Lipid raft localization regulates the cleavage specificity of protease activated receptor 1 in endothelial cells.
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.


OBJECTIVE
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-alpha-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.
Bae
J-S
JS
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO 63104, USA.
Yang
L
L
Rezaie
A R
AR
D000469:Alkaline Phosphatase / Q000378:metabolism; D017209:Apoptosis; D001779:Blood Coagulation Factors / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D008055:Lipids / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry*; D008954:Models, Biological; D010539:Permeability; D011486:Protein C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D044463:Receptor, PAR-1 / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D001779:Blood Coagulation Factors; D008055:Lipids; D011486:Protein C; D044463:Receptor, PAR-1; D011956:Receptors, Cell Surface; C091963:activated protein C receptor; D000469:Alkaline Phosphatase; D013917:Thrombin