Skip to Main Content
 

Global Search Box

 
 
 

ETD Abstract Container

Abstract Header

The Tethered Ligand Activation Mechanism of Protease-Activated Receptor 4

Han, Xu
http://orcid.org/0000-0002-1977-1046
http://rave.ohiolink.edu/etdc/view?acc_num=case1619211697957082

Abstract Details

2021, Doctor of Philosophy, Case Western Reserve University, Pharmacology.
Protease-activated receptors (PARs) are G-protein coupled receptors (GPCRs) that have a unique activation mechanism. Unlike other GPCRs that are activated by free ligands, PARs are activated by a tethered ligand, which is a part of their N-terminus that is unmasked by proteolysis. The fourth member of the family, PAR4 mediates sustained thrombin signaling in platelets and other cells. The structural basis for PAR4 activation and the location of its ligand-binding site (LBS) are unknown. Using hydrogen/deuterium exchange (HDX), computational modeling and signaling studies, I determined the molecular mechanism for tethered ligand-mediated PAR4 activation. HDX identified that the LBS is composed by transmembrane domain 3 (TM3) and TM7. Unbiased computational modeling and signaling studies further determined Thr153 from the LBS was critical for the receptor function. HDX and modeling also showed that extracellular loop 3 (ECL3) serves as a gatekeeper for the interaction between the tethered ligand and LBS. A naturally occurring sequence variant (P310L, rs2227376) and two experimental mutations (S311A and P312L) determined the rigidity conferred by prolines in ECL3 are essential for PAR4 activation. Next, the role of the polymorphism at position 310 in venous thromboembolism (VTE) was examined using the INVENT consortium multi-ancestry GWAS meta-analysis. Individuals with the PAR4 Leu310 allele had a 15% relative risk reduction for VTE compared to the Pro310 allele. Finally, to determine how the hypo-reactive PAR4 sequence variant in ECL3 impacts platelet function, a point mutation was introduced into the mouse PAR4 gene, F2rl3, via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by measuring αIIbβ3 integrin activation and P-selectin surface expression. P322L-PAR4 greatly reduced platelet responsiveness to AYPGKF and physiological-dose of thrombin. The response to ADP and convulxin was unchanged among genotypes. In conclusion, I have uncovered the structural basis for PAR4 activation and identified a previously unrecognized association of for PAR4 in VTE. Our new mouse model provides a great foundation to further evaluate the role of PAR4 in VTE development and other diseases.
Marvin Nieman (Advisor)
Derek Taylor (Committee Chair)
Ruth Keri (Committee Member)
Evi Stavrou (Committee Member)
Beata Jastrzebska (Committee Member)
242 p.
Biomedical Research; Pharmacology
Protease-activated receptors, PAR, Thrombin, Amide-HDX, Platelets, Thrombosis and Hemostasis

Recommended Citations

Citations

  • Han, X. (2021). The Tethered Ligand Activation Mechanism of Protease-Activated Receptor 4 [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1619211697957082

    APA Style (7th edition)

  • Han, Xu. The Tethered Ligand Activation Mechanism of Protease-Activated Receptor 4 . 2021. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1619211697957082.

    MLA Style (8th edition)

  • Han, Xu. "The Tethered Ligand Activation Mechanism of Protease-Activated Receptor 4 ." Doctoral dissertation, Case Western Reserve University, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1619211697957082

    Chicago Manual of Style (17th edition)

case1619211697957082
68
© 2021, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.
Release 3.2.12