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Proteinase-mediated signaling: Proteinase-activated receptors (PARs) and much more
Life Sciences 74 (2003) 237 – 246 www.elsevier.com/locate/lifescie
Proteinase-mediated signaling: Proteinase-activated receptors (PARs) and much more Morley D. Hollenberg * Department of Pharmacology and Therapeutics, University of Calgary Faculty of Medicine, 3330 Hospital Drive North West, Calgary, AB, Canada T2N 4N1
Abstract Quite apart from their ability to generate active polypeptides from hormone precursors and to function as digestive enzymes, proteinases are now known to play a hormone-like role by triggering signal transduction pathways in target cells. The best understood example of proteinase-mediated signaling can be seen in the action of thrombin, which in addition to triggering the coagulation cascade, regulates platelet and endothelial cell function via its serine proteinase activity. The discovery of the G-protein-coupled ‘receptor’ responsible for these cellular actions of thrombin (Proteinase-activated Receptor-1, or PAR1) represents one of the more intriguing signal transduction stories elucidated over past decade or so. It is the objective of this brief review to provide an overview of the discovery and molecular pharmacology of the PAR family and to indicate the widespread roles these receptor systems can play in a variety of tissues. Further, the article (1) illustrates the utility of employing receptorselective PAR-activating peptides to determine the potential physiological roles these receptors play in vivo and (2) describes how these agonists have identified receptors other than the PARs. Finally, the mechanisms other than via the PARs by which proteinases can generate cellular signals are summarized. D 2003 Published by Elsevier Inc. Keywords: Proteinase; Thrombin; Trypsin; PAR; signaling
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 * Tel.: +1-403-220-6931; fax: +1-403-270-0979. E-mail address: [email protected] (M.D. Hollenberg). 0024-3205/$ - see front matter D 2003 Published by Elsevier Inc. doi:10.1016/j.lfs.2003.09.010
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Fig. 1. Schematic model of the activation of human proteinase-activated receptor 1 (PAR1) by thrombin and by a receptorselective PAR-activating peptide. The receptor is depicted as a putative 7-transmembrane structure, with an N-terminal domain containing the cryptic receptor-activating sequence, SFLLRNP. . ., preceded by the thrombin target motif, LDPR. The putative ‘fourth’ intracellular loop of the receptor, formed by palmitoylation of the intracellular C-terminal tail, is not shown. (A) Upon cleavage by thrombin (scissors), the revealed sequence, SFLLRNP, as a ‘tethered ligand’ is believed to dock at least in part with extracellular domain-2 to trigger a receptor-driven signal (arrow). (B) In the absence of thrombin cleavage, a receptorselective PAR1-activating peptide, TFLLR-amide is shown to mimic the tethered ligand, thereby generating an intracellular signal.
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
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the pharmacology of the PAR-activating peptides has led to the synthesis of PAR-selective receptoractivating 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 (NakanishiMatsui 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.
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 AYPGKFamide, for PAR4. As mentioned above, PAR3 has been found not to signal on its own. Since the thrombinrevealed sequences of PAR3 (TFRGAP, for human; SFNGGP for murine) predict an activation of either PAR2 (TFRGAP) or both PAR1 and PAR2 (SFNGGP), the use of the putative PAR3-activating peptides would unfortunately yield misleading and inaccurate results in tissue or cell systems. Nonetheless, with the use of the selective PAR1-activating probes, it has become clear that, as expected, PAR1 is a key regulator of human platelet aggregation and secretion as well as of endothelial cell function (Coughlin, 2000). In vascular tissue, PAR1 activation can lead to an endothelium-dependent nitric oxide mediated vasodilatation in conduit vessels, like the rat aorta (Muramatsu et al., 1992; Hollenberg et al., 1997). Alternatively, in rat aorta tissue, activation of PAR1 in an endothelium-free preparation can cause a calcium-dependent contractile response (Laniyonu and Hollenberg, 1995). What might not have been anticipated is that PAR1 can also play an important role in embryonic development via its impact on endothelial cell function (Griffin et al., 2001). PAR1 is also of importance in the re-stenosis process after vascular injury (Andrade-Gordon et al., 2001), very likely due to the activation of PAR1 in both the vascular endothelial and smooth muscle cells. Since PAR1 is widely distributed in a variety of tissues (Table 1) it is not surprising that the use of the receptor-activating probes has revealed a multitude of effects of PAR1 activation in sites that range from the vasculature, as already described, to the brain, lung and gastrointestinal tract. As well as having cardiovascular effects, PAR1 can also play an important role in causing a neurogenic inflammatory response (Cocks and Moffatt, 2000; de Garavilla et al., 2001; summarized by Macfarlane et al., 2001 and Vergnolle et al., 2001). In the setting of cancer, PAR1 appears to play an important role as a potential oncogene (Martin et al., 2001) and as a stimulus for cell invasion (Even-Ram et al., 1998; 2001; Henrikson et al., 1999; Kamath et al., 2001). Finally, it has unexpectedly turned out that PAR1 can play a role not only in a neurogenic inflammatory response, but also in promoting analgesia (Asfaha et al., 2002). Thus, there are many settings in which PAR1 may play a role, as revealed by the use of its receptor-selective agonist. PAR2, like PAR1 can also play a role in the cardiovascular system, as revealed by the use of its selective PAR-AP, SLIGRL-amide. One of the first effects described for PAR2 activation in a conduit
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Table 1 Localization and potential roles of proteinase-activated receptors PAR1
PAR2
PAR3
PAR4
Tissue distribution (Northern blot analysis)
Brain, lung, heart, stomach, colon, kidney, testis
Prostate, small intestine, colon, liver, kidney, pancreas, trachea
Heart, kidney, pancreas, thymus, small intestine, stomach, lymph node, trachea
Cellular expression
Platelets, endothelium, vascular smooth muscle, leukocytes, GI tract epithelium, fibroblasts, neurons, mast cells
Endothelium, leukocytes, GI tract epithelium and lung, airway and vascular smooth muscle, neurons, mast cells, keratinocytes, lung fibroblasts, renal tubular cells
Airway smooth muscle, platelets
Lung, pancreas, thyroid, testis, small intestine, placenta, skeletal muscle, lymph node, adrenal gland, prostate, uterus, colon Platelets, megakaryocytes
Known physiological role Potential physiological roles
Platelet activation
a
Regulation of vascular tone, Pro-inflammatory, modulator of nociception, embryonic development,
Platelet activation Regulation of vascular tone, pro-inflammatory, mediator of nociception, airway protection,
Cofactor for PAR4
Platelet activation
GI, gastrointestinal. a Information for PAR2, PAR3, and PAR4 is provided for human tissues; for PAR1, distribution is recorded in rat tissues, for which more extensive data are available than in human tissues. (adapted from Hollenberg and Compton, 2002, with permission).
vessel was the demonstration of an endothelium-dependent, nitric oxide-mediated relaxation in a preconstricted rat aorta ring (Al-Ani et al., 1995). Also, as for PAR1, PAR2 is found in an extensive variety of tissues (Table 1), with a conspicuous presence in the gastrointestinal tract (stomach, ileum, colon) and kidney. Not surprisingly, use of a PAR2-AP as a probe for receptor function has revealed that this receptor is involved in regulating gastric smooth muscle function (Yang et al., 1992) and in affecting gastric acid secretion (Kawao et al., 2002; also see article by Kawabata and colleagues in this issue of Life Sciences). In contrast with PAR1, which can have both an inflammatory and anti-inflammatory role (Vergnolle et al., 1999), PAR2 appears to play primarily a pro-inflammatory role in the periphery, promoting both neurogenic inflammation and hyperalgesia (summarized by Vergnolle et al., 2001). In the lung, however, PAR2 activation can be protective, in terms of triggering the release of bronchorelaxant prostanoids from the lung epithelium (Cocks and Moffatt, 2000), even though PAR2 activation, via the intravenous administration of a PAR2-AP can lead to bronchoconstriction via a neural mechanism (Ricciardolo et al., 2000). In the kidney, use of the PAR-activating peptides has shown that PAR2 stimulation can cause a vasodilator effect by both an NO-dependent and an NO-independent mechanism (Gui et al., 2003). In the setting of renal function, it can be noted that the role for PAR2 (vasodilatation and increase in the glomerular filtration rate) is quite distinct from that of PAR1 (vasoconstriction and a reduction in the glomerular filtration rate) (Fig. 2). Thus, it is very likely that, as
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Fig. 2. Bi-directional effects on renal vascular flow of activating PAR2 and PAR1. Perfusion flow (RPF, ml/min/mg tissue) was monitored in an isolated perfused rat kidney. The preparation was first treated with the PAR1 agonist, TFLLR-amide to cause vasoconstriction and a reduction in flow (first arrow). The PAR2 agonist, SLIGRL-amide was then added, resulting in a vasodilatation and an increase in flow. The figure illustrates the bi-directional effect on renal perfusion flow caused by the activation of PARs 1 and 2 (adapted from Gui, 2003, with permission).
for the effects seen in the kidney, PARs 1 and 2 will be found to play bi-directional roles in a number of tissues (Gui et al., 2003). It is a given that, as more work is done using the PAR2-selective agonists to evaluate the consequence of activating this receptor in different tissues, many more physiological roles for this receptor will be found. The function of PAR4, as determined by the actions of its receptor-activating peptide, AYPGKFamide, has been explored primarily in the setting of platelet stimulation. In human platelets, both PAR1 and PAR4 can trigger aggregation (summarized by Coughlin, 2000). In rodents, as opposed to humans, PAR4, with the participation of PAR3 as a co-receptor, is the receptor primarily responsible for platelet aggregation. That PAR1 was not the functional receptor for thrombin in rat and rabbit platelets was heralded by the observation that a PAR1-activating peptide was not able to cause aggregation in platelets from these species (Kinlough-Rathbone et al., 1993). In rat platelets, the activation of PAR4 can have quite distinct effects, leading either to aggregation or to the production of an anti-angiogenic factor (Ma et al., 2001). Use of the PAR4 and PAR1 activating peptides has also established differences between the roles of these two receptors in regulating human platelets (Chung et al., 2002). Given that PAR4 is also widely expressed (Table 1), it is to be anticipated that this receptor will be found to play much wider physiological roles than simply regulating platelet function. Importantly, there appear to be marked differences between species in the distribution of PAR4. These differences will need to be taken into consideration when exploring the pharmacology of the PAR4APs in different animals and generalizing findings obtained in one species to another. Preliminary work indicates that PAR4 also has effects on the vascular and gastrointestinal systems (Hollenberg et al., 1999). No doubt, the use of the PAR4-activating peptide and its related antagonist (Hollenberg and Saifeddine, 2001) will shed further light on the pathophysiology of this most recent addition to the PAR family. Apart from its recognized function in platelets, the diverse physiological roles that PAR4 may play represents a fruitful topic for further investigation.
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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 AM, 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 perform a limited structureactivity evaluation for the response system in question. The partial reverse-sequence peptides (FSLLRamide, for PAR1; LSIGRL-amide for PAR2), which are unable to activate either receptor, have charge and compositional properties that are the same as those of the parent receptor-activating peptides. The same can be said for the control complete reverse sequence peptides, like RLLFT-amide for PAR1 and LRGILS-amide for PAR2. These reverse-sequence peptides are essential controls for any study employing the PAR-APs. Even when the precautions outlined in the previous paragraph are taken, and even in response systems where the reverse peptides have no activity, it is still worth exploring the structure-activity relationships for a series of PAR-AP analogues. For instance, in a study of the ability of PAR-agonist peptides to regulate intestinal transport in a rat jejunal preparation, a series of PAR2AP analogues showed an order of agonist potencies that was distinct from the one originally observed in PAR2-expressing cells. Using criteria established some time ago by Ahlquist (1948), it was possible to conclude that the stimulation of intestinal transport by the PAR2-derived peptides was due to the activation of a receptor other than PAR2 (Vergnolle et al., 1998). In a similar vein, use of the PAR2-activating peptide, trans-cinnamoyl-LIGRLOamide uncovered actions in mast cells and in murine mesenteric arteries could not be accounted for by the activation of PAR2 (McGuire et al., 2002; Stenton et al., 2002). Added to these observations, further work with vascular preparations using the PAR2 peptide agonists demonstrated that, via a mechanism not involving PAR2, the peptides caused the release of a diffusible endothelial-derived contracting factor (Roy et al., 1998; Saifeddine et al., 1998). Thus, the PAR-activating peptides have proved of use not only in elucidating the potential pathophysiological roles that these receptors can play in a number of tissues, but also in revealing the existence of novel peptide receptors which may or may not turn out to be PARs. One looks forward with interest to the identification of the receptors responsible for the actions of the PAR-APs that cannot be attributed to the known PAR family members.
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 a-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 the mechanism whereby trypsin (or another serine proteinase) activates the insulin receptor (that is, via the removal of tonic inhibition of receptor dimerization and cross-phosphorylation) will also be found to apply to other growth factor receptors. In addition to thrombin’s ability to aggregate platelets (an activity that requires thrombin to be proteolytically active), enzymatically inactive thrombin has been found to exhibit chemotactic and
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growth-promoting effects on cells of the monocyte/macrophage lineage. This activity of thrombin can be attributed to a sequence of about 60 amino acid residues in its non-catalytic domain (Bar-Shavit et al., 1986). A different polypeptide sequence of about 30 residues that can be cleaved from thrombin also has growth-promoting effects in cultured cells (Glenn et al., 1988). Due to its catalytic activity, thrombin can also activate other enzymes, like pro-matrix metalloproteinase-2 (Lafleur et al., 2001), to affect cell behaviour. In addition, apart from its action on PAR1, thrombin can modulate human platelet function via a glycoprotein Ib signaling mechanism that appears to act synergistically with PARs 1 and 4 (Soslau et al., 2001). It is highly likely that other proteinases will be found to act via non-PAR mechanisms such as the ones described above for trypsin and thrombin. In this regard, the utility of the PAR-APs to serve as reference agonists for the actions of different proteinases and to assess the ability of various proteinases to trigger or dis-arm the PARs (Kawabata et al., 1999) can be seen to be of great value. It will be of importance in future work to search for other proteolytic mechanisms, like the disruption of integrin-matrix interactions, that may regulate cell behaviour without involving the PARs.
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 play a role equal to that of the proteinases by regulating the paracrine/autocrine/endocrine actions of the enzymes they inhibit. The emergence of the role of proteinases as signaling molecules points to two very important directions for the future. First, it is a key issue to define the roles that the PARs play in normal and abnormal physiology. In this regard the use of the PAR-activating peptides, selective PAR antagonists and studies of model murine systems from animals deficient in one or more of the PARs will be of particular value. Further, it now becomes of great interest to determine the up-regulation and PAR target specificity of both known and to-be-discovered serine proteinases that may play roles in concert with the PARs (or via non-PAR mechanisms) in a variety of pathophyisiological settings. Clearly only the first chapter has so far been written for the long story to be told dealing with proteinase-mediated signalling, that involves the PARs and much more. 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 Foundation of Canada, a Johnson and Johnson Focused Giving Grant and from an Rx&D/CIHR University-Industry grant, supported in conjunction with funding from the Institute de Recherches Internationales Servier/ Servier Canada.
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Vergnolle, N., Hollenberg, M.D., Wallace, J.L., 1999. Pro-and Anti-Inflammatory Actions of Thrombin: A Distinct Role for Proteinase-Activated Receptor-1 (Par1). Brit. J. Pharmacol. 126, 1262 – 1268. Vergnolle, N., Wallace, J.L., Bunnett, N.W., Hollenerg, M.D., 2001. Protease-activated receptors in inflammation, neuronal signaling and pain. Trends in Phamacol. Sci. 22, 146 – 152. Vu, T.K., Hung, D.T., Wheaton, V.I., Coughlin, S.R., 1991. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64, 1057 – 1068. Yang, S.-G., Laniyonu, A., Saifeddine, M., Moore, G.J., Hollenberg, M.D., 1992. Actions of thrombin and thrombin receptor peptide analogues in gastric and aortic smooth muscle: development of bioassays for structure-activity studies. Life Sci. 51, 1325 – 1332.
Proteinase-mediated signaling: Proteinase-activated receptors (PARs) and much more Morley D. Hollenberg * Department of Pharmacology and Therapeutics, University of Calgary Faculty of Medicine, 3330 Hospital Drive North West, Calgary, AB, Canada T2N 4N1
Abstract Quite apart from their ability to generate active polypeptides from hormone precursors and to function as digestive enzymes, proteinases are now known to play a hormone-like role by triggering signal transduction pathways in target cells. The best understood example of proteinase-mediated signaling can be seen in the action of thrombin, which in addition to triggering the coagulation cascade, regulates platelet and endothelial cell function via its serine proteinase activity. The discovery of the G-protein-coupled ‘receptor’ responsible for these cellular actions of thrombin (Proteinase-activated Receptor-1, or PAR1) represents one of the more intriguing signal transduction stories elucidated over past decade or so. It is the objective of this brief review to provide an overview of the discovery and molecular pharmacology of the PAR family and to indicate the widespread roles these receptor systems can play in a variety of tissues. Further, the article (1) illustrates the utility of employing receptorselective PAR-activating peptides to determine the potential physiological roles these receptors play in vivo and (2) describes how these agonists have identified receptors other than the PARs. Finally, the mechanisms other than via the PARs by which proteinases can generate cellular signals are summarized. D 2003 Published by Elsevier Inc. Keywords: Proteinase; Thrombin; Trypsin; PAR; signaling
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 * Tel.: +1-403-220-6931; fax: +1-403-270-0979. E-mail address: [email protected] (M.D. Hollenberg). 0024-3205/$ - see front matter D 2003 Published by Elsevier Inc. doi:10.1016/j.lfs.2003.09.010
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Fig. 1. Schematic model of the activation of human proteinase-activated receptor 1 (PAR1) by thrombin and by a receptorselective PAR-activating peptide. The receptor is depicted as a putative 7-transmembrane structure, with an N-terminal domain containing the cryptic receptor-activating sequence, SFLLRNP. . ., preceded by the thrombin target motif, LDPR. The putative ‘fourth’ intracellular loop of the receptor, formed by palmitoylation of the intracellular C-terminal tail, is not shown. (A) Upon cleavage by thrombin (scissors), the revealed sequence, SFLLRNP, as a ‘tethered ligand’ is believed to dock at least in part with extracellular domain-2 to trigger a receptor-driven signal (arrow). (B) In the absence of thrombin cleavage, a receptorselective PAR1-activating peptide, TFLLR-amide is shown to mimic the tethered ligand, thereby generating an intracellular signal.
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
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the pharmacology of the PAR-activating peptides has led to the synthesis of PAR-selective receptoractivating 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 (NakanishiMatsui 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.
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 AYPGKFamide, for PAR4. As mentioned above, PAR3 has been found not to signal on its own. Since the thrombinrevealed sequences of PAR3 (TFRGAP, for human; SFNGGP for murine) predict an activation of either PAR2 (TFRGAP) or both PAR1 and PAR2 (SFNGGP), the use of the putative PAR3-activating peptides would unfortunately yield misleading and inaccurate results in tissue or cell systems. Nonetheless, with the use of the selective PAR1-activating probes, it has become clear that, as expected, PAR1 is a key regulator of human platelet aggregation and secretion as well as of endothelial cell function (Coughlin, 2000). In vascular tissue, PAR1 activation can lead to an endothelium-dependent nitric oxide mediated vasodilatation in conduit vessels, like the rat aorta (Muramatsu et al., 1992; Hollenberg et al., 1997). Alternatively, in rat aorta tissue, activation of PAR1 in an endothelium-free preparation can cause a calcium-dependent contractile response (Laniyonu and Hollenberg, 1995). What might not have been anticipated is that PAR1 can also play an important role in embryonic development via its impact on endothelial cell function (Griffin et al., 2001). PAR1 is also of importance in the re-stenosis process after vascular injury (Andrade-Gordon et al., 2001), very likely due to the activation of PAR1 in both the vascular endothelial and smooth muscle cells. Since PAR1 is widely distributed in a variety of tissues (Table 1) it is not surprising that the use of the receptor-activating probes has revealed a multitude of effects of PAR1 activation in sites that range from the vasculature, as already described, to the brain, lung and gastrointestinal tract. As well as having cardiovascular effects, PAR1 can also play an important role in causing a neurogenic inflammatory response (Cocks and Moffatt, 2000; de Garavilla et al., 2001; summarized by Macfarlane et al., 2001 and Vergnolle et al., 2001). In the setting of cancer, PAR1 appears to play an important role as a potential oncogene (Martin et al., 2001) and as a stimulus for cell invasion (Even-Ram et al., 1998; 2001; Henrikson et al., 1999; Kamath et al., 2001). Finally, it has unexpectedly turned out that PAR1 can play a role not only in a neurogenic inflammatory response, but also in promoting analgesia (Asfaha et al., 2002). Thus, there are many settings in which PAR1 may play a role, as revealed by the use of its receptor-selective agonist. PAR2, like PAR1 can also play a role in the cardiovascular system, as revealed by the use of its selective PAR-AP, SLIGRL-amide. One of the first effects described for PAR2 activation in a conduit
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Table 1 Localization and potential roles of proteinase-activated receptors PAR1
PAR2
PAR3
PAR4
Tissue distribution (Northern blot analysis)
Brain, lung, heart, stomach, colon, kidney, testis
Prostate, small intestine, colon, liver, kidney, pancreas, trachea
Heart, kidney, pancreas, thymus, small intestine, stomach, lymph node, trachea
Cellular expression
Platelets, endothelium, vascular smooth muscle, leukocytes, GI tract epithelium, fibroblasts, neurons, mast cells
Endothelium, leukocytes, GI tract epithelium and lung, airway and vascular smooth muscle, neurons, mast cells, keratinocytes, lung fibroblasts, renal tubular cells
Airway smooth muscle, platelets
Lung, pancreas, thyroid, testis, small intestine, placenta, skeletal muscle, lymph node, adrenal gland, prostate, uterus, colon Platelets, megakaryocytes
Known physiological role Potential physiological roles
Platelet activation
a
Regulation of vascular tone, Pro-inflammatory, modulator of nociception, embryonic development,
Platelet activation Regulation of vascular tone, pro-inflammatory, mediator of nociception, airway protection,
Cofactor for PAR4
Platelet activation
GI, gastrointestinal. a Information for PAR2, PAR3, and PAR4 is provided for human tissues; for PAR1, distribution is recorded in rat tissues, for which more extensive data are available than in human tissues. (adapted from Hollenberg and Compton, 2002, with permission).
vessel was the demonstration of an endothelium-dependent, nitric oxide-mediated relaxation in a preconstricted rat aorta ring (Al-Ani et al., 1995). Also, as for PAR1, PAR2 is found in an extensive variety of tissues (Table 1), with a conspicuous presence in the gastrointestinal tract (stomach, ileum, colon) and kidney. Not surprisingly, use of a PAR2-AP as a probe for receptor function has revealed that this receptor is involved in regulating gastric smooth muscle function (Yang et al., 1992) and in affecting gastric acid secretion (Kawao et al., 2002; also see article by Kawabata and colleagues in this issue of Life Sciences). In contrast with PAR1, which can have both an inflammatory and anti-inflammatory role (Vergnolle et al., 1999), PAR2 appears to play primarily a pro-inflammatory role in the periphery, promoting both neurogenic inflammation and hyperalgesia (summarized by Vergnolle et al., 2001). In the lung, however, PAR2 activation can be protective, in terms of triggering the release of bronchorelaxant prostanoids from the lung epithelium (Cocks and Moffatt, 2000), even though PAR2 activation, via the intravenous administration of a PAR2-AP can lead to bronchoconstriction via a neural mechanism (Ricciardolo et al., 2000). In the kidney, use of the PAR-activating peptides has shown that PAR2 stimulation can cause a vasodilator effect by both an NO-dependent and an NO-independent mechanism (Gui et al., 2003). In the setting of renal function, it can be noted that the role for PAR2 (vasodilatation and increase in the glomerular filtration rate) is quite distinct from that of PAR1 (vasoconstriction and a reduction in the glomerular filtration rate) (Fig. 2). Thus, it is very likely that, as
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Fig. 2. Bi-directional effects on renal vascular flow of activating PAR2 and PAR1. Perfusion flow (RPF, ml/min/mg tissue) was monitored in an isolated perfused rat kidney. The preparation was first treated with the PAR1 agonist, TFLLR-amide to cause vasoconstriction and a reduction in flow (first arrow). The PAR2 agonist, SLIGRL-amide was then added, resulting in a vasodilatation and an increase in flow. The figure illustrates the bi-directional effect on renal perfusion flow caused by the activation of PARs 1 and 2 (adapted from Gui, 2003, with permission).
for the effects seen in the kidney, PARs 1 and 2 will be found to play bi-directional roles in a number of tissues (Gui et al., 2003). It is a given that, as more work is done using the PAR2-selective agonists to evaluate the consequence of activating this receptor in different tissues, many more physiological roles for this receptor will be found. The function of PAR4, as determined by the actions of its receptor-activating peptide, AYPGKFamide, has been explored primarily in the setting of platelet stimulation. In human platelets, both PAR1 and PAR4 can trigger aggregation (summarized by Coughlin, 2000). In rodents, as opposed to humans, PAR4, with the participation of PAR3 as a co-receptor, is the receptor primarily responsible for platelet aggregation. That PAR1 was not the functional receptor for thrombin in rat and rabbit platelets was heralded by the observation that a PAR1-activating peptide was not able to cause aggregation in platelets from these species (Kinlough-Rathbone et al., 1993). In rat platelets, the activation of PAR4 can have quite distinct effects, leading either to aggregation or to the production of an anti-angiogenic factor (Ma et al., 2001). Use of the PAR4 and PAR1 activating peptides has also established differences between the roles of these two receptors in regulating human platelets (Chung et al., 2002). Given that PAR4 is also widely expressed (Table 1), it is to be anticipated that this receptor will be found to play much wider physiological roles than simply regulating platelet function. Importantly, there appear to be marked differences between species in the distribution of PAR4. These differences will need to be taken into consideration when exploring the pharmacology of the PAR4APs in different animals and generalizing findings obtained in one species to another. Preliminary work indicates that PAR4 also has effects on the vascular and gastrointestinal systems (Hollenberg et al., 1999). No doubt, the use of the PAR4-activating peptide and its related antagonist (Hollenberg and Saifeddine, 2001) will shed further light on the pathophysiology of this most recent addition to the PAR family. Apart from its recognized function in platelets, the diverse physiological roles that PAR4 may play represents a fruitful topic for further investigation.
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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 AM, 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 perform a limited structureactivity evaluation for the response system in question. The partial reverse-sequence peptides (FSLLRamide, for PAR1; LSIGRL-amide for PAR2), which are unable to activate either receptor, have charge and compositional properties that are the same as those of the parent receptor-activating peptides. The same can be said for the control complete reverse sequence peptides, like RLLFT-amide for PAR1 and LRGILS-amide for PAR2. These reverse-sequence peptides are essential controls for any study employing the PAR-APs. Even when the precautions outlined in the previous paragraph are taken, and even in response systems where the reverse peptides have no activity, it is still worth exploring the structure-activity relationships for a series of PAR-AP analogues. For instance, in a study of the ability of PAR-agonist peptides to regulate intestinal transport in a rat jejunal preparation, a series of PAR2AP analogues showed an order of agonist potencies that was distinct from the one originally observed in PAR2-expressing cells. Using criteria established some time ago by Ahlquist (1948), it was possible to conclude that the stimulation of intestinal transport by the PAR2-derived peptides was due to the activation of a receptor other than PAR2 (Vergnolle et al., 1998). In a similar vein, use of the PAR2-activating peptide, trans-cinnamoyl-LIGRLOamide uncovered actions in mast cells and in murine mesenteric arteries could not be accounted for by the activation of PAR2 (McGuire et al., 2002; Stenton et al., 2002). Added to these observations, further work with vascular preparations using the PAR2 peptide agonists demonstrated that, via a mechanism not involving PAR2, the peptides caused the release of a diffusible endothelial-derived contracting factor (Roy et al., 1998; Saifeddine et al., 1998). Thus, the PAR-activating peptides have proved of use not only in elucidating the potential pathophysiological roles that these receptors can play in a number of tissues, but also in revealing the existence of novel peptide receptors which may or may not turn out to be PARs. One looks forward with interest to the identification of the receptors responsible for the actions of the PAR-APs that cannot be attributed to the known PAR family members.
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 a-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 the mechanism whereby trypsin (or another serine proteinase) activates the insulin receptor (that is, via the removal of tonic inhibition of receptor dimerization and cross-phosphorylation) will also be found to apply to other growth factor receptors. In addition to thrombin’s ability to aggregate platelets (an activity that requires thrombin to be proteolytically active), enzymatically inactive thrombin has been found to exhibit chemotactic and
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growth-promoting effects on cells of the monocyte/macrophage lineage. This activity of thrombin can be attributed to a sequence of about 60 amino acid residues in its non-catalytic domain (Bar-Shavit et al., 1986). A different polypeptide sequence of about 30 residues that can be cleaved from thrombin also has growth-promoting effects in cultured cells (Glenn et al., 1988). Due to its catalytic activity, thrombin can also activate other enzymes, like pro-matrix metalloproteinase-2 (Lafleur et al., 2001), to affect cell behaviour. In addition, apart from its action on PAR1, thrombin can modulate human platelet function via a glycoprotein Ib signaling mechanism that appears to act synergistically with PARs 1 and 4 (Soslau et al., 2001). It is highly likely that other proteinases will be found to act via non-PAR mechanisms such as the ones described above for trypsin and thrombin. In this regard, the utility of the PAR-APs to serve as reference agonists for the actions of different proteinases and to assess the ability of various proteinases to trigger or dis-arm the PARs (Kawabata et al., 1999) can be seen to be of great value. It will be of importance in future work to search for other proteolytic mechanisms, like the disruption of integrin-matrix interactions, that may regulate cell behaviour without involving the PARs.
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 play a role equal to that of the proteinases by regulating the paracrine/autocrine/endocrine actions of the enzymes they inhibit. The emergence of the role of proteinases as signaling molecules points to two very important directions for the future. First, it is a key issue to define the roles that the PARs play in normal and abnormal physiology. In this regard the use of the PAR-activating peptides, selective PAR antagonists and studies of model murine systems from animals deficient in one or more of the PARs will be of particular value. Further, it now becomes of great interest to determine the up-regulation and PAR target specificity of both known and to-be-discovered serine proteinases that may play roles in concert with the PARs (or via non-PAR mechanisms) in a variety of pathophyisiological settings. Clearly only the first chapter has so far been written for the long story to be told dealing with proteinase-mediated signalling, that involves the PARs and much more. 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 Foundation of Canada, a Johnson and Johnson Focused Giving Grant and from an Rx&D/CIHR University-Industry grant, supported in conjunction with funding from the Institute de Recherches Internationales Servier/ Servier Canada.
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