Expression and Purification of Protease-Activated Receptor 4 (PAR4) and Analysis with Histidine Hydrogen-Deuterium Exchange.

Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolysis 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. PAR4 has nine histidines that are spaced throughout the protein, allowing a global view of solvent accessible and nonaccessible regions. Peptides containing each of the nine His residues were used to determine the t1/2 for each His residue in apo or thrombin-activated PAR4. The thrombin-cleaved PAR4 exhibited 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, the rate of thermolysin proteolysis following stimulation with the PAR4 activation peptide was the same as that of unstimulated 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 biophysically and shed light on the overall conformational changes that follow activation of PARs by a protease.

Identification and expression patterns of members of the protease-activated receptor (PAR) gene family during zebrafish development.

Protease-activated receptors (PARs) play critical roles in hemostasis in vertebrates including zebrafish. However, the zebrafish gene classification appears to be complex, and the expression patterns of par genes are not established. Based on analyses of genomic organization, phylogenetics, protein primary structure, and protein internalization, we report the identification of four zebrafish PARs: par1, par2a, par2b, and par3. This classification differs from one reported previously. We also show that these genes have distinct spatiotemporal expression profiles in embryos and larvae, with par1, par2a, and par2b expressed maternally and ubiquitously during gastrula stages and their expression patterns refined at later stages, and par3 expressed only in 3-day-old larvae. Notably, the expression patterns of zebrafish par1 and par2b resemble those of their mammalian counterparts, suggesting that receptor function is conserved among vertebrates. This conservation is supported by our findings that Par1 and Par2b are internalized following exposure to thrombin and trypsin, respectively.

Expression and purification of the human thrombin receptor.

The human thrombin receptor has been overexpressed in Sf9 (Spodoptera frugiperda) insect cells using a baculovirus vector. Cell surface expression of the receptor was confirmed by immunocytochemistry with polyclonal antibodies raised against the extracellular domain of the receptor. The expressed receptor was functional; both thrombin and the thrombin receptor agonist peptide produced increases in intracellular calcium in transfected cells. The concentration of thrombin causing the half-maximal increase (EC50) in intracellular calcium was 3.9 nM, whereas the EC50 for the agonist peptide was 2.7 microM. However, the observed maximum increase in intracellular calcium concentration with the agonist peptide (547 nM) was twofold greater than that observed with thrombin (258 nM). The recombinant receptor was purified by immunoaffinity chromatography using a monoclonal antibody raised against the receptor extracellular domain. The purified preparation contained two species with apparent molecular masses of 48 and 90 kDa, both of which were recognized by mono- and polyclonal antibodies against the thrombin receptor. The yield of the purified receptor was 0.78 mg/liter of insect cells suspension culture (10(6) cells/ml). The purified thrombin receptor will be useful in future structural and functional studies.

Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2.

We previously reported the molecular cloning of a mouse guanosine-nucleotide-binding-protein-coupled receptor similar to the thrombin receptor. Since the physiological agonist was unknown, the receptor was named proteinase-activated receptor 2. We describe here the cloning and functional expression of the gene encoding the corresponding human receptor. The gene is divided into two exons separated by about 14 kb intronic DNA. The deduced protein sequence is 397 amino acids long and 83% identical to the mouse receptor sequence. Within the extracellular amino terminus, the residues predicted to form the tethered agonist ligand differ between the two receptors; of the first six residues only four are conserved. At positions five and six, a lysine residue and a valine residue, respectively, have replaced arginine and leucine residues found in the mouse sequence. When the human receptor is expressed in Chinese hamster ovary cells, it can be activated by low nanomolar concentrations of the serine proteinase trypsin and by peptides made from the receptor sequence. Northern-blot analysis of receptor expression showed that the receptor transcript is widely expressed in human tissues with especially high levels in pancreas, liver, kidney, small intestine and colon. Moderate expression was detected in many organs but none in brain or skeletal muscle. By fluorescence in situ hybridization, the human proteinase-activated receptor 2 gene was mapped to chromosomal region 5q13, where, previously, the related thrombin receptor gene has been located.