Analysis of Protease Activated Receptor 4 (PAR4) By Histidine Hydrogen Deuterium Exchange
Authors: Nieman Marvin T. , de la Fuente Maria , Miyagi Masaru 
Published: October 2019

Abstract
Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that have central roles in the cardiovascular system. Numerous studies have focused on the signaling pathways mediated by PARs; however, the structural rearrangements that initiate these pathways are unknown. PARs are activated when proteolytic cleavage exposes a tethered ligand that interacts with a binding pocket on the receptor. Since the ligand is attached to the receptor, there are likely substantial conformational changes that occur during PAR activation. We now describe the first biophysical studies examining this unique mode of activation using PAR4. PAR4 is one of the thrombin receptors on platelets and is required for sustained platelet activation. Recent data from our laboratory and others suggest that PAR4 has the potential to be a therapeutic target. To examine the tethered ligand activation mechanism in general and further examine PAR4 as a target, we have expressed and purified PAR4 that does not contain additional stabilizing sequences from Sf9 cells for biophysical studies. The current study uses histidine-hydrogen deuterium exchange (His-HDX). His-HDX is a structural mass spectrometry technique that relies on the slow exchange of the C2 carbon on the imidazole ring of histidine. PAR4 has nine histidine residues that are well spaced throughout the protein, which gives us a global view of solvent accessible and non-accessible regions. Unique peptides were generated that contained each of the histidine residues (His136, His159, His180, His229, His240, His269, His280, His306, and His380). These peptides were used to determine the t1/2 for each His residue in full length or thrombin cleaved PAR4. The t1/2 values for His159 and His269 were greater than 40 hours in both states indicating that these two histidine residues are not exposed to solvent in either case. In contrast, the thrombin cleaved PAR4 had a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380) demonstrating that these regions have a decrease in solvent accessibility upon thrombin treatment. These residues are spread throughout the protein, which indicates that PAR4 undergoes a global conformational change and the overall structure becomes more rigid. In agreement with these data, thrombin cleaved PAR4 is more resistant to protease digestion by chymotrypsin. The half-life was extended 4-fold over the uncleaved PAR4. We are also able to make conclusions regarding specific regions of PAR4. His229 is located in extracellular loop 2 near the proposed ligand-binding site and has a 2-fold increase in t1/2 following thrombin treatment. These data suggest that the tethered ligand may be binding to this region and protecting it from exchange. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member using biophysical approaches. These studies shed light on the overall conformational changes that follow activation of PARs by a protease. Finally, the His-HDX used for purified PAR4 lays important groundwork that will allow us to examine PAR4 and other platelet GPCRs in their native membrane environment using this and related mass spectrometry approaches.

Disclosures
No relevant conflicts of interest to declare.