Protease-activated receptor-1 cleaved at R46 mediates cytoprotective effects
| Autor: | Miriam Ender, Matthias Riewald, Paolo Galli, Jerzy Madon, Reto A. Schuepbach |
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| Přispěvatelé: | University of Zurich, Schuepbach, R A |
| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
Indazoles
Protein Conformation Molecular Sequence Data 2720 Hematology 610 Medicine & health 030204 cardiovascular system & hematology Cleavage (embryo) Arginine Transfection Article Antibodies 03 medical and health sciences Structure-Activity Relationship 0302 clinical medicine Thrombin medicine Staurosporine Humans Urea Receptor PAR-1 Amino Acid Sequence Binding site 030304 developmental biology 0303 health sciences Chemistry HEK 293 cells Endothelial Cells Hematology Molecular biology Peptide Fragments 3. Good health Cell biology Protease-Activated Receptor 1 HEK293 Cells 10022 Division of Surgical Research Cytoprotection cardiovascular system RNA Interference Receptors Thrombin Binding Sites Antibody Signal transduction 10023 Institute of Intensive Care Medicine medicine.drug Protein C Signal Transduction |
| Zdroj: | Journal of thrombosis and haemostasis : JTH |
| Popis: | Summary. Background: Activated protein C (aPC) mediates powerful cytoprotective effects through the protease-activated receptor-1 (PAR1) that translate into reduced harm in mouse injury models. However, it remains elusive how aPC-activated PAR1 can mediate cytoprotective effects whereas thrombin activation does the opposite. Objectives: We hypothesized that aPC and thrombin might induce distinct active conformations in PAR1 causing opposing effects. Methods: We analyzed antibody binding to, and cleavage and signalling of PAR1 in either endogenously expressing endothelial or overexpressing 293T cells. Results: In thrombin-cleaved PAR1 neither the tethered ligand nor the hirudin-like domain were available for anti-PAR1 ATAP2 and WEDE15 binding unless the tethered ligand was quenched. In contrast, aPC irreversibly prevented ATAP2 binding while not affecting WEDE15 binding. Reporter constructs with selective glutamine substitutions confirmed R41 as the only thrombin cleavage site in PAR1, whereas aPC preferentially cleaved at R46. Similarly, we report distinct cleavage sites on PAR3, K38 for thrombin and R41 for aPC. A soluble peptide corresponding to R46-cleaved PAR1 enhanced the endothelial barrier function and reduced staurosporine toxicity in endothelial as well as in 293T cells if PAR1 was expressed. Overexpression of PAR1 variants demonstrated that cleavage at R46 but not R41 is required for cytoprotective aPC signaling. Conclusions: We provide a novel concept on how aPC and thrombin mediate distinct effects. We propose that the enzyme-specific cleavage sites induce specific conformations which mediate divergent downstream effects. This unexpected model of PAR1 signaling might lead to novel therapeutic options for the treatment of inflammatory diseases. |
| Databáze: | OpenAIRE |
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