Identification of exosite residues of factor Xa involved in recognition of PAR-2 on endothelial cells
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Published on: May 24, 2024
Chandrashekhara Manithody1, Likui Yang, Alireza R Rezaie
1Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, United States.
Abstract:
Recent results have indicated that factor Xa (FXa) cleaves protease-activated receptor 2 (PAR-2) to elicit protective intracellular signaling responses in endothelial cells. In this study, we investigated the molecular determinants of the specificity of FXa interaction with PAR-2 by monitoring the cleavage of PAR-2 by FXa in endothelial cells transiently transfected with a PAR-2 cleavage reporter construct in which the extracellular domain of the receptor was fused to cDNA encoding for alkaline phosphatase. Comparison of the cleavage efficiency of PAR-2 by a series of FXa mutants containing mutations in different surface loops indicated that the acidic residues of 39-loop (Glu-36, Glu-37, and Glu-39) and the basic residues of 60-loop (Lys-62 and Arg-63), 148-loop (Arg-143, Arg-150, and Arg-154), and 162-helix (Arg-165 and Lys-169) contribute to the specificity of receptor recognition by FXa on endothelial cells. This was evidenced by significantly reduced activity of mutants toward PAR-2 expressed on transfected cells. The extent of loss in the PAR-2 cleavage activity of FXa mutants correlated with the extent of loss in their PAR-2-dependent intracellular signaling activity. Further characterization of FXa mutants indicated that, with the exception of basic residues of 162-helix, which play a role in the recognition specificity of the prothrombinase complex, none of the surface loop residues under study makes a significant contribution to the activity of FXa in the prothrombinase complex. These results provide new insight into mechanisms through which FXa specifically interacts with its macromolecular substrates in the clotting and signaling pathways.
Insights
Factor Xa (FXa) specifically binds protease-activated receptor 2 (PAR-2) via surface loops, influencing intracellular signaling in endothelial cells. Mutations in these loops reduce FXa
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Factor Xa (FXa) cleaves protease-activated receptor 2 (PAR-2), initiating protective intracellular signals in endothelial cells.
- Understanding the molecular basis of FXa-PAR-2 interaction specificity is crucial for elucidating signaling pathways.
Purpose of the Study:
- To investigate the molecular determinants governing the specificity of Factor Xa (FXa) interaction with protease-activated receptor 2 (PAR-2).
- To identify specific residues and surface loops of FXa critical for PAR-2 recognition and cleavage in endothelial cells.
Main Methods:
- Utilized a PAR-2 cleavage reporter construct in endothelial cells, fusing the extracellular domain of PAR-2 to alkaline phosphatase.
- Generated and analyzed FXa mutants with alterations in surface loops (39-loop, 60-loop, 148-loop, 162-helix) to assess cleavage efficiency and signaling activity.
- Evaluated the impact of FXa mutations on prothrombinase complex activity.
Main Results:
- Acidic residues in the 39-loop and basic residues in the 60-loop, 148-loop, and 162-helix of FXa are critical for PAR-2 recognition specificity.
- Mutations in these loops significantly reduced FXa's ability to cleave PAR-2 and elicit downstream intracellular signaling.
- The identified surface loop residues, except for the 162-helix basic residues, did not significantly affect FXa's prothrombinase complex activity.
Conclusions:
- Specific surface loops and residues of FXa dictate its precise interaction with PAR-2 on endothelial cells, modulating signaling pathways.
- These findings offer novel insights into how FXa selectively binds its macromolecular substrates in both clotting and signaling contexts.
- The study highlights the distinct roles of FXa surface loops in substrate recognition for different biological functions.
