Proteinase-mediated signaling: Proteinase-activated receptors (PARs) and much more
Introduction
The discovery of the G-protein coupled proteinase-activated receptor (PAR) family, activated by the proteolytic unmasking of a receptor-tethered ligand (Fig. 1), represents one of the more intriguing signal transduction stories elucidated over the past decade or so. A search for the cell surface receptor responsible for thrombin signaling led two groups independently to clone a G-protein coupled receptor responsible for thrombin-mediated calcium signaling and platelet aggregation Rasmussen et al., 1991, Vu et al., 1991. The unique feature of the first receptor discovered to be responsible for these actions of thrombin (now termed proteinase activated receptor-1 or PAR1), relates to the cleavage by thrombin at arginine 41 of the amino-terminal domain of the human receptor, to reveal a sequence: SFLLRNPN…., that remaining tethered, then binds to and activates the receptor, as depicted in Fig. 1A. A second novel feature discovered about this receptor family by the Coughlin laboratory, as illustrated in Fig. 1B, was that without proteolytic cleavage, the receptor can nonetheless be activated by short peptides (PAR-activating peptides, or PAR-APs) with sequences modeled on the revealed ‘tethered ligand’ (Vu et al., 1991). Cloning of the PAR1 receptor for thrombin has led ultimately to the discovery of three other members of this novel receptor family, PARs 2, 3, and 4 (summarized by Coughlin, 2000, Dery et al., 1998, Macfarlane et al., 2001, Hollenberg and Compton, 2002). Work on the pharmacology of the PAR-activating peptides has led to the synthesis of PAR-selective receptor-activating agonists. By using the peptide, TFLLR-amide for activating PAR1 and SLIGRL-amide to activate PAR2 it has been possible to evaluate the impact of activating these PARs in cell, tissue or intact animals to determine the potential actions that the PAR-activating enzymes might have. Use of the PAR-APs eliminates the confounding effects that the proteinases could potentially have by mechanisms other than via the PARs. One enigma that remains relates to the third member of the family, PAR3. Unlike other family members, PAR3 does not appear to signal in response to its cognate activating peptide, but rather appears to function as a co-factor for the activation of PAR4 Nakanishi-Matsui et al., 2000, Sambrano et al., 2001. However, the independent differential expression of PAR3 and PAR4 in different tissues raises the possibility that PAR3 may play a functional role that has yet to be determined.
Section snippets
Receptor-activating peptides as probes for par function
Once receptor-selective PAR agonists became available, it was possible to use these compounds as probes for receptor function in a variety of settings, both in vitro and in vivo. The selective agonists that have proved of particular value are: TFLLR-amide, for PAR1, SLIGRL-amide, for PAR2 and AYPGKF-amide, for PAR4. As mentioned above, PAR3 has been found not to signal on its own. Since the thrombin-revealed sequences of PAR3 (TFRGAP, for human; SFNGGP for murine) predict an activation of
Actions of par-activating peptides via receptors other than the PARs
The above paragraphs illustrate well, the utility of the PAR-activating peptides as probes to evaluate the possible pathophysiological roles of the receptors. That said, it must be pointed out that these peptides are active in the concentration range from 10 to 100 μM, such that an interaction with receptors other than the PARs is feasible and must be ruled out if possible. The most straightforward way to assess the validity of the effects of the peptides as surrogate PAR activators is to
Proteinase signalling via mechanisms other than PARs
Early work using a rat diaphragm bioassay for the action of insulin revealed that the proteolytic enzymes, pepsin and pepsinogen exhibit insulin-like activity (Rieser, 1967), as does trypsin (summarized by Hollenberg, 1996). This insulin-like action of trypsin can be attributed to the cleavage from the insulin receptor of a domain in its α-subunit that represses the receptor's tyrosine kinase activity and that participates in the binding of insulin (Shoelson et al., 1988). It is probable that
Conclusions and future directions
The information summarized in the sections above cast in a new light, the potential signalling properties that can be ascribed to proteinases. Clearly, enzymes like trypsin and thrombin can in a way join the ranks of other hormonal regulators like insulin and adrenaline. It is well recognized that the synthesis of thrombin and members of the trypsin family can occur at sites as various as those that harbor the PARs. Further, endogenous proteinase inhibitors, like the serpins, may be found to
Acknowledgements
Work in the author's laboratory is aided substantially by two group grants from the Canadian Institutes of Health Research (CIHR): The Proteinases and Inflammation Network (PAIN) and the Group on the Regulation of Vascular Contractility. In addition, essential funds for the author's work referenced in this article have come from operating grants provided by the Canadian Institutes of Health Research, from project grants provided by the Kidney Foundation of Canada, the Heart and Stroke
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