Doctor of Philosophy
Physiology and Pharmacology
Proteinase activated receptor 4 (PAR4) is a G-protein-coupled receptor with an important role in the platelet response to vascular injury. In platelets, PAR4 activation, by thrombin-cleavage of its N-terminus and unmasking of a tethered ligand, leads to G-protein- and beta-arrestin-mediated intracellular signalling pathways which result in platelet activation, shape change, and ultimately, platelet aggregation. As an important platelet thrombin receptor, PAR4 is an interesting target for the development of anti-platelet therapeutics. However, molecular determinants of PAR4 activation, signalling, and signal regulation remain poorly understood. In this thesis, mechanisms of PAR4 activation and signalling were studied through determination of the molecular basis of agonist binding at the extracellular surface of the receptor that result in G-protein signalling and beta-arrestin recruitment and evaluation of the role of Helix-8 and C-terminal tail residues on effector interaction and signalling. In our first series of experiments, we evaluated the impact of single amino acid substitutions to the parental PAR4 agonist peptide, AYPGKF-NH2, in an effort to understand the chemical characteristics enabling activity at the PAR4 receptor. We identified key residue characteristics contributing to agonist peptide activation of PAR4. We further showed that different chemical modifications affect G-protein and beta-arrestin signalling through PAR4, providing leads for further development of biased ligands of PAR4. Additionally, using in silico receptor modelling and peptide docking, we identify a key residue in the ECL2 of the receptor that is required for receptor activation by either peptide or enzyme-revealed tethered ligand. Subsequent studies evaluated the role of the Helix-8 and C-terminal tail motifs in PAR4 and the related receptor, PAR2, on intracellular effector interaction and signalling. PAR4 was compared to PAR2 since several key Class A GPCR regulatory sites are missing in PAR4 and are retained in PAR2. These studies have revealed residues and structural features that are involved in PAR4 interaction with G-proteins and beta-arrestin. Importantly, we also identified key differences in residues that are important for signalling following enzyme- versus peptide-activation of PARs. Taken together, this body of work enhances our understanding of how PAR4 engages and is activated by agonists to promote signalling and cellular function.
Summary for Lay Audience
This thesis studies the molecular basis of signalling from Proteinase activated Receptor-4 (PAR4), a thrombin-activated GPCR that is an important regulator of the innate immune response to injury or infection. When blood vessels become damaged, they release signals to communicate with blood cells, called platelets. Proteins expressed on the surface of the platelet, called receptors, receive the signals from the damaged blood vessel and signal platelets to become active and change shape – making them sticky and forming a blood clot. This is the first step in repairing a damaged vessel. Sometimes this clotting response becomes overactive when a person is at significant risk of heart attacks or stroke, thus, a lot of drug discovery efforts have gone into blocking this process. A group of these cellular receptors, called G-protein-coupled receptors (GPCRs), become activated by signals sent from the damaged blood vessel leading to platelets becoming activated. Thus, studies investigating how this process happens and how to therapeutically block it have been very important for drug development. In recent years, a GPCR named Proteinase Activated Receptor 4 (PAR4) has become an increasingly attractive target for blocking platelet activation. It is believed that blocking PAR4 activation will allow for appropriate blood clotting to occur through other receptors (such as PAR1) but will block the formation of larger, more dangerous, blood clots. Unfortunately, there is a lot we don’t know about how PAR4 becomes activated and how it communicates these signals in the platelet. In a series of studies, we investigated how the receptor becomes activated through drug binding (receiving the signal) and interaction with proteins inside the cell (how it communicates the signal into the cell). We have identified properties of drugs that can selectively target PAR4. We have also identified key parts of the PAR4 receptor that control PAR4 signalling. Together, these studies uncover new insights into PAR4 signalling and aid development of therapies targeting PAR4.
Thibeault, Pierre E., "Molecular Mechanisms Regulating the Platelet Thrombin Receptor PAR4" (2020). Electronic Thesis and Dissertation Repository. 7479.
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Available for download on Saturday, January 01, 2022