The Interaction between the Endothelial Cell Protein C Receptor and Protein C Is Dictated by the γ-Carboxyglutamic Acid Domain of Protein C

• Published in: The Journal of biological chemistry Vol. 272; no. 42; p. 26279
• Main Authors: Lisa M. Regan, Jeffery S. Mollica, Alireza R. Rezaie, Charles T. Esmon
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 17-10-1997
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.42.26279

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 125 I-labeled chimera ( K
d = 176 n m ) and 125 I-APC ( K
d = 65 n m ) 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 ( K
i ≈ 139 n m ) similarly to protein C ( K
i ≈ 75 n m ). 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 (IC 50 = 2 μ m ). 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.