos/metabolismo , Receptores de Trombina/metabolismo , beta-Arrestinas/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Motivos de Aminoácidos , Animais , Células HeLa , Humanos , Megacariócitos/citologia , Camundongos , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Trombina/genética , beta-Arrestinas/genéticaRESUMO
Thrombin, the key effector protease of the coagulation cascade, drives fibrin deposition and activates human platelets through protease-activated receptor-1 (PAR1). These processes are critical to the progression of thrombotic diseases. Thrombin is the main target of anticoagulant therapy, and major efforts have led to the discovery of new oral direct inhibitors of thrombin. Dabigatran is the first oral anticoagulant licensed for the prevention of thromboembolisms associated with orthopedic surgery and stroke prevention in atrial fibrillation. Dabigatran is a direct thrombin inhibitor that effectively blocks thrombin's catalytic activity but does not preclude thrombin's exosites and binding to fibrinogen. Thus, we hypothesized that catalytically inactive thrombin retains the capacity to bind to PAR1 through exosite-I and may modulate its function independent of receptor cleavage and activation. Here, we report that dabigatran at clinically relevant concentrations is an effective and acute inhibitor of thrombin-induced PAR1 cleavage, activation, internalization, and ß-arrestin recruitment in vitro. Interestingly, prolonged exposure to catalytic inactive thrombin incubated with dabigatran at 20-fold higher therapeutic concentration resulted in increased PAR1 cell-surface expression, which correlated with higher detectable levels of ubiquitinated receptor. These findings are consistent with ubiquitin function as a negative regulator of PAR1 constitutive internalization. Increased PAR1 expression also enhanced agonist-induced phosphoinositide hydrolysis and endothelial barrier permeability. Thus, catalytically inactive thrombin appears to modulate PAR1 function in vitro by stabilizing receptor cell-surface expression; but given the high clearance rate of thrombin, the high concentration of dabigatran required to achieve this effect the in vivo physiologic relevance is unknown.