The interaction between the endothelial cell protein C receptor and protein C is dictated by the gamma-carboxyglutamic acid domain of protein C.
The endothelial cell protein C receptor (EPCR) binds to both protein C and activated protein C (APC) with similar affinity. Removal of the Gla domain of protein C results in the loss of most of the binding affinity. This observation is compatible with at least two models: 1) the Gla domain of protein C interacts with phospholipid on cell surfaces to stabilize interaction with EPCR or 2) the Gla domain of protein C makes specific protein-protein interactions with EPCR. The latter model predicts that chimeric proteins containing the protein C Gla domain should interact with EPCR. To test this, we constructed a prothrombin chimera in which the Gla domain and aromatic stack of prothrombin were replaced with the corresponding region of protein C. The 125I-labeled chimera (Kd = 176 nM) and 125I-APC (Kd = 65 nM) both bound specifically to 293 cells stably transfected with EPCR, but both bound poorly to sham-transfected cells. The chimera also blocked APC binding to EPCR-transfected cells in a dose-dependent fashion (Ki approximately 139 nM) similarly to protein C (Ki approximately 75 nM). Chimera binding to EPCR-transfected cells was blocked by soluble EPCR, demonstrating direct protein-protein interaction between the chimera and EPCR. Consistent with this conclusion, the isolated Gla domain of protein C blocked APC binding to EPCR-transfected cells (IC50 = 2 microM). No inhibition was observed with the isolated prothrombin Gla domain. A protein C chimera with the prothrombin Gla domain and aromatic stack failed to bind to EPCR detectably. These data suggest that the Gla domain of protein C is responsible for much of the binding energy and specificity of the protein C-EPCR interaction.
Regan
L M
LM
Howard Hughes Medical Institute, Oklahoma City, Oklahoma 73104, USA.
Mollica
J S
JS
Rezaie
A R
AR
Esmon
C T
CT
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D001779:Blood Coagulation Factors; D002460:Cell Line; D006801:Humans; D007457:Iodine Radioisotopes; D011485:Protein Binding; D011486:Protein C / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*
D001779:Blood Coagulation Factors; D007457:Iodine Radioisotopes; D011486:Protein C; D011956:Receptors, Cell Surface; C091963:activated protein C receptor; D015055:1-Carboxyglutamic Acid