Expression and purification of Protease Activated Receptor 4 (PAR4) and analysis with histidine hydrogen deuterium exchange.

Abstract

Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that are activated by proteolyis of the N-terminus, which exposes a tethered ligand that interacts with the receptor. Numerous studies have focused on the signaling pathways mediated by PARs. However, the structural basis for initiation of these pathways is unknown. Here, we describe a strategy for the expression and purification of PAR4. This is the first PAR family member to be isolated without stabilizing modifications for biophysical studies. We monitored PAR4 activation with histidine-hydrogen deuterium exchange (His-HDX). PAR4 has 9 histidines that are spaced throughout the protein allowing a global view of solvent accessible and non-accessible regions. Peptides containing each of the 9 His residues were used to determine the t1/2 for each His residue in apo or thrombin activated PAR4. 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 decreased solvent accessibility upon thrombin treatment. In agreement, thrombin cleaved PAR4 also was resistant to thermolysin digestion. In contrast, activation with the PAR4 agonist peptide was digested at the same rate as apo PAR4. Further analysis showed the C-terminus is protected in thrombin activated PAR4 compared to uncleaved or agonist peptide treated PAR4. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member using biophysically and shed light on the overall conformational changes that follow activation of PARs by a protease.