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1.
Proc Natl Acad Sci U S A ; 118(49)2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34873055

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/genética
2.
RNA ; 26(10): 1464-1480, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32631843

RESUMO

Many eukaryotes use RNA processing, including alternative splicing, to express multiple gene products from the same gene. The budding yeast Saccharomyces cerevisiae has been successfully used to study the mechanism of splicing and the splicing machinery, but alternative splicing in yeast is relatively rare and has not been extensively studied. Alternative splicing of SKI7/HBS1 is widely conserved, but yeast and a few other eukaryotes have replaced this one alternatively spliced gene with a pair of duplicated, unspliced genes as part of a whole genome doubling (WGD). We show that other examples of alternative splicing known to have functional consequences are widely conserved within Saccharomycotina. A common mechanism by which alternative splicing has disappeared is by replacement of an alternatively spliced gene with duplicate unspliced genes. This loss of alternative splicing does not always take place soon after duplication, but can take place after sufficient time has elapsed for speciation. Saccharomycetaceae that diverged before WGD use alternative splicing more frequently than S. cerevisiae, suggesting that WGD is a major reason for infrequent alternative splicing in yeast. We anticipate that WGDs in other lineages may have had the same effect. Having observed that two functionally distinct splice-isoforms are often replaced by duplicated genes allowed us to reverse the reasoning. We thereby identify several splice isoforms that are likely to produce two functionally distinct proteins because we find them replaced by duplicated genes in related species. We also identify some alternative splicing events that are not conserved in closely related species and unlikely to produce functionally distinct proteins.


Assuntos
Processamento Alternativo/genética , Proteoma/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Evolução Molecular , Duplicação Gênica/genética , Genoma/genética , Isoformas de Proteínas/genética
3.
J Biol Chem ; 291(35): 18453-64, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27402844

RESUMO

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/genética
4.
J Biol Chem ; 291(5): 2223-36, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26635365

RESUMO

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that undergoes proteolytic irreversible activation by coagulant and anti-coagulant proteases. Given the irreversible activation of PAR1, signaling by the receptor is tightly regulated through desensitization and intracellular trafficking. PAR1 displays both constitutive and agonist-induced internalization. Constitutive internalization of PAR1 is important for generating an internal pool of naïve receptors that replenish the cell surface and facilitate resensitization, whereas agonist-induced internalization of PAR1 is critical for terminating G protein signaling. We showed that PAR1 constitutive internalization is mediated by the adaptor protein complex-2 (AP-2), whereas AP-2 and epsin control agonist-induced PAR1 internalization. However, the mechanisms that regulate PAR1 recycling are not known. In the present study we screened a siRNA library of 140 different membrane trafficking proteins to identify key regulators of PAR1 intracellular trafficking. In addition to known mediators of PAR1 endocytosis, we identified Rab11B as a critical regulator of PAR1 trafficking. We found that siRNA-mediated depletion of Rab11B and not Rab11A blocks PAR1 recycling, which enhanced receptor lysosomal degradation. Although Rab11A is not required for PAR1 recycling, depletion of Rab11A resulted in intracellular accumulation of PAR1 through disruption of basal lysosomal degradation of the receptor. Moreover, enhanced degradation of PAR1 observed in Rab11B-deficient cells is blocked by depletion of Rab11A and the autophagy related-5 protein, suggesting that PAR1 is shuttled to an autophagic degradation pathway in the absence of Rab11B recycling. Together these findings suggest that Rab11A and Rab11B differentially regulate intracellular trafficking of PAR1 through distinct endosomal sorting mechanisms.


Assuntos
Endossomos/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica , Receptor PAR-1/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Autofagia , Biotinilação , Membrana Celular/metabolismo , Ensaio de Imunoadsorção Enzimática , Biblioteca Gênica , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Lisossomos/metabolismo , Microscopia de Fluorescência , Fagossomos/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Trombina/farmacologia
5.
Mol Pharmacol ; 88(1): 95-105, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25934730

RESUMO

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.


Assuntos
Antitrombinas/farmacologia , Arrestinas/metabolismo , Benzimidazóis/farmacologia , Receptor PAR-1/metabolismo , Trombina/metabolismo , beta-Alanina/análogos & derivados , Dabigatrana , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , beta-Alanina/farmacologia , beta-Arrestinas
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