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Lipid raft localization regulates the cleavage specificity of protease activated receptor 1 in endothelial cells
Author(s) -
BAE J.S.,
YANG L.,
REZAIE A. R.
Publication year - 2008
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2008.02924.x
Subject(s) - lipid raft , microbiology and biotechnology , proteases , transfection , chemistry , thrombin , cleavage (geology) , endothelial protein c receptor , biology , signal transduction , biochemistry , enzyme , paleontology , platelet , fracture (geology) , gene , immunology
Summary. Background: The endothelial protein C receptor (EPCR)‐dependent cleavage of protease activated receptor 1 (PAR‐1) by either activated protein C (APC) or thrombin in lipid rafts initiates protective signaling responses in endothelial cells. Objectives: To investigate the mechanism by which APC and thrombin interact with and cleave PAR‐1 in lipid rafts. Methods: We constructed two types of PAR‐1 cleavage reporter constructs in which a secreted alkaline phosphatase (ALP) was fused to the extracellular domain of PAR‐1. The first construct has a transmembrane domain capable of uniformly anchoring the fusion protein to the membrane surface, while the second construct has the recognition sequence for targeting the fusion protein to lipid rafts/caveolae in transfected cells. Results: Both APC and the Gla‐domainless (GD)‐APC cleaved the PAR‐1 exodomain with similar efficiency in HUVECs transfected with the first construct. Unlike APC, GD‐APC did not cleave PAR‐1 in cells transfected with the second construct; however, prior treatment of cells with S195A mutants of either protein C or thrombin led to the GD‐APC cleavage of PAR‐1 with a comparable or higher catalytic efficiency. The same results were obtained if the cellular signaling properties of APC and GD‐APC were monitored in the TNF‐α‐induced endothelial cell apoptosis and permeability assays. Conclusions: The lipid raft localization renders the scissile bond of the PAR‐1 exodomain unavailable for interaction with coagulation proteases. The binding of either the Gla‐domain of protein C to EPCR or exosite‐1 of thrombin to the C‐terminal hirudin‐like sequence of PAR‐1 changes the membrane localization and/or the conformation of the PAR‐1 exodomain to facilitate its recognition and subsequent cleavage by these proteases.