Proteolysis of the Exodomain of Recombinant Protease-Activated Receptors:  Prediction of Receptor Activation or Inactivation by MALDI Mass Spectrometry

Abstract

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce activation of platelets and vascular cells, while PAR2 is preferentially cleaved by trypsin. In pathological situations, other proteolytic enzymes may be generated in the circulation and could modify the responses of PARs by cleaving their extracellular domains. To assess the ability of such proteases to activate or inactivate PARs, we designed a strategy for locating cleavage sites on the exofacial NH(2)-terminal fragments of the receptors. The first extracellular segments of PAR1 (PAR1E) and PAR2 (PAR2E) expressed as recombinant proteins in Escherichia coli were incubated with a series of proteases likely to be encountered in the circulation during thrombosis or inflammation. Kinetic and dose-response studies were performed, and the cleavage products were analyzed by MALDI-TOF mass spectrometry. Thrombin cleaved PAR1E at the Arg41-Ser42 activation site at concentrations known to induce cellular activation, supporting a native conformation of the recombinant polypeptide. Plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3 cleaved at multiple sites and would be expected to disable PAR1 by cleaving COOH-terminal to the activation site. Cleavage specificities were further confirmed using activation site defective PAR1E S42P mutant polypeptides. Surface plasmon resonance studies on immobilized PAR1E or PAR1E S42P were consistent with cleavage results obtained in solution and allowed us to determine affinities of PAR1E-thrombin binding. FACS analyses of intact platelets confirmed the cleavage of PAR1 downstream of the Arg41-Ser42 site. Mass spectrometry studies of PAR2E predicted activation of PAR2 by trypsin through cleavage at the Arg36-Ser37 site, no effect of thrombin, and inactivation of the receptor by plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3. The inhibitory effect of elastase was confirmed on native PAR1 and PAR2 on the basis of Ca(2+) signaling studies in endothelial cells. It was concluded that none of the main proteases generated during fibrinolysis or inflammation appears to be able to signal through PAR1 or PAR2. This strategy provides results which can be extended to the native receptor to predict its activation or inactivation, and it could likewise be used to study other PARs or protease-dependent processes.

N-Linked Glycosylation Regulates Human Proteinase-activated Receptor-1 Cell Surface Expression and Disarming via Neutrophil Proteinases and Thermolysin

“…In addition, our data also revealed that cathepsin G at 100 nM disarms N62Q and N75Q. Like us, others have also reported that cathepsin G at 30 nM could disarm PAR 1 (30 (4) showed that the preferential cathepsin G cleavage site for PAR 1 is the Phe 55 -Trp 56 site. Moreover, our finding with the cathepsin G mutant is in accord with a previous report that has demonstrated that Phe 55 -Trp 56 is the main region cleaved within the receptor N terminus by cathepsin G (12).…”

“…This just confirmed that trypsin disarms the mutant receptor, and the disarmed mutant receptors still remained on the cell surface but could not be activated by thrombin anymore because the tethered ligand had been removed; however, TFLLR will still activate those mutant receptors by binding to receptor ECL2. Trypsin can activate PAR 1 by cleaving at the activating cleavage site Arg 41 -Ser 42 to expose the tethered ligand (2,4,29). Moreover, trypsin has also been reported to cleave PAR 1 at residues Arg 70 -Leu 71 and Lys 82 -Gln 83 , which amputates the tethered ligand from the receptor (4) (Fig.…”

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

“…Because elastase cleaves PAR 2 distal to the trypsin cleavage site, elastase pretreatment can prevent subsequent activation by trypsin, and thereby "disarm" the receptor (49,56). We found that pretreatment with elastase prevented trypsin signals in oocytes, which confirms this mechanism.…”

“…Elastase cleavage of PAR 2 was first reported in vitro in Escherichia coli expressing the N-terminal domain of the receptor (56). Because elastase cleaves PAR 2 distal to the trypsin cleavage site, elastase pretreatment can prevent subsequent activation by trypsin, and thereby "disarm" the receptor (49,56).…”

The Roles of Proteinase-Activated Receptors in the Vascular Physiology and Pathophysiology

“…2,[25][26][27][28] Plasmin either activates or disarms PAR1, 6,29,30 whereas it inactivates PAR2 and activates PAR4. 31,32 The activated protein C (APC) has been shown to activate PAR1 and PAR2, [33][34][35] although its functional role still remains controversial. 36 In the following sections, I would like to discuss the role of PARs in regulating the vascular functions and in the pathophysiology of vascular diseases, with some focus on thrombin and its major receptor PAR1.…”