Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Print
Document related concepts
Subventricular zone wikipedia , lookup
Optogenetics wikipedia , lookup
Neuromuscular junction wikipedia , lookup
NMDA receptor wikipedia , lookup
Synaptogenesis wikipedia , lookup
Stimulus (physiology) wikipedia , lookup
Molecular neuroscience wikipedia , lookup
Channelrhodopsin wikipedia , lookup
Endocannabinoid system wikipedia , lookup
Clinical neurochemistry wikipedia , lookup
Transcript
Physiol Rev 84: 579 – 621, 2004; 10.1152/physrev.00028.2003. Protease-Activated Receptors: Contribution to Physiology and Disease VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT Departments of Surgery and Physiology, University of California, San Francisco, California Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 I. Introduction II. Protease-Activated Receptors: Mechanisms of Activation by Cell-Surface Proteolysis A. Discovery and structure B. Proteases cleave PARs to expose a tethered ligand domain C. Protease binding to PARs facilitates cleavage and activation D. There are functional interactions between PARs E. Protease binding to other membrane proteins can facilitate PAR cleavage and activation F. Multiple proteases activate PARs III. Protease-Activated Receptors: Transduction of the Signal A. Tethered ligands interact with extracellular domains to initiate signal transduction B. Structure-activity relations of tethered ligand domains C. PARs activate multiple signaling cascades IV. Protease-Activated Receptors: Termination of the Signal A. Cell-surface proteolysis can disable PARs B. PAR signaling is attenuated by receptor desensitization C. PARs are downregulated by intracellular proteases D. Sustained signaling by proteases requires mobilization and synthesis of new receptors V. Protease-Activated Receptors: Contribution to Physiological and Pathophysiological Control Mechanisms A. Protease signaling in the circulatory and cardiovascular systems B. Protease signaling to the nervous system C. Protease signaling in the gastrointestinal system D. Protease signaling in the airway E. Protease signaling in the skin VI. Protease-Activated Receptors: Mechanisms of Disease and Targets for Therapy A. Cardiovascular disease B. Inflammatory disease C. Cancer VII. Conclusions and Future Perspectives 580 581 581 582 583 584 585 587 589 589 590 590 594 594 594 596 597 598 599 601 604 607 609 610 610 611 611 612 Ossovskaya, Valeria S., and Nigel W. Bunnett. Protease-Activated Receptors: Contribution to Physiology and Disease. Physiol Rev 84: 579 – 621, 2004; 10.1152/physrev.00028.2003.—Proteases acting at the surface of cells generate and destroy receptor agonists and activate and inactivate receptors, thereby making a vitally important contribution to signal transduction. Certain serine proteases that derive from the circulation (e.g., coagulation factors), inflammatory cells (e.g., mast cell and neutrophil proteases), and from multiple other sources (e.g., epithelial cells, neurons, bacteria, fungi) can cleave protease-activated receptors (PARs), a family of four G protein-coupled receptors. Cleavage within the extracellular amino terminus exposes a tethered ligand domain, which binds to and activates the receptors to initiate multiple signaling cascades. Despite this irreversible mechanism of activation, signaling by PARs is efficiently terminated by receptor desensitization (receptor phosphorylation and uncoupling from G proteins) and downregulation (receptor degradation by cell-surface and lysosomal proteases). Protease signaling in tissues depends on the generation and release of proteases, availability of cofactors, presence of protease inhibitors, and activation and inactivation of PARs. Many proteases that activate PARs are produced during tissue damage, and PARs make important contributions to tissue responses to injury, including hemostasis, repair, cell survival, inflammation, and pain. Drugs that mimic or interfere with these processes are attractive therapies: selective agonists of PARs may facilitate www.prv.org 0031-9333/04 $15.00 Copyright © 2004 the American Physiological Society 579 580 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT healing, repair, and protection, whereas protease inhibitors and PAR antagonists can impede exacerbated inflammation and pain. Major future challenges will be to understand the role of proteases and PARs in physiological control mechanisms and human diseases and to develop selective agonists and antagonists that can be used to probe function and treat disease. I. INTRODUCTION FIG. 1. Mechanisms by which cell-surface proteolysis regulates signal transduction. Proteases can regulate signaling by cleaving ligands (A–C) or receptors (D and E). A: cell-surface proteases can induce shedding of membranebound signaling molecules; e.g., tumor necrosis factor-␣ (TNF-␣)-converting enzyme or TACE, a member of the ADAM (a disintegrin and metalloproteinase) family of cell-surface proteases, liberates soluble TNF-␣ from the cell surface and thus releases a soluble cytokine. B: cellsurface peptidases can generate biologically active peptides; e.g., angiotensin converting enzyme or ACE converts the decapeptide angiotensin I (ANG I) to the octapeptide ANG II, the principal active form. C: cell-surface peptidases can degrade and inactivate neuropeptides; e.g., neutral endopeptidase (NEP) cleaves substance P (SP) at multiple sites to form inactive fragments. D: soluble proteases can cleave proteaseactivated receptors (PARs) to expose a tethered ligand that binds and activates the cleaved receptor; e.g., the coagulation factor thrombin cleaves PAR1 to activate the receptor. E: soluble proteases can cleave PARs to remove the tethered ligand, generating a disabled receptor; e.g., cathepsin G from neutrophils cleaves PAR1 to remove the tethered ligand and thereby prevent activation by thrombin. Physiol Rev • VOL 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 Proteolytic enzymes are estimated to comprise 2% of the human genome (266). It is not surprising, therefore, that proteases participate in a diverse array of biological processes, ranging from degradation of dietary proteins in the lumen of the gastrointestinal tract to the control of the cell cycle. This review concerns the importance of proteolytic events at the cell surface in signal transduction, physiological control, and disease. Proteases that are anchored to the plasma membrane or that are soluble in the extracellular fluid can cleave ligands or receptors at the surface of cells to either initiate or terminate signal transduction (Fig. 1). Thus cellsurface proteases can release or generate active ligands, or degrade and inactivate receptor agonists. For example, tumor necrosis factor (TNF)-␣-converting enzyme or TACE cleaves the precursor of TNF-␣ at the plasma membrane, thereby releasing a soluble form of this proinflammatory cytokine. In a similar manner, angiotensin converting enzyme (ACE), which is also an integral membrane protein, converts angiotensin I to angiotensin II in the extracellular fluid to generate the principal active form of this hormone. In contrast, neutral endopeptidase degrades and inactivates the neuropeptide substance P (SP) in the vicinity of its receptors and thus terminates the biological effects of SP. Certain soluble and membrane-bound proteases cleave G protein-coupled receptors (GPCRs) at the cell surface to activate or inactivate receptors. For example, the coagulation factor thrombin cleaves protease-activated receptor 1 (PAR1) on platelets, which activates the receptor to induce platelet aggregation and hemostasis. Conversely, cathepsin G from neutrophils cleaves PAR1 at a different site from thrombin to 581 PROTEASE-ACTIVATED RECEPTORS II. PROTEASE-ACTIVATED RECEPTORS: MECHANISMS OF ACTIVATION BY CELL-SURFACE PROTEOLYSIS A. Discovery and Structure 1. PAR1 PAR1, formerly known as the thrombin receptor, was cloned by two laboratories by the strategy of expressing RNA from thrombin-responsive cells of humans and hamsters in oocytes from Xenopus (233, 305). Clones were identified that encoded a protein of 425 residues with 7 hydrophobic domains of a typical GPCR. The deduced sequence of human PAR1 contained an amino-terminal signal sequence, and extracellular amino-terminal domain of 75 residues, and a potential cleavage site for thrombin within the amino-tail (LDPR412S42FLLRN, where 2 denotes cleavage) (Fig. 2). 2. PAR2 PAR1 remained the only member of the family until the serendipitous discovery of PAR2. PAR2 was identified by screening a mouse genomic library using degenerate primers to the second and sixth transmembrane domains of the bovine neurokinin 2 receptor (214, 215). A clone was found that encoded a protein of 395 residues with the 1. A summary of PAR activating proteases, disabling proteases, activating peptides, localization, and phenotypes of PAR-deficient mice TABLE PAR1 Activating proteases Thrombin FXa APC Granzyme A Gingipains-R Trypsin Inactivating proteases Cathepsin G Plasmin Elastase Proteinase-3 Trypsin LDPR41 2 S42FLLRN SFLLRN TFLLRN Platelets (human), endothelium, epithelium, fibroblasts, myocytes, neurons, astrocytes Partial embryonic lethality, vascular matrix deposition after injury Cleavage site AP Localization Phenotype of knockout PAR2 PAR3 PAR4 Trypsin Tryptase FVIIa FXa MT-SP1 Proteinase-3 Acrosien Der P3 D9 Gingipains-R Elastase Cathepsin G Thrombin SKGR34 2 S35LIGKV SLIGKV LPIK38 2 T39FRGAP None Epithelium, endothelium, fibroblasts, myocytes, neurons, astrocytes Platelets (mouse), endothelium, myocytes, astrocytes PAPR47 2 G48YPGQV GYPGQV AYPGKF Platelets (human), endothelium, myocytes, astrocytes Impaired leukocyte migration; impaired allergic inflammation of airway, joints, kidney Protection against thrombus formation/ pulmonary embolism Protection against thrombus formation/ pulmonary embolism Cathepsin G References are provided in the text. PAR, protease-activated receptor; AP, activating peptide. Physiol Rev • VOL 84 • APRIL 2004 • Thrombin Trypsin Cathepsin G Gingipains-R www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 generate a disabled receptor that cannot respond to thrombin, which could impede blood clotting. These proteolytic events are critically important for normal physiological control: the conversion of angiotensin I to angiotensin II is central to the reflex regulation of blood pressure and volume, and thrombin-induced aggregation of platelets is vital for normal hemostasis. However, these processes are also of great interest in understanding mechanisms of disease and for the development of effective therapies. Thus inhibitors of ACE are widely used to treat hypertension and congestive heart failure, and antagonists of PARs are being developed to treat thrombotic and inflammatory diseases. The focus of this review is on the PAR family GPCRs. Four PARs have been identified by molecular cloning (Table 1). A wide range of proteases cleave and activate PARs, including proteases from the coagulation cascade, inflammatory cells, and the digestive tract. Receptor activation initiates an array of signaling events in many cell types with diverse consequences, ranging from hemostasis to pain transmission. We review the mechanisms by which cell-surface proteolysis activates PARs to initiate signal transduction, discuss the mechanisms that terminate signaling by PARs, review the importance of PARs in physiological control in major systems, and speculate on the contribution of PARs to disease. There are several comprehensive reviews of PARs (68, 108, 182, 217). 582 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT typical characteristics of a GPCR and with ⬃30% amino acid identity to human PAR1. The extracellular amino terminus of 46 residues contained a putative trypsin cleavage site SKGR342S35LIGKV (Fig. 2). 3. PAR3 The discovery of PAR2 provided the impetus for attempts to identify other receptors of this type. Indeed, the existence of additional receptors for thrombin was suggested by the observation that platelets derived from PAR1-deficient mice still responded to thrombin, whereas fibroblasts were unresponsive (64). PAR3 was subsequently cloned using degenerate primers to conserved domains of PAR1 and PAR2 to screen RNA from rat platelets (135). The human and murine forms of PAR3 were subsequently cloned, and human PAR3 was found to share ⬃28% sequence homology to human PAR1 and PAR2. Like PAR1 and PAR2, PAR3 is a typical GPCR with a thrombin cleavage site within the extracellular amino terminus at LPIK382T39FRGAP (Fig. 2). groups subsequently used these sequences to clone a new GPCR, PAR4 (143, 317). Human PAR4 is a 385-amino acid protein, with a potential cleavage site for thrombin and trypsin in the extracellular amino-terminal domain: PAPR472G48YPGQV (Fig. 2). PAR4 is ⬃33% homologous to the other human PARs, but with some distinct differences in the amino- and carboxy-terminal domains. B. Proteases Cleave PARs to Expose a Tethered Ligand Domain The general mechanism by which proteases cleave and activate PARs is the same: proteases cleave at specific sites within the extracellular amino terminus of the receptors; this cleavage exposes a new amino terminus that serves as a tethered ligand domain, which binds to conserved regions in the second extracellular loop of the cleaved receptor, resulting in the initiation of signal transduction (Fig. 2). There is no known function of the amino-terminal fragment of the receptor that is removed by proteolysis. 4. PAR4 1. PAR1 Two laboratories identified PAR-like sequences by searching expressed sequence tag (EST) libraries, and both The mechanism by which thrombin activates PAR1 has been investigated in detail. Thrombin cleaves PAR1 at Physiol Rev • VOL 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 FIG. 2. Structural and functional domains of PARs. The figure shows alignment of domains of human PAR1, PAR2, PAR3, and PAR4. A and B: mechanism of cleavage and interaction of the tethered ligand with extracellular binding domains. C: functionally important domains in the amino terminus, second extracellular loop, and carboxy terminus. Conserved residues in loop II are in bold. [Adapted from Derian et al. (89) and Macfarlane et al. (182).] PROTEASE-ACTIVATED RECEPTORS 2. PAR2 Similar observations indicate that trypsin cleaves PAR2 at R342S35LIGKV to reveal the amino-terminal tethered ligand SLIGKV in humans (213–215). Mutation of the trypsin site prevents trypsin cleavage and activation of PAR2. Synthetic peptides corresponding to the tethered ligand domain (SLIGKV) activate PAR2 without the need for receptor cleavage. Exposure of cells to trypsin results in loss of immunoreactivity to an antibody against an amino-terminal epitope, which indicates that trypsin cleaves intact PAR2 at the cell surface (23). 3. PAR3 Thrombin cleaves PAR3 at K382T39FRGAP, and mutation of the cleavage site to one that would be resistant to thrombin prevents activation (135). Cleavage by thrombin exposes a new amino terminus (TFRGAP) that may interact with the receptor as a tethered ligand. However, in marked contrast to PAR1, PAR2, and PAR4, synthetic peptides corresponding to this putative tethered ligand do not activate PAR3. The reason for this discrepancy is unknown, although differences in affinity, steric hindrances, and the possibility that cleavage releases conformation of the receptor constrained by the uncleaved amino-terminal region could explain these unexpected results. Another unexpected and unexplained observation is that mouse PAR3 is unable to signal when expressed alone, without other PARs (143). Physiol Rev • VOL 4. PAR4 Thrombin and trypsin cleave PAR4 at R472G48YPGQV, and peptides corresponding to the tethered ligand domain GYPGQV can directly activate PAR4 (143, 317). Mutation of the cleavage site prevents activation by thrombin and trypsin, but not by the synthetic peptide, which confirms the importance of proteolytic cleavage for receptor activation. C. Protease Binding to PARs Facilitates Cleavage and Activation Thrombin can activate PAR1 and PAR3 by a two-step mechanism: first the protease binds and then it cleaves the receptor (Fig. 3A). The process of binding and activation has been most thoroughly studied for thrombin and PAR1 (306). The extracellular amino terminus of human PAR1 contains a sequence of charged residues (D51KYEPF56) that is distal to the thrombin cleavage site. This charged domain binds to an anion binding site on thrombin, thereby temporarily concentrating the protease at the surface of the receptor. This negatively charged region of PAR1 resembles a domain of the leech anticoagulant hirudin, which inhibits thrombin by binding its anion site. The importance of the hirudin-like domain is emphasized by the finding that its deletion markedly diminishes the capacity of thrombin to activate PAR1, whereas substitution of this region with the corresponding domain of hirudin allows a full recovery of activity. Moreover, ␥-thrombin, which lacks an anion site, is 100fold less potent than ␣-thrombin, which has the site, in cleaving PAR1 at the activation site (25). Furthermore, platelets respond to low concentrations of ␣-thrombin but not ␥-thrombin, because the latter cannot bind to the PAR1 (260). Protease binding thereby increases the efficiency of activation, probably by concentrating the protease on the surface of the receptor or by altering the conformation of the receptor to facilitate cleavage. PAR3 also contains a hirudin-like site (FEEFP) that is distal to the thrombin cleavage site, which interacts with thrombin (135). Thus ␥-thrombin is 100-fold less potent than ␣-thrombin for activating PAR3, and alanine substitutions within the hirudin site of PAR3 attenuate activation of PAR3 by thrombin. PAR4, in contrast to the other thrombin receptors, lacks a hirudin-like binding site for thrombin (143, 317). It is for this reason that both ␣-thrombin and ␥-thrombin can activate PAR4 with similar potency (317). The lack of a thrombin binding site also accounts for the observation that ␣-thrombin activates PAR4 with a potency that is 50-fold less than for activation of PAR1. Thus PAR4 is a low-affinity receptor for thrombin, whereas PAR1 and PAR3 are high-affinity thrombin receptors. Protease binding sites have not been identified for other PARs. 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 R412S42FLLRN to expose the tethered ligand SFLLRN, which binds and activates the cleaved receptor, resulting in signal transduction. Several observations support this mechanism of activation. Mutation of the cleavage site prevents thrombin cleavage and signaling, indicating the importance of this site for activation of PAR1 (305). The general importance of proteolytic activation is indicated by the finding that replacement of thrombin site with an enterokinase site generates a receptor responsive to enterokinase (306). A synthetic peptide that mimics the tethered ligand domain (S42FLLRNPNDKYEPF) directly activates intact PAR1, without the requirement for hydrolysis by thrombin (305), and peptides as short as six residues (S42FLLRN) are also fully active (253) (see sect. IIIB). Such synthetic agonists, referred to as activating peptides (AP), are useful tools for investigating PAR functions. Direct physical proof that thrombin cleaves PAR1 is provided by the findings that exposure of platelets to thrombin reduces binding an antibody directed against the cleavage site of PAR1 but does not alter binding of an antibody directed to a domain distal to the cleavage site (212). Moreover, exposure to thrombin increases the electrophoretic mobility of epitope-tagged PAR1, indicating a reduction of molecular weight by proteolytic cleavage (304). 583 584 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT D. There Are Functional Interactions Between PARs A common theme of signaling by GPCRs is that there are frequently several different receptors for a single ligand and often several ligands for one receptor. Thus thrombin can activate PAR1, PAR3, and PAR4, with different potencies, and trypsin, tryptase, and certain coagulation factors can activate PAR2. This complexity becomes particularly interesting because PARs are frequently coexpressed, and interactions between receptors in the same cell can have important functional consequences. 1. Dual receptor systems Human platelets express two thrombin receptors: PAR1 and PAR4. There are marked differences in the mechanisms of activation and inactivation of these receptors that have important consequences for thrombin signaling to platelets. PAR1, by virtue of a hirudin-like site, is able to bind thrombin and thus responds to low concentrations of enzyme. PAR4 lacks the hirudin site and can Physiol Rev • VOL respond only to high thrombin concentrations (142). However, whereas PAR1 responses are rapidly shut-off, probably as a result of phosphorylation of residues in the carboxy terminus and uncoupling from G proteins (see sect. IV), PAR4 responses are sustained and thus desensitize slowly (263). The coexpression of two receptors with different potencies and kinetics of desensitization may have important functional consequences. Thus PAR1 mediates rapid and transient increases in intracellular Ca2⫹ concentration ([Ca2⫹]i) in human platelets to low concentrations of thrombin, whereas PAR4 mediates delayed and sustained increases in [Ca2⫹]i to higher thrombin concentrations (70). This prolonged signal is important for the late phases of platelet aggregation. Similar dual receptor systems probably exist on other cells with important functional consequences. 2. PAR3 as a cofactor for PAR4 In some instances, protease binding to one receptor can facilitate cleavage of another receptor that is expressed on the same cell. This appears to be the situation 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 FIG. 3. Mechanisms of activation and intermolecular cooperation of PARs. A: thrombin activates PAR1 and PAR3 in a two-step process. First, thrombin binds to a hirudin-like domain; second, thrombin cleaves to expose the tethered ligand, which binds and activates the cleaved receptor. B: PAR3 is a cofactor for PAR4 in murine platelets. Thrombin binds to the hirudin site of PAR3, but PAR3 does not signal in mouse platelets. The PAR3bound thrombin then cleaves and activates PAR4. C: intermolecular signaling of PAR2 and PAR1 in endothelial cells. The exposed tethered ligand domain of PAR1 can bind and activate PAR2, resulting in intermolecular signaling. [Adapted from Coughlin (68).] PROTEASE-ACTIVATED RECEPTORS 3. Intermolecular PAR signaling GPCRs can form homodimers and heterodimers with important consequences for signal transduction. Although the principal mechanism of PAR activation is intramolecular (i.e., the unmasked tethered ligand binds to the cleaved receptor), there are several examples of intermolecular interactions between different PAR molecules. Intermolecular signaling, by which a cleaved receptor can activate an uncleaved receptor, was first demonstrated for PAR1 (43). The approach was to express the amino terminus of PAR1 (including the tethered ligand domain) attached to the transmembrane domain of CD8 (to anchor the fragment at the cell surface) with truncated PAR1 lacking the amino terminus, either alone or together. When expressed alone, there was no response to thrombin. However, when coexpressed, cells were able to respond to thrombin, suggesting that thrombin cleaves the amino terminus of PAR1 to reveal the tethered ligand domain, which then interacts with the truncated receptor, which transduces the signal. Thus at least in a reconstituted system, it is possible for the tethered ligand of cleaved PAR1 to activate an uncleaved receptor. There is Physiol Rev • VOL also evidence of intermolecular signaling between different PARs (Fig. 3C). Peptides corresponding to the tethered ligand of PAR1 (SFLLRN) can also activate PAR2, but not vice versa (21). To examine intermolecular signaling, a signaling defective mutant of PAR1 and wild-type PAR2 were expressed alone or together (218). When expressed alone, neither receptor responded to thrombin, but when coexpressed, there was a robust thrombin response. Moreover, in endothelial cells that naturally express PAR1 and PAR2, a PAR1 antagonist blocked only 75% of the response to thrombin, whereas desensitization of PAR2 blocked the remaining 25% of the response. These results suggest that the tethered ligand domain of PAR1 can interact with uncleaved PAR2 to transduce the signal. This novel form of intermolecular signaling between different PARs clearly requires the close association of receptors at the cell surface, which could be influenced by levels of expression or by anchoring proteins that may affect mobility of receptors in the membrane. E. Protease Binding to Other Membrane Proteins Can Facilitate PAR Cleavage and Activation As discussed, thrombin binding to a PAR can facilitate cleavage and activation of that receptor or a different PAR. In addition, binding to nonreceptor proteins can concentrate proteases at the cell surface and thereby facilitate PAR activation. For example, glycoprotein I␣ is a cell-surface platelet protein with a high-affinity binding site for ␣-thrombin. Disruption of this interaction impedes the capacity of ␣-thrombin to cleave PAR1 and cause aggregation of platelets, suggesting that glycoprotein I␣ is a cofactor for PAR1 that promotes PAR1mediated platelet aggregation (84). Thrombin may also interact with other proteins on platelets (118). This theme of anchoring proteins serving as cofactors for proteolytic activation of PARs is particularly important for signaling by coagulation factors (F), notably FVIIa and FXa that act upstream of thrombin (reviewed in Refs. 68, 240). Coagulation proceeds by two processes: an extrinsic and an intrinsic pathway. The extrinsic pathway is initiated by tissue damage and requires tissue factor (TF) or thromboplastin, an integral membrane protein that is normally expressed by extravascular cells (e.g., myocytes, keratinocytes), and which is expressed by endothelial cells and monocytes during inflammation. During coagulation, TF binds FVIIa, and the TF-FVIIa complex interacts with the zymogen FX to form active FXa. FXa then interacts with FVa, a cofactor for FXa-mediated conversion of prothrombin to thrombin. Thrombin plays a critical role in hemostasis by converting fibrinogen to fibrin and by cleaving PARs on platelets to induce aggregation. The intrinsic pathway is triggered by exposure of blood to collagen underlying damaged endothelium, and 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 for PAR3 and PAR4, the thrombin receptors on murine platelets (mouse platelets lack PAR1) (199) (Fig. 3B). Observations in mice deficient in PAR3 indicate that PAR3 is required for the response of platelets to low but not to high concentrations of thrombin, which is attributable to PAR4. These findings could be explained by the fact that PAR3 contains a thrombin binding site, and can thus respond to low concentrations of thrombin (135), whereas PAR4 lacks a binding site and thus mediates responses to high thrombin concentrations (143, 317). However, the situation is complicated by the finding that murine PAR3 expressed in COS cells does not signal, for reasons that are not fully understood. This finding suggests that, in murine platelets, PAR3 somehow facilitates the activation of PAR4 by low concentrations of thrombin. In support of this hypothesis, the potency of thrombin signaling to PAR4 in COS cells is increased 6- to 15-fold by coexpression of PAR3, whereas responses to PAR4 AP are unaffected (199). Moreover, the expression of a construct of the amino terminus of PAR3 (including the hirudin-like site) attached to the transmembrane domain of CD8 (to anchor the construct at the cell surface) with PAR4 also facilitates thrombin signaling. Together, these findings suggest that PAR3 is a cofactor for PAR4 in murine platelets: the hirudin-like site of PAR3 binds and concentrates thrombin at the cell surface, and thereby promotes thrombin cleavage of PAR4. At high concentrations, thrombin can directly cleave and activate PAR4, even though it lacks a thrombin binding domain. This novel mechanism of cooperative interaction between PAR3 and PAR4 adds to the complexity of interactions between GPCRs. 585 586 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT the presence of FX. These results suggest the local formation of FXa at the plasma membrane results in PAR2 activation (34). The receptor that mediates FXa signaling depends on the cell in question. Observations made using cells prepared from PAR-deficient animals indicate that FXa signaling to endothelial cells is mostly mediated by PAR2 but also by PAR1, whereas PAR1 solely mediates FXa signaling to fibroblasts (35). These mechanisms of signaling by FVIIa and FXa may be of particular importance during septic shock when upstream coagulation proteases make a substantial contribution to the inflammatory response and when the expression of TF is upregulated on several cell types. The role of anchoring proteins in PAR signaling has also been extended to the anticoagulant pathway (Fig. 4B). Low levels of thrombin are normally present in the circulation, and when vessels remain intact these low levels of thrombin contribute to the anticoagulant protein C (APC) pathway. The endothelial protein C receptor (EPCR) binds protein C to the surface of endothelial cells. Thrombomodulin also binds thrombin to the endothelial cell surface, where thrombin converts protein C to APC. When APC is released from the EPCR, it acts as a circu- FIG. 4. Formation of coagulation and anticoagulation complexes promotes activation of PARs. A: tissue factor (TF) anchors FVIIa to the cell surface and thereby facilitates activation of PAR1 and PAR2. A ternary complex of TF-FVIIaFXa efficiently cleaves and activates PAR1 and PAR2. B: thrombomodulin (TM) concentrates low concentrations of thrombin to the surface of endothelial cells. Endothelial protein C receptor (EPCR) binds protein C (PC), and thrombin converts PC to activated PC (APC). APC, still bound to EPCR, then activates PAR1. Physiol Rev • VOL 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 begins with conversion of FXII to FXIIa, which is catalyzed by kallikrein and kininogen. This conversion then initiates a cascade of events involving activation of FXI, FX, and converging with the extrinsic pathway and the FXa-dependent conversion of prothrombin to thrombin. In addition to zymogen cleavage, FVIIa and FXa can signal to PARs. FVIIa signals to cells when allosterically associated with TF for full activity, in part through PAR1 and PAR2 (34) (Fig. 4A). Thus, although FVIIa, even at high concentrations, does not signal to Xenopus oocytes expressing PAR1 or PAR2, FVIIa robustly signals to oocytes expressing TF together with PAR1 or PAR2. Similarly, soluble FXa weakly activates PAR1, PAR2, or PAR4, but when FXa is associated with a complex comprising TF-FVIIa-FXa, it potently activates PAR1 and PAR2 (34, 239). Moreover, although the TF-FVIIa complex can activate PAR2, it does so with far lower efficiency than the TF-FVIIa-FXa complex, in which locally generated FXa induces signaling. Thus FVIIa can signal to fibroblasts expressing PAR2 and TF with a potency of ⬃4 nM, but this concentration exceeds the estimated plasma concentration of FVIIa (50 pM). However, the potency of FVIIa is reduced by almost three orders of magnitude (to 8 pM) in PROTEASE-ACTIVATED RECEPTORS F. Multiple Proteases Activate PARs Although many proteases have been found to cleave and activate PARs in cultured cells, the capacity of a protease to signal in intact tissues depends on many factors. First, proteases must be generated or released in sufficient concentrations to activate PARs. Although the catalytic properties of proteases would ensure that even very low concentrations could eventually cleave all receptors on the surface of the cell, it is the rate of hydrolysis of PARs that determines the magnitude of the resulting cellular signal (137). Second, efficient hydrolysis and activation of PARs may require the presence of accessory cofactors, for example, TF and EPCR in the case of FVIIa-FXa and APC, respectively (34, 46, 238, 239). Third, the capacity of a protease to signal will depend on the availability of protease inhibitors that serve to dampen the effects of many proteases in vivo. Thus, although trypsin is a very potently activated PAR2 in cultured cells, trypsin inhibitors are widely expressed and may well limit the capacity of trypsin to signal in tissues. With these caveats in mind, many proteases have been identified that are capable, at least theoretically, of activating PARs. Physiol Rev • VOL 1. Coagulation factors Serine proteases of the coagulation cascade are perhaps the best characterized activators of PARs (68, 108, 240). As discussed in section IIE, proteases that mediate coagulation and anticoagulation by cleaving zymogens or active enzymes themselves can also signal to several cell types by cleaving and activating PARs. Thus thrombin activates PAR1, PAR3, or PAR4 at the surface of platelets, resulting in aggregation, which contributes to hemostasis (68). The TF-FVIIa-FXa complex signals by cleaving PAR1 and PAR2 on a variety of cell types, including endothelial cells, which is of particular importance in inflammation (34, 239). Thrombin can also exert anti-inflammatory effects that depend on the local generation of APC and consequent activation of PAR1 on endothelial cells (46, 238). 2. Pancreatic and extrapancreatic trypsins Trypsins potently activate PAR2 and PAR4. There are at least three distinct trypsin genes in humans: trypsin I, II, and mesotrypsin; trypsin IV is a splice variant of mesotrypsin. The potential of trypsins to signal to cells by cleaving PAR2 or PAR4 depends on the release of the zymogen trypsinogen, the presence of enteropeptidase, which activates trypsinogen, and the existence of the large array of endogenous trypsin inhibitors. During feeding, trypsinogens I and II are secreted from the pancreas into the lumen of the small intestine, where they are activated by enteropeptidase. In feeding rats, luminal trypsin attains concentrations (1 M) that are more than capable of strongly activating PAR2 at the apical surface of enterocytes (EC50 ⬃5 nM) (167). Pancreatic trypsinogens are also prematurely activated in the inflamed pancreas where they are released into the interstitial fluid and vasculature and could activate PAR2 in pancreatic acini, duct cells, or nerves (205). However, trypsins are widely distributed enzymes that are expressed by many extrapancreatic cells, including endothelial cells (169), epithelial cells, the nervous system (168), and in tumors (165, 166). However, despite this widespread distribution, almost nothing is known about the control of secretion or activation of extrapancreatic trypsins or their potential functions as PAR activators. Trypsin II isolated from conditioned medium from colon cancer cell lines can cleave and activate PAR2; since these cells also express PAR2 it is theoretically possible that trypsin II could regulate cells in an autocrine manner (6, 94). A tryptic-like serine protease purified from rat brain, designated P22, degrades matrix and can signal to cells by activating PAR2 (252). P22 could be of particular interest in brain injury, where there appears to be enhanced secretion. Trypsin IV is also a potential PAR activator. Trypsinogen IV is invariably coexpressed in epithelial cell lines, endothelial cells, and human colonic mucosa with PAR2, and trypsin IV cleaves 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 lating anticoagulant protein by degrading FVa and FVIIa. However, when retained at the cell surface, the EPCRAPC complex promotes activation of PAR1 (238). Thus APC can signal to fibroblasts only when they coexpress EPCR and PAR1, and APC signaling to endothelial cells is blocked by an active site blocked form of APC, which competes for EPCR, and by antibodies that block PAR1 cleavage. Gene expression analysis of endothelial cells indicates that this mechanism of APC-induced activation of PAR1 mediates in large part the anti-inflammatory effects of APC. Thus PAR1 mediates the effects of low concentrations of APC on gene expression in endothelial cells. In addition to its anticoagulant activity, APC is an anti-inflammatory agent that reduces organ damage in animal models (111) and reduces mortality of patients with sepsis (20). PAR1 can contribute to the anti-inflammatory actions of APC. Thus APC prevents hypoxic-induced apoptosis of brain endothelial cells through transcriptionally dependent inhibition of tumor suppressor gene p53, regulation of Bax/Bcl2 proteins, and reduction of caspase-3 signaling (46). Blockade of PAR1 with an antibody against the activation site prevents APC-mediated cytoprotection in cultured endothelial cells, indicating a crucial role for PAR1 in this protection. In a model of focal ischemic stroke in mice, APC markedly reduces brain infarction volumes. This effect is attenuated in EPCR-deficient mice, and the neuroprotective role of APC is abolished by PAR1 blocking antibodies. Thus EPCR and PAR1 play a major role in the protective effects of APC. 587 588 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT and activates PAR2 and PAR4 (N. W. Bunnett, unpublished observations). Of particular interest, trypsin IV is resistant to most proteinaceous trypsin inhibitors that effectively inhibit trypsins I and II (146). Together, these results raise the intriguing possibility that trypsin IV can signal for prolonged periods in tissues by autocrine or paracrine activation of PAR2 and PAR4. 3. Mast cell proteases Physiol Rev • VOL 4. Leukocyte proteases Proteases from leukocytes are released at sites of inflammation and may serve as PAR activators under these conditions. Neutrophils store a variety of proteases (cathepsin G, elastase, proteinase 3) in azurophil granules, which may be released on activation. Cathepsin G is released from activated neutrophils and causes aggregation of platelets. This effect of cathepsin G may be mediated by PAR4 (249). Thus cathepsin G increases [Ca2⫹]i in PAR4-transfected fibroblasts, PAR4-expressing oocytes, and human platelets, and a PAR4-blocking antibody inhibits activation of platelets by cathepsin G and prevents Ca2⫹ signaling in platelets induced by activation of neutrophils. Thus cathepsin G may activate platelets and other cells at sites of injury and inflammation by cleaving PAR4. However, some of the effects of cathepsin G are not PAR mediated, since cathepsin G also signals to cardiac myocytes in a PAR-independent manner (245). Proteinase 3 is present in neutrophil secretory granules and at the cell surface and is also expressed by other inflammatory cells. Proteinase 3 cleaves peptide fragments of PAR2 at the activation site, and proteinase 3-induced Ca2⫹ responses in oral epithelial cells are suppressed by desen- 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 There has been considerable interest in mast cell tryptase as an activator of PAR2. Tryptase is the most abundant protease of human mast cells; it comprises up to 25% of the total cellular proteins and is expressed by almost all subsets of human mast cells (38). However, there are distinct interspecies differences in the expression of proteases in mast cells (reviewed in Ref. 190). Thus, whereas tryptase appears to be abundant in the guinea pig (188), it is a less prominent protease in mast cells of rats and mice. Many of the proinflammatory and mitogenic effects of tryptase are mimicked by PAR2 APs, suggesting that tryptase exerts its effects through this receptor. However, there is some unresolved controversy about the effectiveness of tryptase as a PAR2 activator. On one hand, human tryptase from a variety of sources (purified from human lung, skin, and mast cell lines) can cleave PAR2 to expose the tethered ligand domain and signals to transfected cells as well as many cell types that naturally express PAR2 at physiological levels (2, 18, 19, 66, 67, 193, 196, 235, 254, 256, 268, 270). These signals appear to be mediated by tryptase, for they are abolished by selective inhibitors, and are PAR2 dependent because they are absent from nontransfected cells and are diminished by selective downregulation of PAR2. The general consensus of these studies is that tryptase is a PAR2 activator but that it is considerably less potent than trypsin. However, most of the preparations used in these studies likely contain several forms of tryptase. Human mast cells express at least five distinct tryptase genes: ␣, I, II, III, and transmembrane tryptase, and splice variants also exist. Thus the molecular form of tryptase responsible for these effects is unknown. Observations using recombinant tryptase are less clear. Although recombinant ␣ and II tryptase have been reported to activate PAR2 in BaF3 cells (193), another report found no activity of I tryptase as a PAR activator (130). The reason for this discrepancy is not known, but there are several possible explanations. One possibility is that a combination of tryptases in the purified preparations is responsible for PAR2 cleavage and activation. Another is that a posttranslational modification of PAR2 influences tryptase activation. PAR2 contains sites for N-linked glycosylation: Asn30, 6 residues proximal to the cleavage and activation site, and Asn222 in extracellular loop II. The potency with which tryptase (but not trypsin) activates PAR2 is dramat- ically increased by mutation Asn30, by enzymatic deglycosylation, or by expression of PAR2 in glycosylationdefective cells (62, 63). Thus glycosylation of the receptor at a site close to the activation site markedly impairs the capacity of tryptase to signal. Although the reason for this finding is not known, glycosylation could impede access of the amino terminus of PAR2 to the active site of tryptase. Tryptase is a 134-kDa tetrameric protease in the form of a flat ring that is composed of four monomers whose active sites face the center of the ring (228). One can speculate that a large, glycosylated structure may not be accessible to the active sites. It will be important to determine whether PAR2 is similarly glycosylated in tissues and to know if there are mechanisms that deglycosylate the receptor. However, can tryptase activate PAR2 in vivo? On balance, the evidence is in favor of an important role for tryptase in PAR2 activation under conditions of inflammation and mast cell activation when large amounts of tryptase are released close to PAR2 expressing cells. Thus injected tryptase has proinflammatory and hyperalgesic actions in conscious mice that are not observed in PAR2-deficient animals (40, 296). Because tryptase is a large and poorly diffusible protease, it is likely then that tryptase signals in a paracrine manner to cells that are in close proximity to mast cells, such as sensory nerves that express PAR2, which participate in inflammation and pain (267, 270, 296). Other proteases from mast cells have not been shown to activate PARs, although chymase can suppress thrombin signaling to keratinocytes, suggesting that it disables PAR1 (254). PROTEASE-ACTIVATED RECEPTORS sitization of PAR2, suggesting that proteinase 3 is a PAR2 activator (292). 5. Cell-surface proteases 6. Nonmammalian proteases An intriguing observation is that a number of nonmammalian proteases from mites, bacteria, and fungi have been found to signal to mammalian cells by cleaving and activating PARs. The dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae produce a series of proteases (cysteine proteases, trypsins, chymotrypsins, collagenases) that are allergens in the airway epithelium. The cysteine and serine proteases Der P1 and Der P9 stimulate cytokine release from human airway epithelial cells and induce mobilization of Ca2⫹, effects reminiscent of PAR activation (162). Indeed, Der P3 and Der P9 can cleave fragments of PAR2 at the activation site, and desensitization experiments suggest that the effects of these proteases on airway epithelial cells are mediated in part by PAR2 (273). Bacterial proteases can also signal through PARs. Porphyromonas gingivallis is a major mediator of periodontitis in humans, and bacterial arginine-specific gingipains-R (RgpB and HRgpA) have been implicated in this disease. RgpB can activate PAR1and PAR2-transfected cells and signals to an oral epithelial cell line to induce release of the powerful proinflammatory cytokine interleukin-6 (178, 179). RgpB and HRgpA can also signal to transfected cells expressing PAR1 and PAR4, and low concentrations of both proteases mobilize Ca2⫹ in platelets and induce aggregation with a similar potency to thrombin (180). Cross-desensitization studies and use of antibodies that block PAR cleavage and activation suggest that the effects of gingipains-R on platelets are mediated by PAR1 and PAR4. These interesting results reveal a novel mechanism by which bacteria influence mammalian cells and could explain an emerging link between periodontitis and cardiovascular disorders. Certain fungi are also allergens in the airway, and proPhysiol Rev • VOL teases from extracts of several species affect cytokine production from airway epithelial cells (147). Some of these effects are reminiscent of the activation of PAR2, although the protease and receptor that are principally responsible remain to be determined. III. PROTEASE-ACTIVATED RECEPTORS: TRANSDUCTION OF THE SIGNAL A. Tethered Ligands Interact With Extracellular Domains to Initiate Signal Transduction Analyses of mutant and chimeric receptors and of analogs of APs have allowed identification of the critical residues of the tethered ligand domains that interact with binding domains of the PARs and which are thus essential for signal transduction. The consensus of these experiments is that conserved regions of extracellular loop II and the amino terminus interact with the tethered ligands of PAR1 and PAR2. Interactions between the tethered ligand and PAR1 have been investigated by analysis of chimeras of human and Xenopus PAR1 (105). The tethered ligands of human (SFLLRN) and Xenopus (TRFIFD) PAR1 are specific for their respective receptors and thus discriminate between the receptors. Replacement of the extracellular amino terminus and the second extracellular loop of the Xenopus receptor with the corresponding domains of human PAR1 confers the chimera with selectivity for peptides corresponding to the human tethered ligand. Moreover, substitution of only two residues of Xenopus PAR1 with corresponding residues of the human receptor (Phe87 for Asn in the extracellular amino terminus and Glu260 for Leu in the second extracellular loop) also confers selectivity for the human peptide. Thus these two residues in the extracellular amino terminus and extracellular loop 2 are critical for interaction with the tethered ligand domain. Further mutational analysis of human PAR1 and study of analogs of the PAR1 AP suggest that Glu260 of extracellular loop 2 interacts with Arg5 of the tethered ligand SFLLRN (201). Substitution of eight residues from the second extracellular loop of the Xenopus receptor to human PAR1 generates a receptor that is constitutively active in the absence of ligand, suggesting that alteration of the conformation of extracellular loop II is sufficient to transduce a signal across the plasma membrane (202). Interactions between the tethered ligand of PAR2 and the cleaved receptor have been similarly examined by studying chimeras of PAR1 and PAR2 (175). These studies also reveal the importance of extracellular loop II for the activation of PAR2. The important role of residues in extracellular loop II of PAR2 for interaction with tethered ligand peptides has also been revealed by the study of mutant receptors and analogs of the PAR2 AP (4). An 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 Given that anchoring proteins serve as cofactors that facilitate the capacity of certain proteases to activate PARs, is it possible that proteases that are themselves integral membrane proteins can activate PARs? One such protease, membrane-type serine protease 1 (MT-SP1), has been identified. MT-SP1 is a type II integral membrane protein with an extracellular protease domain (276). Analysis of the substrate specificity of MT-SP1 suggests PAR2 as a potential substrate, and both MT-SP1 and PAR2 are coexpressed at the surface of certain cell types (e.g., PC-3 cells). Indeed, solubilized MT-SP1 signals to oocytes expressing PAR2, but not to cells expressing PAR1, PAR3, or PAR4. It remains to be determined if the membrane-bound MT-SP1 can activate PAR2 under more physiological circumstances. 589 590 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT acidic region (PEE) that is just distal to a highly conserved domain (CHDVL) makes an important contribution to determining the selectivity of PAR2 agonists. B. Structure-Activity Relations of Tethered Ligand Domains Physiol Rev • VOL C. PARs Activate Multiple Signaling Cascades In common with most GPCRs, PARs couple to multiple signaling pathways and can thereby regulate many cellular functions. The mechanisms of PAR1 and PAR2 signaling have been extensively investigated and reviewed (89, 182). 1. Concentration-response relationships The catalytic nature of PAR activation means that even a very low concentration of an enzyme would eventually cleave every receptor molecule at the cell surface. How then do proteases generate graded responses in cells? For neurotransmitter receptors, graded concentrations of agonists induce graded responses through differing degrees of receptor occupancy. For PARs, graded responses depend on the rate of receptor cleavage (137). Thus thrombin induces cumulative phosphoinositide hydrolysis that correlates with cumulative receptor cleavage, suggesting that each cleavage event generates a “quantum” of phosphatidylinositol signal. This signaling is 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 The observation that synthetic peptides corresponding to the tethered ligand domain of PAR1 are agonists for the receptor without the need for proteolysis had three important consequences. First, it enabled the use of synthetic peptides as probes for PAR function, thereby avoiding the sole use of proteases, which have many biological effects not related to PARs. These APs are now widely used to investigate the physiological functions of PARs in vitro and in vivo. Second, it permitted convenient structure activity studies of the tethered ligand domain through functional analyses of synthetic peptides (187, 253). Such studies have provided important information about critical residues of the tethered ligand and facilitated the development of more selective agonists (123). Third, analogs of the APs have been used as templates for the development of antagonists of PAR1 (9). However, there are certain caveats to the use of APs as PAR agonists. Unfortunately, APs are weak agonists compared with proteases, often requiring concentrations in excess of 1 M to activate most PARs. The low potency of APs is mostly attributed to the inefficient presentation of these soluble peptides to the binding domains of the receptor, compared with the tethered peptide. In addition, these peptides are readily inactivated by proteolysis. Because APs are used at such high concentrations, they may have nonspecific effects that are unrelated to PAR activation. The use of control peptides, such as inactive scrambled peptides or reversed sequences, is thus essential, although these too can have activity (296). In addition, APs are not always specific for a particular PAR; for example, the PAR1 AP SFLLRN also activates PAR2 (21). Finally, there are no effective APs for PAR3. The characteristics of PAR APs and their utility as templates for design of antagonists have been recently reviewed in detail (182) and are only summarized here. The first described PAR1 AP was a 14-residue peptide (305), although it was soon realized that the hexapeptide SFLLRN was fully functional (253). Although SFLLRN is a PAR1 agonist, it also activates PAR2 (21). However, replacement of Ser1 with Thr1 (corresponding to the sequence in Xenopus PAR1) generates an agonist (TFLLRN) that is selective for PAR1 and that does not activate PAR2. Analysis of analogs of SFLLRN in which individual residues are substituted for Ala (alanine scanning), together with site-directed mutagenesis of the tethered ligand, indicates that critical residues within this domain include Phe2, Leu4, and Arg5. Substitution of other residues can be tolerated, depending on the type of substitution. For instance, changes in Ser1 maintain function provided the amino group is maintained, whereas removal of the amino group abolishes activity. Detailed structure-function analyses of analogs of the PAR1 AP, together with analyses by NMR and other techniques, have facilitated the design of penta- and tetrapeptides with enhanced potencies. For example, H-Ala-(pF)Phe-Arg-Cha-hARg-Tyr-NH2 induces platelet aggregation with an EC50 of 10 nM, and an iodinated form of this peptide can be used in binding assays (100). A peptidomimetic antagonist of PAR1 has been developed that is selective for PAR1 over the other PARs (9). Such antagonists have been successfully used in nonhuman primates to suppress thrombus formation and vascular occlusion (88) and could be of promise to treat human disease. Analyses of analogs of the PAR2 AP have similarly identified the residues that are essential for biological activity. In general, the rat/mouse peptide SLIGRL is slightly more potent than the human agonist SLIGKV (21). Analysis by alanine scanning indicates that Leu2 and Arg5 are essential for activity. Replacing Ser1 or Arg5 with Ala also reduces activity, whereas substitution of Gly4 or Leu6 has only a slight effect on PAR2 activation. The PAR4 AP GYPGKF is neither as efficacious as thrombin in activating PAR4 nor as potent as APs for PAR1 or PAR2 in activating the corresponding receptors, and is thus of limited use for probing receptor function. However, the analog AYPGKF is ⬃10-fold more potent than GYPGKF and is as efficacious as thrombin in activating PAR4 (98). AYPGKF is also relatively specific for PAR4. This specificity depends on Tyr2, since replacement with Phe generates an agonist of PAR1 and PAR4. PROTEASE-ACTIVATED RECEPTORS rapidly terminated despite the continued presence of an exposed tethered ligand, by mechanisms of desensitization that are discussed in section IVB. Thus cells sense the rate of receptor cleavage, which is related to the concentration of protease. In other words, high concentrations of thrombin rapidly cleave many PAR1 molecules, permitting the accumulation of inositol 1,4,5-trisphosphate (InsP3) and signal transduction. Proteases that inefficiently cleave PARs could not rapidly generate a sufficient quantity of second messenger for signal transduction to occur. 2. PAR1 signaling important role in coupling since thrombin signaling in fibroblasts and platelets that express PAR1 is attenuated by the microinjection of Gq␣ antibodies (13, 16). Moreover, platelets derived from Gq␣-deficient mice exhibit markedly diminished thrombin-induced aggregation and degranulation (222). These mice have increased bleeding times, probably due to impaired thrombin signaling to platelets. Gq11␣ activates phospholipase C-1, leading to generation of InsP3, which mobilizes intracellular Ca2⫹, and diacylglycerol (DAG), which activates protein kinase C (PKC). Thus thrombin-stimulated InsP3 generation and Ca2⫹ mobilization are blunted in mutant fibroblasts that express low levels of phospholipase C-1 but normal levels of the other phospholipases (99). Thrombin-induced activation of phospholipase A2 and phospholipase D is diminished in this cell line, suggesting that phospholipase C-1 activates PKC-␣, which may be required for phospholipase A2 and phospholipase D activation. Together, Ca2⫹ and PKC activate numerous pathways, including Ca2⫹-regulated protein kinases and mitogen-activated protein (MAP) kinases. PAR1 also couples to G12␣ and G13␣ in platelets and FIG. 5. Summary of PAR1 signal transduction. PAR1 couples to Gi␣, G12/ 13␣, and Gq11␣. Gi␣ inhibits adenylyl cyclase (AC) to reduce cAMP. G12/13␣ couples to guanine nucleotide exchange factors (GEF), resulting in activation of Rho, Rho-kinase (ROK), and serum response elements (SRE). Gq11␣ activates phospholipase C (PLC) to generate inositol trisphosphate, which mobilizes Ca2⫹, and diacylglycerol (DAG), which activates protein kinase C (PKC). PAR1 can activate the mitogen-activated protein kinase cascade by transactivation of the EGF receptor, through activation of PKC, phosphatidylinositol 3-kinase (PI3K), Pyk2, and other mechanisms. G␥ subunits couple PAR1 to other pathways, such as activation of G proteins receptor kinases (GRKs), potassium channels (Ki), and nonreceptor tyrosine kinases (TK). [Modified from Coughlin (68).] Physiol Rev • VOL 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 Signal transduction commences with coupling of PARs to heterotrimeric G proteins at the plasma membrane. Considerable progress has been made in identifying the subtypes of G proteins that interact with PAR1, and the role of these subunits in signaling and physiological regulation has been studied in knockout mice (reviewed in Ref. 219). PAR1 interacts with several ␣-subunits, in particular Gq11␣, G12/13␣ and Gi␣, which accounts for the pleotropic action of its ligands (Fig. 5). A major pathway of coupling is through Gq␣ (131). Gq␣ plays an 591 592 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT Physiol Rev • VOL which in turn phosphorylates MAP kinase (serine-threonine kinase). MAP kinases in turn regulate multiple substrates in the cytoplasm and nucleus. The MAPK ERK1/2 module plays a critical role in cell proliferation and differentiation. Most information about the regulation of this module derives from study of tyrosine kinase receptors, such as the epidermal growth factor (EGF) receptor. EGF binding to its receptor results in receptor dimerization and autophosphorylation. The phosphotyrosine on the intracellular domain of the receptor binds through an SH2 domain to the adaptor protein Shc, which recruits the Grb2-SOS complex to exchange GDP for GTP on p21ras. This initiates a cascade of phosphorylation events: p21ras phosphorylates the serine-threonine Raf-1 kinase, a MAP kinase kinase kinase, which phosphorylates MEK1/2 (MAP kinase kinase), which in turn phosphorylates ERK1/2 (MAP kinase). There are several mechanisms by which PAR1 can couple to this pathway. In astrocytes, PAR1 activation of ERK1/2 and proliferation depend on a pertussis toxin-sensitive pathway mediated by G␥, PI 3-kinase and ras, and a pertussis toxin-insensitive pathway involving PKC and raf (310). Another mechanism may involve transactivation of the EGF receptor. Indeed, activation of PAR1 induces phosphorylation of the EGF receptor in enterocytes, and PAR1-induced Cl⫺ secretion is suppressed by inhibition of EGF receptor kinase (30). Possible mechanisms of transactivation include activation of the Ca2⫹-dependent kinase Pyk-2, and PAR1 agonists activate Pyk-2 in endothelial cells (161). Additionally, some GPCRs activate matrix metalloproteases, which induce shedding of EGF receptor ligands from the cell surface (231). PAR1 can also activate the MAP kinase p38 module in fibroblasts by a mechanism involving EGF receptor transactivation by Src family kinases (246). The observation that mitogenic effects of PAR1 activators require tyrosine kinase activity, yet PAR1 does not possess intrinsic tyrosine kinase activity, led to the investigation of the role of the janus family of tyrosine kinases (JAKs) in PAR1 signaling and mitogenesis. JAKs tyrosine phosphorylate STAT proteins (signal transducers and activators of transcription), which dimerize and translocate to the nucleus to direct gene transcription. In vascular smooth muscle cells, thrombin activates JAK2, resulting in nuclear translocation of the transcription factors STAT2 and STAT3 (183). Inhibition of JAK2 suppresses thrombin-induced ERK2 activity and proliferation, suggesting that JAK2 is upstream of the Ras/Raf/MEK/ERK pathway. 3. PAR2 signaling Less is known about the signaling mechanisms of PAR2 than for PAR1. Activators of PAR2 stimulate generation of InsP3 and mobilization of Ca2⫹ in PAR2-transfected cell lines, enterocytes, keratinocytes, myocytes, 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 astrocytoma cells (10, 220). Thus thrombin stimulates incorporation of a GTP analog into G12␣ and G13␣ in platelets, and thrombin-stimulated DNA synthesis is blocked by microinjection of antibodies to G12␣ in astrocytoma cells. G12/13␣ plays a major role in control of cell shape and migration. Upon stimulation with thrombin, platelets change from a discoid to a spheroid shape and extrude pseudopodia, which is believed to be a prerequisite for full activation. Although platelets from Gq␣-deficient animals show impaired thrombin-induced aggregation and degranulation (222), they undergo normal shape changes in response to thrombin (163). The effects of thrombin on the shape of these platelets is mediated by G12/13␣ (163). G12/13␣ interacts with Rho guanine-nucleotide exchange factors (GEFs), permitting Rho-mediated control of cell shape and migration. For example, in platelets G12/13␣ mediate activation of Rho-kinase and myosin light-chain kinase, which participate in thrombin-induced shape changes. Fibroblasts from G13␣-deficient mice show diminished migratory responses to thrombin, and these animals also exhibit impaired ability of endothelial cells to develop into an organized vascular system, resulting in intrauterine death (221). In human umbilical vein endothelial cells, PAR1 activators induce stress fiber formation, accumulation of cortical actin, and cell rounding (303). Inhibition of Rho partially attenuates thrombininduced cell rounding, whereas dominant-negative Rac blocks the response to thrombin. Thus Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin. PAR1 also couples to Gi␣ proteins, which inhibit adenylyl cyclase and suppress formation of cAMP. Activation of PAR1 in fibroblasts inhibits cAMP generation in a pertussis toxin-sensitive fashion, suggesting involvement of a Gi-like protein (132). Expression of a mutant of Gi-2 in CHO cells suppresses thrombin-stimulated arachidonic acid release, suggesting that Gi-2 couples PAR1 to cytoplasmic phospholipase A2 (315). G␥ subunits of heterotrimeric G proteins couple PAR1 to may other pathways, notably activation of phosphatidylinositol (PI) 3-kinase. PI 3-kinase thus links PAR1 to changes in cytoskeletal structure, cell motility, survival, and mitogenesis. For instance, in astrocytes, the effects of PAR1 agonists on activation of extracellular signal response kinases (ERKs) 1/2 and proliferation are strongly inhibited by wortmannin, which blocks PI 3-kinase (310). There has been considerable interest in understanding the mechanisms by which PAR1 couples to the MAP kinase cascades, given the important mitogenic role of thrombin. Several MAP kinase “signaling modules” have been characterized in mammalian cells (reviewed in Ref. 314) with a common organization: a MAP kinase kinase kinase (a serine-threonine kinase) phosphorylates and activates MAP kinase kinase (threonine-tyrosine kinase), PROTEASE-ACTIVATED RECEPTORS neurons, astrocytes and tumor cells (24, 66, 67, 167, 213, 214, 251, 290). Therefore, it is likely that PAR2 couples to Gq␣. Indeed, PAR2 signaling is unaffected by pertussis toxin in PAR2-transfected cells and in enterocytes, suggesting that PAR2 does not signal through Gi proteins (86). In enterocytes and transfected epithelial cells, activation of PAR2 also stimulates arachidonic acid release and rapid generation of prostaglandins E2 and F1␣, suggesting activation of phospholipase A2 and cyclooxygenase-1 (167). PAR2 activators also strongly activate MAP kinases ERK1/2 and weakly stimulate the MAP kinase p38, although c-jun amino-terminal kinase is not activated (15, 86, 319). 593 Recent observations indicate that -arrestins play a major role in PAR2-induced activation of ERKs. -Arrestins are cytosolic proteins that interact with activated GPCRs at the plasma membrane and in the cytosol (see sect. IV). Activation of PAR2 stimulates the assembly of a MAP kinase signaling module, a large-molecular-weight complex containing PAR2, -arrestins, raf-1, and activated ERK1/2, which forms at the plasma membrane or in early endosomes (86) (Fig. 6C). The module retains activated ERK1/2 in the cytosol where they cannot induce proliferation. However, in cells expressing a mutant PAR2, which is unable to interact with -arrestins, the module is unable to form. In these cells PAR2 agonists activate ERK1/2 by Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 FIG. 6. Desensitization, downregulation, and trafficking of PARs. Details vary between PARs, as discussed in the text. A: receptor cleavage (1) activates PARs, which couple to heterotrimeric G proteins (2). Activation induces membrane translocation of GRKs (GRK3 and 5 for PAR1), which phosphorylate the receptor (3) and promote membrane translocation and interaction with -arrestins, which mediate uncoupling and desensitization (4). B: receptor cleavage and interaction with -arrestins (PAR2, not PAR1) (1) induces clathrin- and dynamin-dependent endocytosis (2) and rab5a-mediated trafficking to early endosomes (3). PARs are then sorted to lysosomes (4) by a mechanism involving sorting nexin 1 (SNX1 for PAR1). Resensitization requires mobilization of intracellular pools of PARs (5). The pools are replenished by new PAR synthesis or by constitutive endocytosis of unactivated PARs (6). C: in the case of PAR2, activation induces recruitment of -arrestins that serve as molecular scaffolds to recruit and organize components of the mitogen-activated protein kinase (MAPK) pathway to coated pits (1) or early endosomes (2). -Arrestins interact with PAR2, raf-1, MEK1/2, and activated ERK1/2 to specify the subcellular location and function of activated ERK1/2. Physiol Rev • VOL 84 • APRIL 2004 • www.prv.org 594 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT alternate pathways that allow nuclear translocation and stimulation of proliferation. Therefore, -arrestins are molecular scaffolds that promote the formation of an ERK1/2 module at the plasma membrane or in endosomes. The scaffold retains activated ERKs in these locations, where they may regulate cytosolic targets. PAR2 agonists also activate the NFB pathway in keratinocytes and myocytes, emphasizing the importance of PAR2 in inflammation (28, 145). IV. PROTEASE-ACTIVATED RECEPTORS: TERMINATION OF THE SIGNAL Proteases that remove or destroy the tethered ligand or cleave the binding domain in extracellular loop II would generate receptors that are unresponsive to activating proteases. Many proteases can disable PARs in this manner (Table 1). However, the same criteria that should be applied to consideration of the physiological relevance of activating proteases (see sect. IIF) must also be applied to any potential inactivating enzymes. Disabling proteases may serve to dampen signaling by activating proteases and could thereby be an additional mechanism for terminating protease signaling. A portion of proteolytically activated PAR1 can recycle to the cell surface in some cell types, although it is usually targeted for degradation (129). Proteolytically activated and recycled PAR1 can continue to signal at the cell surface (283). In this case, proteases that cleave or remove the exposed tethered ligand would arrest signaling. Proteases from inflammatory cells, including neutrophils and mast cells, can cleave and disable PARs. Neutrophil cathepsin G, elastase, and proteinase 3 cleave PAR1 to remove the activation site and thereby abolish thrombin signaling (197, 236). PAR1 AP still signals to cells exposed to these proteases, suggesting that the binding domain is preserved. Chymase, an abundant mast cell protease, also renders keratinocytes unresponsive to thrombin, suggesting that it too inactivates PAR1 (254). Elastase and cathepsin G cleave PAR2 in transfected cells and airway epithelial cell lines, removing amino-terminal epitopes and thereby generating receptors that are unresponsive to trypsin but normally responsive to PAR2 AP (95). However, elastase and cathepsin G can signal to fibroblasts to induce release of interleukin-8 and monocyte chemoattractant protein 1, possibly by activation of PAR2 (291). Cathepsin G and elastase also abolish signaling by thrombin to PAR3-transfected cells, and thus disable PAR3 (74). Interestingly, this process of inactivation occurs without the loss of binding of monoclonal antibodies that recognize sites flanking the tethered ligand domain, suggesting inactivation does not involve removal of Physiol Rev • VOL B. PAR Signaling Is Attenuated by Receptor Desensitization Proteases activate PARs by an irreversible mechanism: cleavage exposes the tethered ligand domain that is always available to interact with the cleaved receptor. Thus activation would result in prolonged signaling unless there were efficient mechanisms to attenuate the response. The principal mechanism that terminates signaling by PARs is broadly similar to the classical pathway of desensitization that has been described in detail for many other GPCRs, in particular rhodopsin and the 2- 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 A. Cell-Surface Proteolysis Can Disable PARs the tethered ligand. The mechanism of this inactivation is unknown but could involve cleavage of tethered ligand binding domains. Some proteases can cleave PARs at several sites, including activation and disabling sites, and the net result depends on the efficiency of cleavage at different locations. For example, although cathepsin G can cleave PAR1 at the activation site Arg41-Ser42, the major cleavage site is at Phe55-Trp56, which removes the tethered ligand and disables the receptor (197). Although trypsin has been reported to activate PAR1, it also efficiently cleaves PAR1 at distal sites that would remove the tethered ligand domain. Indeed, in endothelial cells, trypsin inactivates PAR1, generating a receptor that is unresponsive to thrombin (200). In addition to cleavage at the activation site (Arg34-Ser35), tryptase also cleaves PAR2 at the Lys41Val42 site, which could inactivate the receptor (196). In the case of tryptase, the activating cleavage is more important since tryptase activates PAR2. The consequences of exposing cells to proteases depends on the repertoire of PARs expressed by a cell and whether the proteases activate or disable particular receptors. For example, cathepsin G disables PAR1 but activates PAR4. Thus in human platelets, which express PAR1 and PAR4, cathepsin G can induce aggregation through PAR4 even though it disables PAR1 and thereby prevents signaling by low concentrations of thrombin (249). In contrast, in fibroblasts and endothelial cells that do not express PAR4, cathepsin G abolishes thrombin signaling through PAR1. In addition, proteolysis can impair the ability of PAR3 to act as a cofactor for PAR4 (74). In murine platelets, PAR3 binds thrombin but does not signal. Instead, it concentrates thrombin in the vicinity of PAR4 and thus serves as cofactor for PAR4 signaling in response to low concentrations of thrombin. Cathepsin G does not cause aggregation of murine platelets but prevents aggregation to low concentrations of thrombin, indicating that cathepsin G abolishes this cofactor role of PAR3. It is very likely that proteases that destroy other nonreceptor cofactors, such as TF, would also have profound effects on PAR function. PROTEASE-ACTIVATED RECEPTORS 1. PAR1 PAR1 activators trigger rapid phosphorylation of the receptor in transfected cell lines (136, 302, 304). Both GRKs and second messenger kinases could mediate this phosphorylation. Although activation of PKC stimulates PAR1 phosphorylation, thrombin-stimulated phosphorylation persists after inhibition or downregulation of PKC, suggesting other mechanisms of agonist-induced phosphorylation (136). One principal mediator of agonist-stimulated phosphorylation of PAR1 is GRK3, since overexpression of GRK3 enhances agonist-induced PAR1 phosphorylation and suppresses thrombin signaling, whereas GRK2 is considerably less effective. The importance of GRK3 in desensitization of PAR1 has been confirmed in vivo by observations in transgenic mice overexpressing GRK3 in the myocardium (133). Although signaling of the 2-adrenergic receptor is unaltered in these animals, since this receptor is mostly regulated by GRK2, thrombininduced signaling is markedly attenuated by overexpression of GRK3. However, the observation that signaling of GRK3-insensitive mutants of PAR1 is still rapidly attenuated suggests the existence of additional mechanisms, perhaps involving other GRKs (136). GRK5 mediates PAR1 desensitization in endothelial cells, where GRK5 expression suppresses thrombin signaling and a dominant negative GRK5 mutant prolongs thrombin signals (278). Physiol Rev • VOL -Arrestins are cofactors for GRKs; they interact with GRK-phosphorylated GPCRs at the cell surface and disrupt their association with heterotrimeric G proteins to terminate the signal. -Arrestins also mediate desensitization of PAR1 (225). Desensitization of PAR1 is markedly diminished in embryonic fibroblasts prepared from mice lacking both -arrestin-1 and -2, and also in fibroblasts lacking only -arrestin-1, indicating a major role of -arrestin-1 in desensitization of PAR1 in fibroblasts. This observation is the first to show a differential role of -arrestin isoforms in desensitization of a GPCR. Several observations suggest that modifications of the tethered ligand domain of cleaved PAR1 may also contribute to desensitization. One mechanism may involve cleavage of the tethered ligand domain. Exposure of platelets to PAR1 AP induces release of a PAR1 fragment by a process inhibited by soybean trypsin inhibitor, suggesting that a trypsinlike enzyme cleaves activated PAR1 (223). In rat astrocytes, thrombin exposure strongly desensitizes Ca2⫹ mobilization to a second exposure to thrombin and PAR1 AP (289). In contrast, exposure to PAR1 AP desensitizes the response to subsequent challenge with thrombin but not to AP. These findings led to the hypothesis that after activation with AP, the tethered ligand domain is proteolytically destroyed, rendering the receptor unresponsive to thrombin but not AP. Two observations support this hypothesis. First, as predicted, exposure to thermolysin, which removes the tethered ligand, generates a receptor that responds to PAR1 AP but not to thrombin. Second, treatment with soybean trypsin inhibitor enhances responses to thrombin and PAR1 AP, suggesting the existence of an exogenous protease that contributes to desensitization. The identity of the protease remains to be determined. The potential importance of modifications of the exposed tethered ligand is also illustrated by a study of PAR1 signaling in Sf9 insect cells. PAR1 signals are prolonged in these cells, even after washout of agonist (44). However, treatment with thermolysin, which removes the tethered ligand, attenuates the otherwise sustained Ca2⫹ response. 2. PAR2 Responses to PAR2 activators are rapidly attenuated and strongly desensitized to repeated exposure, indicating the existence of powerful mechanisms that control signaling, which have not been fully characterized. PKC appears to play an important role in regulating PAR2 (23, 86). Thus activation of PKC with phorbol esters abolishes PAR2-induced Ca2⫹ signaling in transfected cell lines and enterocytes that naturally express this receptor, whereas PKC inhibitors magnify responses to PAR2 activators. Mutation of a putative PKC site in the carboxy tail (ST336/6 3 A) renders the receptor unresponsive to inhibition by phorbol esters (86). This receptor is also de- 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 adrenergic receptor (reviewed in Refs. 22, 181). The general mechanism of desensitization of these receptors is as follows (Fig. 6A). Ligand occupation of the GPCR induces the translocation of members of the family of G protein receptor kinases (GRKs) from the cytosol to the activated receptor at the cell surface. GRKs are serine-threonine kinases that phosphorylate activated GPCRs, usually within the carboxy terminus or third intracellular loop. Phosphorylation triggers the membrane translocation of arrestins, which interact with the phosphorylated GPCR, disrupt association with heterotrimeric G proteins, and thereby terminate signal transduction. Additional mechanisms may involve phosphorylation by second messenger kinases, which also terminate signaling. However, there remain many critical aspects of desensitization of GPCRs, including PARs, that are unexplored. In many cases, the GRK and arrestin isoforms that are coexpressed with receptors in question and which mediate desensitization to physiological stimuli are unknown. Most information about desensitization derives from studies of transfected receptors in cell lines; although genetically modified animals that are deficient in certain GRKs and arrestins are available, comparatively little is known about mechanisms of desensitization in vivo. Mechanisms of desensitization also vary between different PARs, probably due to structural differences, especially in the intracellular loop III and carboxy terminus. 595 596 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT sensitization defective: Ca2⫹ responses to trypsin and PAR2 AP are exaggerated, and there is diminished desensitization to repeated stimulation. The role of GRKs and -arrestins in PAR2 desensitization is unknown. 3. PAR3 and PAR4 C. PARs Are Downregulated by Intracellular Proteases In addition to processes that regulate coupling of receptors to signaling pathways, cells also determine their responsiveness to agonists by regulating the levels of receptors that are expressed at the plasma membrane and which are thus accessible to agonists in the extracellular fluid. The level of expression of receptors at the cell surface is a balance between removal by endocytosis and replenishment by recycling or mobilization of intracellular pools. Many receptors internalize after binding agonists. However, the fate of endocytosed receptors depends on postendocytic sorting, which varies from receptor to receptor. At one extreme, some GPCRs that are activated in a reversible fashion by agonist binding, for instance the neurokinin-1 receptor, efficiently recycle to the plasma membrane to be reused by the cell (85). At the other extreme, receptors such as the PARs, which, once cleaved, cannot be reused by the cell, are destined for intracellular degradation, which irrevocably terminates signaling (Fig. 6B). For these receptors, recovery requires synthesis or mobilization of new receptors. Of course, all surface receptors have a finite life span and even recycling receptors are downregulated by intracellular degradation after long-term stimulation. The process of receptor downregulation is of considerable importance because defects can result in exaggerated signaling and abnormal phenotypes, for example, transformation in the case of EGF receptors that are not downregulated. PARs are an ideal model to study these postendocytic sorting events since they are invariably targeted for degradation after activation. The molecular mechanisms and pathways of agonist-induced trafficking of PARs vary from receptor to receptor and in different cells. 1. PAR1 PAR1 is rapidly and extensively internalized after activation: stimuli trigger endocytosis of ⬎85% PAR1 Physiol Rev • VOL 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 Little is known about desensitization of PAR3 or PAR4. PAR3 has a very short carboxy tail, with possible implications for desensitization, since truncated receptors frequently exhibit diminished desensitization. PAR4 is not rapidly phosphorylated after activation, possibly due to lack of sites for GRK phosphorylation in the carboxy tail, and in consequence, PAR4 slowly desensitizes (263). The slow kinetics of desensitization may permit PAR4 to signal in a sustained fashion in human platelets. within 1 min in erythroleukemic and megakaryoblastic cell lines, although endocytosis proceeds a little more slowly in other cell types (129). The initial accumulation of PAR1 into coated pits suggests that internalization proceeds by a clathrin-mediated process. In support of this suggestion, activated PAR1 colocalizes with clathrin in PAR1-transfected fibroblasts, and disruption of clathrin with hypertonic sucrose (causes abnormal clathrin polymerization) and monodansylcadaverine (disrupts imagination of clathrin-coated pits) markedly inhibits endocytosis of activated PAR1 (281). The GTPase dynamin mediates detachment of clathrin-coated pits, and expression of a GTPase-defective dynamin mutant (K44E) also inhibits trafficking of activated PAR1. In addition to endocytosis of activated receptors, PAR1 also undergoes constitutive endocytosis in the absence of proteolytic activation. However, whereas activated PAR1 is mostly destined for lysosomal degradation (129), constitutively endocytosed PAR1 recycles to the plasma membrane (261). Indeed, there is a tonic endocytosis and recycling of PAR1 that maintains receptor at the cell surface and in an intracellular pool. Distinct domains exist in the carboxy tail of PAR1 that are required for stimulated and constitutive endocytosis, suggesting that the two types are endocytosis proceeding by distinct mechanisms (262). -Arrestins play a major role in endocytosis of many GPCRs by serving as adaptor proteins that link GRKphosphorylated receptors to clathrin and AP2. However, -arrestins play no role in stimulated endocytosis of PAR1, which proceeds with normal kinetics in fibroblasts lacking -arrestin-1 and -2, whereas agonist-induced endocytosis of the 2-adrenergic receptor is inhibited in this system (225). In addition, constitutive PAR1 trafficking proceeds normally in fibroblasts that lack -arrestins. Different adaptor proteins, such as AP2, may participate in endocytosis of PAR1. Compared with our understanding of the molecular mechanisms of endocytosis, less is known about the mechanisms of postendocytic sorting of internalized GPCRs, either to degradation or recycling pathways. Once internalized, most activated PAR1 is targeted to lysosomes for degradation. However, lysosomal sorting is not complete because in some cells there is considerable recycling (129). Progress has been made in defining the molecular mechanisms that target PAR1 to lysosomes. Sorting nexin-1 is a membrane-associated protein that interacts with the carboxy tail of the EGF receptor and is required for its downregulation. The yeast ortholog of sorting nexin-1, Vps5p, participates in targeting hydrolases to the vacuole, the yeast equivalent of the lysosome. In PAR1-transfected HeLa cells, agonist stimulation induces colocalization of PAR1 in endosomes with sorting nexin-1 (311). Expression of a carboxy-terminal deletion mutant of sorting nexin-1 has no effect on endocytosis of activated PAR1, but induces its accumulation in early PROTEASE-ACTIVATED RECEPTORS 2. PAR2 Like PAR1, activated PAR2 is endocytosed and trafficked to lysosomes (23, 90). Endocytosis of PAR2 proceeds by a clathrin-mediated mechanism since it is inhibited by blockade of clathrin function. Endocytosis of PAR2 also requires dynamin, because GTPase defective dynamin K44E inhibits endocytosis (242). In contrast to PAR1, endocytosis of PAR2 requires -arrestins. Thus activation of PAR2 induces the marked and rapid translocation of -arrestin-1 from the cytosol to the plasma membrane, followed by a prolonged colocalization of PAR2 and -arrestin-1 in early endosomes (86, 90). Eventually, PAR2 and -arrestin-1 are sorted into distinct compartments: PAR2 is targeted to lysosomes, and -arrestin-1 returns to the cytosol. The importance of -arrestins in endocytosis of PAR2 is indicated by two observations. First, expression of a dominant negative mutant of -arrestin, -arrestin318 – 419, which comprises the clathrin binding domain, prevents endocytosis. Second, a PAR2 mutant, ST363/6 3 A, fails to internalize and shows impaired desensitization, because of its inability to interact with -arrestins. Thus PAR2 appears to belong to the class B GPCRs that form high-affinity interactions with arrestins. -Arrestins also play an important role in PAR2mediated activation of ERK1/2 as discussed in section IIIC (86). Progress has been made in defining the mechanisms of intracellular trafficking of PAR2. The rab GTPases mediate many steps of vesicular trafficking of proteins to and from the plasma membrane and between organelles. Rab5a colocalizes with PAR2 in early endosomes within minutes of activation (242). Expression of a GTPase defective mutant, dominant negative rab5aS34N, disrupts early endosomes and inhibits endocytosis of activated PAR2. Thus rab5a mediates distal steps in endocytic trafficking of PAR2 from clathrin-coated pits to early endoPhysiol Rev • VOL somes. Once internalized, PAR2 translocates to lysosomes and is degraded, as determined by Western blotting (Bunnett, unpublished observation). Although the molecular mechanisms that target PAR2 for degradation are unknown, PAR2 is extensively ubiquitinated after activation, determined by receptor immunoprecipitation and Western blotting for ubiquitin (Bunnett, unpublished observation), and ubiquitination of some other GPCRs is a prerequisite for degradation (185). D. Sustained Signaling by Proteases Requires Mobilization and Synthesis of New Receptors PARs are activated by an irreversible mechanism, and once cleaved, most activated PARs are destined for lysosomal degradation. Thus PARs are “one-shot” receptors, and sustained signaling requires the mobilization of intracellular stores of intact receptors or the synthesis of new receptors. The importance of these mechanisms depends on whether cells possess prominent intracellular stores of PAR1. In megakaryoblasts, where most receptors are rapidly internalized after thrombin cleavage, recovery is a slow process that depends on the synthesis of new receptors (26, 129). In contrast, resensitization of responses to PAR1 activators in endothelial cells and fibroblasts depends on mobilization of the prominent stores of PAR1 in the Golgi apparatus (121, 125). Platelets, which lack both the ability to synthesize new receptors and prominent intracellular pools, are unable to repopulate the plasma membrane with new receptors after thrombin exposure and thus do not recover responsiveness to thrombin. Because platelets are only required to respond once to thrombin, by aggregation, the lack of a robust system of resensitization does not impair their function. Moreover, human platelets also express PAR4, which facilitates prolonged signaling by thrombin. Similar mechanisms account for the resensitization of PAR2. There are prominent intracellular pools of PAR2 in many cells, including enterocytes (23). Resensitization of responses to trypsin and the replenishment of the cell surface with intact PAR2 is minimally affected by cycloheximide but attenuated by disruption of the pools with brefeldin A, suggesting that resensitization requires mobilization of pools. Similar mechanisms of PAR2 resensitization have been observed in vascular tissues in vitro, suggesting that these are widespread mechanisms (113). The role of rab GTPases in trafficking of PAR2 from Golgi stores to the cell surface has been examined. Rab11a colocalizes in the Golgi apparatus with PAR2, and PAR2 activators stimulate redistribution of rab11a into vesicles containing PAR2 that migrate to the cell surface (242). Expression of GTPase defective rab11aS25N causes retention of PAR2 in the Golgi apparatus, and overexpression of wild-type rab11a accelerates both re- 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 endosomes and inhibits its degradation of lysosomes. Thus sorting nexin-1 participates in sorting of PAR1 from early endosomes to lysosomes. The domains of PAR1 that specify targeting to lysosomes are not fully defined, although substitution of the carboxy tail of PAR1 with the equivalent domain of the neurokinin-1 receptor (which recycles) alters PAR1 trafficking to the recycling pathway (282, 283). This recycled receptor appears to be tonically active, which emphasizes the importance of lysosomal degradation as a mechanism that unequivocally terminates PAR1 signaling. Conversely, the neurokinin-1 receptor with the carboxy tail of PAR1 is targeted to lysosomes. Thus carboxy-terminal domains of PAR1 are important for lysosomal trafficking. This lysosomal trafficking of PAR1 (and PAR2) proceeds whether the receptor is activated by proteolysis or by AP, and thus is not unique for the cleaved receptor. 597 598 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT covery of PAR2 at the cell surface and resensitization of PAR2 signaling. Thus rab11a plays an important role in PAR2 resensitization. Of interest, rab5a promotes and rab5aS34N impedes resensitization of cells to trypsin. Thus rab5a is required for PAR2 endocytosis and resensitization, whereas rab11a contributes to trafficking of PAR2 from the Golgi apparatus to the plasma membrane. V. PROTEASE-ACTIVATED RECEPTORS: CONTRIBUTION TO PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL CONTROL MECHANISMS FIG. 7. A summary of the potential physiological and pathophysiological roles of proteases and PARs in different cell types. Signaling depends on the availability of proteases, which can activate or disable PARs, protease inhibitors, PARs, and protease cofactors and anchoring proteins. Proteases signal to a wide variety of cells to regulate critically important biological processes, with implications for physiological regulation and disease. Physiol Rev • VOL 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 Although much is known about the potential functions of PARs (Fig. 7), there remain substantial obstacles to understanding the role of proteases and PARs in physiology and disease. First, the system is complex: protease signaling depends on availability of activating or disabling proteases, protease inhibitors, and cofactors or anchoring proteins. Understanding the system requires knowledge of all of these components, and in many situations this information is lacking. Second, most studies rely on administration of activators (proteases or APs). Although such studies provide information about the potential role of PARs, they do not provide direct information about their function in physiology or diseases states. Proteases and certain APs are not selective activators of PARs, and APs are agonists at only very high concentrations (usually ⬎1 M). Peptides at high concentrations can have biological effects that are unrelated to receptor occupancy. For example, inactive analogs of PAR2 AP cause edema upon intraplantar injection (296). Third, selective antagonists of PARs are not widely available. Although antagonists have been developed for PAR1 (9), the intramolecular mechanism of activation of PARs may hamper the identification of antagonists by traditional methods involving screening of large chemical libraries. There are recent reports of peptide-based antagonists of PAR1 and PAR4 PROTEASE-ACTIVATED RECEPTORS A. Protease Signaling in the Circulatory and Cardiovascular Systems The contributions of proteases and their receptors to regulation the cardiovascular system have been recently reviewed (50, 68, 226, 240). 1. Proteases As discussed in section III, coagulation and anticoagulation factors, in particular thrombin, FVIIa, FXa, and APC, can regulate cells by cleaving PARs. Proteases released from inflammatory cells within the circulation and in tissues can also activate PARs in the circulatory and cardiovascular systems. For example, cathepsin G from neutrophils is a potential activator of PAR4 on platelets (249). Endothelial cells also express trypsins (169), which could activate PAR2 and PAR4, although almost nothing is known about the function of endothelial trypsins. 2. PARs PARs are expressed by circulating cells as well as by endothelial and vascular smooth muscle cells. Platelets Physiol Rev • VOL express several receptors for thrombin, although the combination varies between species: human platelets express PAR1 and PAR4 (142), murine platelets express PAR3 and PAR4 (143), and guinea pig platelets express PAR1, PAR3, and PAR4 (8). Neutrophils also express PAR2 (128). Human umbilical vein endothelial cells express PAR1, PAR2, and PAR3 (194, 218), and vascular smooth muscle cells express PAR1 and PAR2 (79). 3. PAR signaling to circulating cells Platelets are circulating cells that are critical mediators of coagulation. Thrombin cleaves PARs on platelets to cause aggregation, granular secretion, and induction of procoagulant activity, which are essential for hemostasis. Thrombin signaling to platelets is enhanced by interaction with surface proteins such as glycoprotein I␣ (84) or by direct binding to PAR1 or PAR3, which possess hirudinlike thrombin-binding domains (135, 306). Thrombin signals by activating PAR1 and PAR4 in human platelets (142) and PAR4 alone in murine platelets, since PAR3 does not signal in murine platelets (199). What is the relative importance of PAR1 and PAR4 in human platelets? In isolated human platelets, antagonism of PAR1 blocks responses to low concentrations of thrombin, and antagonism of both receptors inhibits responses to low and high thrombin concentrations (142). Activation of PAR1 with AP is as efficacious as thrombin in inducing aggregation, Ca2⫹ influx, and development of procoagulant activity in human platelets, whereas PAR4 AP is less efficacious (7). Although these results could indicate that PAR1 is the primary thrombin receptor on human platelets, with PAR4 functioning in reserve, they may also reflect a generally lower efficacy of PAR4 AP as an agonist of that receptor. PAR4 may also contribute to sustained actions of thrombin on platelets and thereby mediate the late phase of platelet activation (70). In addition, PAR4 could act as a platelet receptor for other proteases, such as cathepsin G (249). In murine platelets, which express PAR3 and PAR4, PAR3 does not signal but instead anchors thrombin to the cell surface by its hirudin-like domain and thereby facilitates thrombin cleavage of PAR4 (199, see sect. IID2). The contributions of PARs to coagulation have also been investigated in vivo by use of antagonists to PAR1. In cynomologus monkeys, whose platelets express PAR1 and PAR4, an antagonist of PAR1 inhibits thrombus formation and vessel occlusion in a model of electrolytic injury of the carotid artery (88). These studies indicate that PAR1 plays a predominant role in platelet regulation in this species. However, the importance of a dual receptor system on murine platelets in vivo is illustrated by the observation that deletion of PAR3 or PAR4 results in prolonged bleeding time and protection from ferric chloride-induced thrombosis of mesenteric arterioles and thromboplastin-induced pulmonary embolism (313). The 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 (termed pepducins) that mimic intracellular domains of these receptors, but they have yet to be widely used (71, 72). Although it is generally more straightforward to develop drugs that inhibit proteases, it can be difficult to attain absolute selectivity between related enzymes. In the absence of selective activators and antagonists, the best current approach to specifically determine the role of PARs in vivo is to study genetically modified animals. Mice have been developed that lack all known PARs (64, 83, 143, 177, 255, 313), and by selective crossing it will be possible to breed animals that lack several receptors. Transgenic animals have also been identified that overexpress PAR2 (255). However, the use of genetically modified mice is also fraught with problems. Effects of deletion on embryonic survival and fertility impede the breeding of animals for experimentation: approximately one-half of PAR1 knockout mice die before birth (64, 83). Compensatory alterations in the expression or activity of other PARs can occur in knockout animals (77), but in general these possibilities have not been thoroughly tested. There are also major differences in biological responses between different strains of mice, and it is essential to study wild-type and knockout animals in the identical genetic background, requiring the use of littermates or extensive back-crossing. Finally, many experimental models using mice are very imperfect models of human disease, requiring caution in extrapolating observations in mice to humans. With these caveats in mind, we will briefly discuss the roles of proteases and their receptors in several systems. 599 600 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT role of PAR4 in vivo has also been examined by use of novel antagonists, the pepducins (71). Peptides derived from the third intracellular loop of PAR1 and PAR4 with attached palmitate groups are potent inhibitors of thrombin-mediated aggregation of human platelets. Treatment of mice with a PAR4 pepducin prolongs bleeding time and protects against systemic platelet activation, consistent with the phenotype of PAR4-deficient mice (72). Thus PAR4 plays an important role in thrombin signaling in mice. 4. PAR signaling to the vessels Physiol Rev • VOL 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 Proteases can signal to endothelial cells and vascular smooth muscle cells to control resistance and blood flow, extravasation of plasma proteins and granulocytes, and angiogenesis. The effects of PAR activators on the contractile state of blood vessels has been extensively examined by classical organ bath pharmacology. The net effect of PAR activators depends on the receptor, the vessel, and whether the effect is mediated by release of agents from the endothelium of by direct actions on vascular smooth muscle. Thrombin and selective PAR1 agonists invariably cause relaxation of precontracted blood vessels, including large conduit-like vessels, for example, human pulmonary artery (116), porcine coronary artery (114), and rat aorta (184), and also of smaller resistance vessels, including human and porcine intramyocardial arteries (117). The consensus of these studies is that PAR1-induced relaxation is prevented by removal of the endothelium and is thus mediated by the secretion of a factor from endothelial cells that relaxes vascular smooth muscle. Relaxation is frequently suppressed by inhibition of nitric oxide (NO) synthase and is thus mediated in part by the release of NO from endothelial cells. However, there is also an NO-independent mechanism. In porcine coronary artery, this NO-independent mechanism involves the release of an endothelial-derived hyperpolarizing factor (114). In human pulmonary arteries, products of cyclooxygenase also contribute to relaxation (116). PAR1 activators can also contract certain vessels by endothelium-dependent and -independent mechanisms (277). Activators of PAR2 also relax vascular tissues. Thus trypsin and PAR2 AP cause relaxation of a wide range of vessels from several species, including rat pulmonary artery (243) and human and porcine coronary and intramyocardial arteries (117). PAR2-mediated relaxation depends on the presence of the endothelium and involves both NO-dependent and -independent mechanisms. PAR2 AP also induces an endothelium-dependent contraction of, for example, rat aorta and renal, mesenteric, and pulmonary arteries (243). However, this contraction may involve a subtype of PAR2 in view of the different potencies with which analogs of the PAR2 AP induce contraction versus relaxation. The existence of a subtype receptor is also suggested by use of a “sandwich assay,” in which a segment of human umbilical vein is sandwiched next to a segment of endothelium-denuded rat aorta that serves as a “reporter” for the release of vasoactive agents from the human tissue (248). PAR2 AP induces release of an unknown factor from human umbilical vein that stimulates contraction of a rat aorta. Notably, trypsin does not have this effect, and the mechanism is thus independent of PAR2. The effects of PAR activators on cardiovascular responses have been investigated in intact animals. In mice, intravenous injection of PAR1-selective AP causes a rapid and sustained hypotension, which is not NO mediated (47). Similarly, in rats and mice, PAR2 activators cause hypotension by NO-dependent and -independent mechanisms, which can be followed by a reflex hypertension (52). The effects of PAR2 activators on blood flow have recently been investigated in humans. Administration of PAR2 AP causes dilation of resistance vessels in the arm with an elevated blood flow, by NO- and prostaglandindependent mechanisms (241). What are the physiological or pathophysiological implications of regulation of blood flow by PARs? Under normal circumstances it is unlikely that PARs regulate blood flow; indeed, there are no obvious abnormalities in the circulatory system in mice lacking PARs. However, PARs may play an important role during inflammation and sepsis, when there are elevated levels of proteases from inflammatory cells, the coagulation cascade and other sources, together with an elevated expression of PARs themselves. Exposure of endothelial cells in culture to inflammatory mediators such as TNF-␣ and lipopolysaccharide induces a selective upregulation of PAR2 but not PAR1 (216). Administration of endotoxin to rats also results in an upregulation of PAR2 in the vascular system that is accompanied by an increased hypotensive response to PAR2 activators (53). Similarly, exposure of segments of human coronary artery to interleukin-1␣ and TNF-␣ upregulates PAR2 and PAR4 and exacerbates PAR2- and PAR4-mediated relaxation of tissues (115). Inflammation is accompanied by marked alterations in the permeability of blood vessels to plasma proteins and cells. Many inflammatory signals trigger the formation of gaps between endothelial cells of postcapillary venules, resulting in plasma extravasation and edema; these stimuli can also induce expression of adhesion molecules that promote the firm adhesion and ultimate infiltration of granulocytes into tissues. Proteases from the coagulation cascade and from inflammatory cells can trigger these events. The effects of thrombin and PAR1 on extravasation of plasma proteins have been studied in cultured endothelial cells and in the intact animal. Thrombin stimulates contraction of endothelial cells from the human umbilical vein, which results in gap formation and PROTEASE-ACTIVATED RECEPTORS Physiol Rev • VOL vascular matrix after injury (48). Activation of PAR2 also stimulates proliferation of endothelial cells (194). In a murine model of limb ischemia, trypsin and PAR2 AP promote angiogenesis and accelerate homodynamic recovery of the limb, suggesting a role for PAR2 in angiogenesis (191). B. Protease Signaling to the Nervous System The role of proteases and PARs in the nervous system is a topic of recent reviews (295, 308). 1. Proteases Protease activators of neuronal PARs may derive from the circulation, from inflammatory cells that are resident in or recruited to neural tissues, or from neurons or astrocytes. Coagulation cascade proteases could signal to the nervous system during trauma and inflammation when there is elevated vascular permeability. However, these proteases are also expressed within the nervous system, although at low levels. Prothrombin transcripts are present in certain regions of the brain that also express PAR1 (91, 312). For example, in the olfactory bulb prothrombin and PAR1 mRNA are coexpressed in the mitral and granular cell layers. Elsewhere in the olfactory bulb and neocortex, the diffuse expression of prothrombin contrasts with the more defined expression of PAR1. Nevertheless, the potential exists for neuronally derived thrombin to activate PAR1 in the nervous system. Proteases from inflammatory cells may also signal to the nervous system. Granzyme A is released from CD4⫹ cytotoxic lymphocytes that infiltrate the brain during experimental allergic encephalomyelitis (272). Granzyme A cleaves a fragment of PAR1 at the activation site, and granzyme A signaling to astrocytes and neurons is inhibited by a PAR1 blocking antibody, suggesting that granzyme A activates PAR1. Neural tissues also express a large number of proteases that potentially activate PARs (252). The biological effects of proteases are tempered by protease inhibitors, and neuronal tissues are replete with an array of inhibitors for proteases that activate PARs. The serpin protease nexin-1 is the most efficient endogenous inhibitor of thrombin. Nexin-1 mRNA is expressed in the brain where it is found in glial cells, synapses, and at high levels in capillaries and vascular smooth muscle in the human cortex (102). Thrombomodulin is a transmembrane glycoprotein that can form complexes with proteases and thereby regulates their activity at the cell surface. Thrombomodulin is expressed by astrocytes in culture, and expression of thrombomodulin is upregulated during trauma and consequent astrogliosis, as well as by activation of PAR1 (230). The upregulation of thrombomodulin by thrombin may thus serve to control activation of PAR1 on astrocytes. 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 increased permeability of confluent cells (174). The intraplantar injection of thrombin and selective PAR1 AP causes edema formation in the rat paw, indicative of plasma extravasation (299). This effect is partly dependent on degranulation of mast cells and release of serotonin and histamine, and partly due to activation of PAR1 on the peripheral endings of primary spinal afferent neurons, and release of SP (87). By use of intravital microscopy it has been shown that thrombin stimulates leukocyte rolling and adhesion when applied topically to postcapillary venules of the rat mesentery (297). However, this response is neither prevented by a PAR1 antagonist nor reproduced by PAR1-selective AP, but may be mediated by PAR4 since PAR4 AP also induces the response. In a similar manner, activation of PAR2 induces edema of the paw and also causes infiltration of granulocytes (298). In this case the edema is mediated in large part by the release of neuropeptides from sensory nerves and is thus dependent on a neurogenic mechanism (270). However, PAR2-induced infiltration of granulocytes is not mediated by the nervous system, but probably by direct effects of PAR2 activators on endothelial cells. Activation of PAR2 also stimulates leukocyte rolling, adhesion, and extravasation in postcapillary venules of the rat mesentery, by a mechanism that depends on release of platelet activating factor (294). Similarly, administration of PAR2 AP to the cremaster muscle of mice induces leukocyte rolling and adherence to venules (177). There is a modest decrease in the surgically induced rolling of neutrophils that is observed in PAR2-deficient mice, suggesting involvement of PAR2 in inflammation. Activation of PAR1 by thrombin and AP stimulates proliferation of endothelial (194) and vascular smooth muscle cells (189). Activation of PAR4 similarly induces proliferation of vascular smooth muscle cells, suggesting that PAR1 and PAR4 may mediate the effects of thrombin (27). The proangiogenic effects of thrombin involve interactions with vascular endothelial growth factor (VEGF), itself a potent angiogenic factor. PAR1 activators upregulate expression of VEGF receptors (KDR and flt-1) by endothelial cells and potentiate the mitogenic effects of VEGF on endothelial cells (285). VEGF itself also promotes thrombin generation. These results suggest the existence of a mechanism of amplification of the angiogenic response involving PAR1 and VEGF receptors. In view of the proangiogenic effects of thrombin, it is not surprising that thrombin and PAR1 may play a role in vascular development. Deletion of prothrombin results in abnormalities of the vasculature of the yolk sac, and deletion of PAR1 results in partial embryonic lethality that is associated with bleeding abnormalities and which can be rescued by overexpression of PAR1 in endothelial cells (110). The role of PAR1 in responses to mechanical damage has also been evaluated in the mouse, which indicates involvement of PAR1 in regulation of the formation of the 601 602 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT 2. PARs 3. PAR signaling in the nervous system The regulation of [Ca2⫹]i in neuronal cells is of fundamental importance in the control of excitability, release of neurotransmitters, and survival, and the signaling of proteases in neurons and astrocytes is commonly studied by measuring generation of second messengers, such as Ca2⫹. All four PARs are expressed in astrocytes (309). Thrombin and PAR1 AP increase [Ca2⫹]i in a glioma cell line and in astrocytes from the rat brain (287, 288). The nature of the response depends on the duration of the stimulus: brief exposure triggers a rapid, transient increase in [Ca2⫹]i, whereas continued exposure induces oscillations or a sustained increase that are attributable to an influx of extracellular Ca2⫹. The influx of extracellular Ca2⫹ and the refilling of internal stores of Ca2⫹, which are both essential for oscillations in [Ca2⫹]i, depend on actiPhysiol Rev • VOL 4. Morphology Innervation depends on the control of neurite outgrowth, and there is great interest in understanding the factors that control this process. Proteases and protease inhibitors regulate the outgrowth of neurites. Neuroblastoma cells and certain primary cultures of neurons rapidly extend neurites when switched from serum-containing to serum-free culture medium. Thrombin inhibits neurite sprouting in serum-free medium (112). Conversely, neurite outgrowth in the presence of serum is stimulated by the thrombin inhibitors protease nexin-1 and hirudin, probably due to inhibition of thrombin in serum. Thrombin similarly inhibits the outgrowth of neurites from dorsal root ganglia (106). Astrocytes contribute to the bloodbrain barrier, and there is considerable interest in understanding the control of astrocytes morphology. Protease and protease inhibitors regulate the morphology of astrocytes (14, 39). When cultured in serum, astrocytes adopt a flattened, fibroblast-like morphology, whereas in serumfree conditions they become stellate and project long processes. Thrombin mimics the effects of serum, whereas protease nexin-1 reverses the effect of thrombin. These effects of thrombin on the morphology of neurons and astrocytes are mimicked by PAR1 AP and are thus likely attributable to activation of PAR1. Rho GTPases regulate the actin-based cytoskeleton and thereby participate in the control of cell shape and migration. Indeed, thrombin-stimulated neurite retraction and rounding of neurons is blocked by treatment of cells with Clostridium botulinum toxin C3 exoenzyme, which ADP-ribosylates and thereby inactivates small Rho GTP-binding proteins, implicating Rho in this action of thrombin and PAR1 (138). 5. Survival and proliferation Proteases and PARs have important effects on the survival of neurons and astrocytes, with major implications for neuronal functions during trauma and inflammation. The effects of thrombin on survival of neurons and astrocytes depend on the concentration of activators and the duration of exposure. Low concentrations of PAR1 activators are protective. Thrombin (⬍10 nM, 48 h) and PAR1 AP markedly protect cultured astrocytes and hypo- 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 Expression of PAR1 mRNA in the rat brain is higher at birth compared with postnatal day 28, and levels increase again in adulthood (206, 207). PAR1 is widely expressed throughout the rat brain, with higher levels of expression in particular regions, where it is likely to be present in neurochemically and functionally distinct cell types (312). Thus PAR1 mRNA is expressed in the neocortex, cingulate/retrosplenial cortex, subiculum, nuclei within the hypothalamus and thalamus, and in discrete layers of the hippocampus, cerebellum, and olfactory bulb. In the hypothalamus, PAR1 is present in dopaminergic neurons, but it is likely to be present in GABAergic granule cells in the olfactory bulb and in glutaminergic neurons of the anterior thalamus. PAR1 is also expressed at low levels in glial cells throughout the brain, and by ependymal cells of the choroid plexus and ventricular lining where it could participate in control of transport of cerebrospinal fluid. Within the spinal cord, PAR1 mRNA is expressed in motoneurons and in preganglionic neurons of the autonomic nervous system. PAR1 is also present in the peripheral nervous system. In dorsal root ganglia, PAR1 colocalizes in neurons with the neuropeptides SP and calcitonin gene-related peptide (CGRP) (87). These are primary spinal afferent neurons that are known to participate in neurogenic inflammation and nociception. PAR1 is present in more than half of the neurons in the myenteric plexus of the guinea pig small intestine, suggesting a role for PAR1 in the control of intestinal motility (67). PAR2 is also expressed within the brain. Thus PAR2 is found within neurons and glial cells of the hypothalamus (79, 265). In the periphery, PAR2 is expressed in primary spinal afferent neurons with SP and CGRP (270) and by a large proportion of neurons in the myenteric and submucosal plexuses of the guinea pig and mouse small intestine (67, 73, 235). vation of protein kinase C by PAR1. The response of astrocytes to thrombin may be mediated by two coexpressed receptors, PAR1 and PAR4 (148). Thus, in primary cultures of human astrocytomas, thrombin as well as PAR1 and PAR4 AP induce elevations in [Ca2⫹]i. Normal brain astrocytes express PAR2 and respond to trypsin and PAR2 AP (290). Selective agonists of PAR1 and PAR2 also signal enteric neurons and primary spinal afferent neurons to increase [Ca2⫹]i, providing functional evidence for expression of these receptors (67, 87, 270). PROTEASE-ACTIVATED RECEPTORS Physiol Rev • VOL in these cells. Thus antagonists of PAR4 could be effective therapies for neurotrauma. 7. Neurogenic inflammation and pain A subpopulation of primary sensory neurons with cell bodies in dorsal root, trigeminal, and vagal (nodose and jugular) ganglia express the neuropeptides SP and CGRP. These neurons have small, dark cell bodies with unmyelinated (C) or thinly myelinated (A-␦) fibers and participate in neurogenic inflammation and nociception. The peripheral projections of these fibers detect thermal, chemical, and high-threshold mechanical stimuli in many tissues, including the skin, gastrointestinal tract, and airway. Stimulation results in the release of SP and CGRP from endings of primary sensory neurons in peripheral tissues, which initiates a variety of responses, collectively referred to as “neurogenic inflammation,” and characterized by arteriolar vasodilatation, extravasation of plasma proteins from postcapillary venules, and adhesion of leukocytes to the venular endothelium. These same stimuli also generate action potentials that are transmitted centrally where they induce the release of neuropeptides within the central nervous system, resulting in the central transmission of nociceptive signals. Proteases that activate PAR1 and PAR2 can signal to these neurons in peripheral and perhaps central tissues to control inflammation and pain (11, 87, 270, 296) (Fig. 8). The role of PAR2 in sensory neurons has been extensively investigated. PAR2 is expressed by ⬃65% of neurons in the dorsal root ganglia of the rat; 25–35% of these PAR2-expressing neurons also express SP and CGRP, respectively, and are thus involved in inflammation and pain (270). Trypsin, tryptase, and PAR2 AP signal to these neurons to elevate [Ca2⫹]i and to stimulate secretion of SP and CGRP from segments of the dorsal horn, bladder, and atria, indicating that activation of PAR2 triggers peptide release from both the central and peripheral projections of these neurons. The intraplantar injection of PAR2 activators causes a sustained (⬎6 h) edema that is mediated by release of SP and CGRP from the peripheral projections of sensory nerves. Activation of PAR2 also induces hyperalgesia (149, 150, 296). Thus intraplantar injection of subinflammatory doses of PAR2 AP causes sustained (⬎24 h) mechanical and thermal hyperalgesia, with increased expression of fos protein in spinal neurons, indicative of neuronal activation. Hyperalgesia is not observed in mice lacking the neurokinin-1 (SP) receptor or preprotachykinin A, which encodes SP and neurokinin A. Neurokinin-1 receptor antagonists also prevent PAR2-induced hyperalgesia, but they are effective only if capable of penetrating the spinal cord or if delivered intrathecally. Thus PAR2-mediated hyperalgesia depends on the release of SP from the central projections of primary spinal afferent neurons. Together, these findings 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 thalamic neurons from death in response to environmental insults, including hypoglycemia and oxidative stress (293). Similarly, thrombin (50 pM, 1 h) and PAR1 AP protect hippocampal neurons in brain slices from death in response to experimental ischemia (271). This protective effect of thrombin is also observed in vivo. Thus brief occlusion of the common carotid arteries of gerbils induces tolerance of hypothalamic CA1 neurons to a more sustained ischemia. Intracerebral injection of the thrombin inhibitor hirudin prevents this protection, indicating involvement of thrombin (271). Thrombin-induced protection of astrocytes from hypoglycemia is attenuated by general inhibitors of serine/threonine kinases and by inhibition of Rho, suggesting involvement of the actin cytoskeleton (92). Another protease that activates PAR1, APC, also protects the brain from ischemic injury by a PAR1-dependent mechanism. Thus the protective actions of APC in a model of ischemic brain injury in mice are markedly attenuated by administration of a PAR1 blocking antibody (46). However, activation of PAR1 also induce neurodegeneration. Prolonged exposure of neurons and astrocytes to high concentrations of thrombin (500 –750 nM) causes apoptosis (93), and thrombin (100 nM, 1 h) decreases survival of hippocampal neurons in brain slices after hypoglycemic and hypoxic stress (271). The same kinase and Rho-dependent pathways that mediate protection also induce apoptosis, the difference resting on the duration and extent of receptor activation. Moreover, whereas low concentrations of thrombin induce transient elevations in [Ca2⫹]i in hippocampal neurons, high concentrations induce large and sustained elevations, which may well trigger apoptosis (271). Supporting this concept, caspase-3 inhibitors prevent PAR1-induced death of motoneurons in the avian embryo (286). Some of the detrimental effects of thrombin and PAR1 APs could be mediated by the N-methyl-D-aspartate (NMDA) receptor system, which contributes to degeneration in the nervous system. Thus PAR1 activation potentiates NMDA currents in hippocampal neurons, raising the possibility that thrombin could exacerbate glutamate-induced neurotoxicity (107). In addition to effects on survival, activation of PAR1 also induces proliferation of astrocytes (229). The dual protective and degenerative effects of PAR1 could have implications for survival of neurons after injury: immediately after trauma and generation of thrombin, PAR1 may serve to protect the brain from damage; thereafter, PAR1 may have deleterious effects. PAR4 may also contribute to inflammation in the brain, with detrimental effects (274). Thus PAR4 AP, but not PAR1 AP, stimulates release of TNF-␣ from brain microglia, an effect that is PAR4 specific because it is prevented by downregulation of PAR4 with antisense. PAR4 AP also activates the inflammatory NFB pathway 603 604 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT suggest that proteases that activate PAR2 can signal to the peripheral projections of primary spinal afferent neurons to trigger release of neuropeptides from their peripheral and central projections; peripheral release causes neurogenic inflammation, and central release induces thermal hyperalgesia. The identity of the PAR2 activating proteases that mediate these effects remains to be determined, but one possibility is tryptase from mast cells. Mast cells containing tryptase are in close proximity to sensory nerve endings in uninflamed and inflamed tissues (267). Indeed, PAR2-deficient mice show diminished thermal hyperalgesia after intraplantar injection of compound 48/80 to degranulate mast cells, suggesting a role for PAR2 in mast cell-dependent hyperalgesia (296). PAR2 activators also induce pain in the pancreas by a neurogenic mechanism that involves activation of pancreatic sensory nerves (124). This observation is relevant to the painful condition of pancreatitis, where trypsinogen is prematurely activated within the pancreas and could cause hyperalgesia by activating PAR2 on pancreatic neurons. The molecular mechanism of PAR2-mediated inflammation and hyperalgesia is unknown. However, it is likely to involve signaling events that regulate activity or expression of ion channels. One candidate is vanilloid receptor-1 (VR1), a member of the transient receptor potential (TRP) family of channels. TRPV1 is a nonselective cation channel that is activated by protons, elevated temperature, and certain lipids, as well as by exogenous vanilloids such as capsaicin (37, 141). TRPV1 plays an important role in inflammatory pain and thermal hyperalgesia, and several Physiol Rev • VOL inflammatory agents transactivate this channel. The TRPV1 antagonist capsezapine antagonizes PAR2-mediated thermal hyperalgesia (152). Moreover, activation of PAR2 enhances TRPV1-induced Ca2⫹ signaling and TRPV1 currents in cell lines and neurons by a PKC-dependent mechanism (Bunnett, unpublished observation) and also potentiates capsaicin-induced release of CGRP (124). These results suggest that activated PAR2 couples PKC, which may phosphorylate and activate TRPV1, thereby inducing hyperalgesia. In a similar manner, activation of PAR1 promotes extravasation of plasma proteins in many tissues by a mechanism that depends on the release of SP from the peripheral projections of afferent neurons (87). However, in contrast to PAR2, PAR1 activation causes somatic analgesia, by mechanisms that remain to be defined (11, 151). C. Protease Signaling in the Gastrointestinal System All PARs are expressed in the gastrointestinal tract, although most studies have focused on PAR2, which appears to be of particular importance in the gut. The role of PARs in the gastrointestinal tract has been recently reviewed (295). 1. Proteases Of all organ systems, the gastrointestinal tract is exposed to the widest array of proteases under physio- 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 FIG. 8. Protease signal to primary spinal afferent neurons. 1) Proteases from mast cells (tryptase), epithelial cells (trypsin), and the circulation (FVIIa, FXa) can cleave PAR2 at the peripheral projections of primary spinal efferent neurons. 2) Cleavage releases calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (NKA). 3) CGRP interacts with the type 1 CGRP receptor [comprised of calcitonin receptor like receptor and receptor activity modifying protein (CRLR/RAMP1)] on arterioles to induce dilation and hyperemia. 4) SP/NKA interact with the neurokinin 1 receptor on endothelial cells of postcapillary venules to induce gap formation, plasma extravasation, and granulocyte infiltration. 5) PAR2 sensitizes TRPV1. 6) SP release within the cord induces thermal hyperalgesia. [Adapted from Steinhoff et al. (270).] PROTEASE-ACTIVATED RECEPTORS 2. PARs PAR1 is present in endothelial cells of the lamina propria of the small intestine, in intestinal epithelial cells, smooth muscle cells, and in neurons of the enteric nervous system (24, 67). PAR2 mRNA is strongly expressed in the small intestine, colon, liver, and pancreas, and more weakly detected in the stomach (24, 213, 214). Immunoreactive PAR2 is localized to the apical and basolateral membrane of enterocytes, in smooth muscle cells, on peripheral terminals of mesenteric afferent nerves, on neurons of the myenteric and submucosal nerve plexuses, to endothelial cells and vascular smooth muscle, and to certain immune cells (neutrophils, mast cells, lymphocytes) (40, 66, 67, 73, 79, 167, 235). PAR3 mRNA is expressed in stomach and small intestine, although the cell types remain to be defined (135). PAR4 is highly expressed in the pancreas, small intestine, and colon, although it is yet to be localized at the cellular level (143, 317). 3. Ion transport Activation of PAR1 and PAR2 stimulates Cl⫺ secretion from the intestinal mucosa. The functional implications of this regulation are twofold. First, stimulation of Cl⫺ secretion would enhance fluid secretion, resulting in diarrhea that is symptomatic of intestinal inflammation. Second, enhanced secretion during intestinal inflammation may serve a protective function by promoting the excretion of toxins. Physiol Rev • VOL The effects of PAR activation on electrolyte secretion by cultured enterocytes and sheets of mucosa have been examined using Ussing chambers, allowing application of agonists to apical (lumen) and basolateral (serosal) surface and measurement of short-circuit current as an index of ion secretion. The role of PAR1 in electrolyte secretion has been examined using SCBN cells, a nontransformed epithelial cell line from the crypts of the human small intestine (30, 31). Application of thrombin or a selective PAR1 AP to the basolateral surface of these cells stimulates an increase in short-circuit current that is attributable to Cl⫺ secretion by a Ca2⫹-dependent mechanism. Although SCBN cells also express the cystic fibrosis transmembrane conductance regulator (CFTR), activation of PAR1 does not raise intracellular cAMP, and thus PAR1 is unlikely to regulate the CFTR. PAR1 agonists induce activation of Src, transactivation of the EGF receptor, activation of ERK1/2, phosphorylation of cytoplasmic phospholipase A2, and stimulation of cyclooxygenase-1 and -2, which induce Cl⫺ secretion. PAR2 signaling in enterocytes has been investigated using hBRIE cells, a nontransformed epithelial cell line from the rat small intestine that retains many characteristics of enterocytes. Trypsin and PAR2 AP stimulate generation of InsP3, mobilization of Ca2⫹, and release of arachidonic acid and prostaglandins E2 and F1␣ (167). The generation of prostaglandins is also stimulated by the application of physiological concentrations of trypsin to the apical membrane of everted sacs of rat jejunum, suggesting expression of PAR2 at the apical membrane. Prostaglandins released from enterocytes may have a protective function and could also regulate fluid and electrolyte secretion. Application of PAR2 AP and activating proteases to the serosal surface of segments of intestine from rat (300), mouse (73), and pig (109) induces an increase in short-circuit current due to stimulation of Cl⫺ secretion, but the mechanisms vary between species and perhaps between different regions of the intestine. In the rat jejunum, Cl⫺ secretion depends on generation of prostaglandins (300). However, in view of the very different potencies of PAR2 agonists in this preparation and a PAR2transfected cell line, Cl⫺ secretion may be due to the activation of a variant of PAR2. In the pig ileum, PAR2stimulated Cl⫺ secretion depends both on eicosanoids and on submucosal neurons, since the stimulation is strongly suppressed by the neuronal toxin saxitoxin (109). Thus application of PAR2 agonists to the serosa may activate submucosal neurons, which then release stimulants of Cl⫺ secretion. In the distal colon of the mouse, application of PAR2 agonists to the serosal surface stimulates Cl⫺ and K⫹ secretion and inhibits amilioride-sensitive Na⫹ absorption (73). These effects are mediated in part by the enteric nervous system. PAR2 is also expressed by epithelial cells of the pancreatic duct, where it regulates ion transport. Trypsin and 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 logical circumstances and during diseases. Digestive proteases from glands in the stomach and intestine as well as from extrinsic sources such as the pancreas bathe the lumen of the gastrointestinal tract during and after feeding. Thus physiological concentrations of trypsin in the lumen of the small intestine can signal to enterocytes by cleaving PAR2 at the apical membrane (167). The intestinal lumen is also replete with bacteria and thus exposed to bacterial proteases. Under normal circumstances, the tight junctions of epithelial cells act as a barrier to prevent the ingress of proteases from the lumen into tissues. However, during stress and inflammation, this barrier is disrupted and proteases from the lumen may penetrate the mucosa and muscle layers of the intestine (318). During inflammation, coagulation proteases and proteases released from inflammatory cells such as mast cells and neutrophils bathe many cell types and can also be detected in the intestinal lumen. For example, high levels of tryptase and trypsin are found in the lumen of the inflamed intestine (32, 232, 250, 258). Many of these enzymes that are normally present in the intestine or that are generated or released during inflammation activate PARs on gastrointestinal cells to influence motility, secretion, and neurotransmission. 605 606 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT PAR2 AP stimulate short-circuit current when added to the basolateral but not apical surface of monolayers of duct cells from the canine pancreas by stimulating Ca2⫹activated Cl⫺ channels and K⫹ channels (205). In this manner PAR2 may play a protective role in pancreatitis, when there is premature activation of trypsinogen and secretion of trypsin into the interstitial fluid. Activation of PAR2 on the basolateral membrane of duct cells could increase ductal secretion and promote clearance of toxins and debris. 4. Motility 5. Exocrine secretion PAR2 regulates pancreatic, salivary, and gastric secretions. PAR2 activation stimulates the release of amylase from isolated rat pancreatic acini (24). Intravenous injection of PAR2 AP in rats and mice also stimulates pancreatic amylase secretion, and the effect in mice is mediated by NO (158). PAR2 is also present in acini of the salivary gland, and PAR2 APs stimulate release of amylase and mucus in rats and mice (158). In mice, the effect on amylase secretion is partially mediated by NO, and the Physiol Rev • VOL 6. Neurotransmission The enteric nervous system comprises a series of ganglionated nerve plexuses that comprise the intrinsic nervous system of the gut. These ganglia include primary afferent neurons, interneurons, and motor neurons, allowing local, reflex regulation of all aspects of gastrointestinal function, including control of motility, exocrine and endocrine secretion, and fluid and electrolyte transport. The expression of PARs in neurons of the myenteric plexus suggests a role for proteases in control of motility. Indeed, PAR1, PAR2, and PAR4 are expressed by a substantial proportion of myenteric neurons in the guinea pig intestine (⬎50% for PAR1, PAR2) (67, 104, 176). Thrombin, trypsin, and tryptase induce a slow and prolonged depolarization in myenteric neurons that is often accompanied by increased excitability (104, 176). Selective APs for PAR1, PAR2 and PAR4 similarly excite these neurons. Neurons of the enteric nervous system can be classified on the basis of their electrophysiological behavior. Two prominent types of neurons are AH, which are the intrinsic primary afferent neurons, and S neurons, which are motor neurons and interneurons. Approximately 50 – 60% of AH neurons and slightly fewer S neurons respond to PAR2 agonists. The PAR2-responsive S neurons also express NO synthase and project axons in an anal direction. These are inhibitory motor neurons, and activation would be expected to suppress motility. PAR2 is also expressed by neurons of the submucosal nerve plexus of the guinea pig small intestine, some of which also express vasoactive intestinal polypeptide and are thus secretomotor neurons that play an important role in the control of fluid and electrolyte transport (235). Trypsin, tryptase, and PAR2 AP evoke transient depolarization of submucosal neurons 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 Activation of PAR1 and PAR2 stimulates contraction of gastric longitudinal muscle from rats and guinea pigs, which is blocked by the cyclooxygenase inhibitor indomethacin and the tyrosine kinase inhibitor genistein (3, 247). In the mouse gastric fundus, activation of PAR1 and PAR2 has a biphasic effect, causing relaxation and then contraction (59). The relaxation is blocked by apamin or ryanodine, indicating that it is mediated by ryanodinesensitive and -insensitive activation of small-conductance Ca2⫹-activated K⫹ channels. In the longitudinal muscle of the rat colon, PAR2 APs inhibit spontaneous contractions (66). PAR1 and PAR2 APs also inhibit contraction of colonic circular muscle, which is prevented by apamin (198). Some muscles express more than one receptor for the same protease, and the net effect on motility depends on which receptor is activated. In the rat esophagus, PAR1 APs stimulate contraction, but PAR4 APs have little effect (154). However, in carbachol-precontracted tissues, PAR1 agonists cause additional contraction, whereas PAR4 agonists induce a strong relaxation. These results suggest that thrombin could have a dual effect in the esophagus, causing contraction by PAR1 and relaxation by PAR4. The dominant effect would depend on the concentration of thrombin: low concentrations preferentially activate PAR1, but high concentrations of thrombin are needed to activate PAR4. The intraperitoneal administration of selective APs of PAR1 and PAR2 to mice accelerates intestinal transit (155). These effects are abolished by verapamil, an inhibitor of the L-type Ca2⫹ channel, but are potentiated by apamin. Thus the net effects of PAR1 and PAR2 activators in vivo are to promote intestinal transit. stimulation of mucin secretion in rats depends on activation of tyrosine kinase (157). Activation of PAR2 stimulates secretion of gastric, but not duodenal, mucus (153). In this manner, PAR2 APs protect the gastric mucosa from injury induced by acid-ethanol or by indomethacin. PAR2induced mucus secretion and gastric protection are suppressed by antagonists of the CGRP and neurokinin-2 receptors and by the TRPV1 antagonist capsezapine, and thus depend on the release of neuropeptides from sensory nerves (152). In contrast, PAR2-mediated salivary secretion is independent of capsaicin-sensitive sensory neurons (156), an observation that illustrates the complexity of the PAR2 signaling system, which varies between tissues. PAR2 is also expressed by the pepsin-secreting chief cells of the stomach, and PAR2 agonists stimulate pepsin secretion in rats (160). Conversely, activation of PAR2 suppresses gastric acid secretion in intact animals (211). Gastric epithelial cells also express PAR1, and PAR1 AP stimulates secretion of mucus and prostaglandin E2, which may be protective (279). PROTEASE-ACTIVATED RECEPTORS 7. Intestinal inflammation Since intestinal inflammation is associated with the generation and release of proteases that are potential activators of PARs, there has been considerable interest in the role of PARs in inflammatory diseases of the intestine. Administration of the trypsin, tryptase, and PAR2 AP into the lumen of the mouse colon provokes a generalized inflammatory reaction in wild-type but not PAR2-deficient mice (40, 41). PAR2-induced inflammation is suppressed by inhibition of NO synthase, by ablation of sensory nerves, and by antagonism of the CGRP type 1 receptor and the neurokinin-1 receptor. Thus proteases induce inflammation in part by a neurogenic mechanism that involves the release of peptides from sensory nerves. Intracolonic PAR2 activators also disrupt the intestinal barrier integrity as observed by the increased paracellular permeability to bacteria. This disruption of tight junctions depends on activation of myosin light-chain kinase (41). Given the large presence and increased activity in the intestinal lumen of trypsin and tryptase, which can activate PAR2, these data have important implications for the pathophysiology of inflammatory bowel diseases. However, PAR2 may also serve a protective function in the intestine. Activation of PAR2 induces release of prostaglandins from enterocytes (167) and stimulates gastric mucus secretion (153), both of which are protective. Administration of PAR2 AP in mice protects against inflammation induced by the rectal administration of dinitrobenPhysiol Rev • VOL zene sulfonic acid, a widely used model of hapten-induced colitis (103). This protective effect is also neurogenic, since it is prevented by ablation of sensory nerves and antagonism of the CGRP 1 receptor. D. Protease Signaling in the Airway The potential involvement of PARs in regulation of the airways and in pulmonary disease has been recently reviewed (58, 127). 1. Proteases During inflammation and trauma, proteases from the coagulation cascade and from inflammatory cells could regulate pulmonary cells by activating PARs. Indeed, there are elevated levels of coagulation proteases (134) and tryptase (139) in bronchoalveolar lavage from patients with chronic inflammatory diseases. Proteases that activate PARs may also arise from cells in the airways. Immunoreactive trypsinogen colocalizes with PAR2 in Clara cells of the epithelium of human bronchioles, whereas in larger bronchioles trypsinogen is expressed by cells just above the basal layer (56, 58). In addition, a trypsinlike enzyme can be detected by zymography in the bronchoalveolar lavage collected from mice treated intranasally with the bacterial endotoxin lipopolysaccharide. Intriguingly, certain nonmammalian proteases such as dust mite allergens can also signal to cells in the airway by cleaving PARs, which may have implications for allergic inflammation (273). 2. PARs The PARs are expressed by many cell types in the airway. There is weak staining for immunoreactive PAR1 in ciliated and basal epithelial cells and in smooth muscle cells of rat trachea and bronchi (49, 127). In rat, mouse, guinea pig, and human, immunoreactive PAR2 is expressed by ciliated and nonciliated epithelial cells, especially at the apical membrane, as well as in glands, airway smooth muscle, and endothelial cells and vascular smooth muscle cells (49, 56, 58, 79, 192, 237, 255). 3. Airway resistance Given the importance of increased airway resistance in asthma, there is great interest in understanding the regulation of contraction of airway smooth muscle. There is evidence that PAR2 activators induce both bronchodilatation, which would be protective, and detrimental bronchoconstriction. The first evidence in favor of a protective role came from the finding that activation of PAR2 induces relaxation of isolated bronchi from several species, including mouse, rat, guinea pig, and human, precontracted with 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 followed by a prolonged hyperexcitability that can last for several hours. This remarkable long-term hyperexcitability mimics that observed after degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, serotonin, prostaglandins). Mast cell degranulation results in release of proteases, which desensitizes neurons to other activators of PAR2. Thus PAR2 AP and proteases from degranulated mast cells induce long-term excitability of submucosal neurons, which could result in enhanced fluid and electrolyte secretion from the mucosa (73, 109). PAR2 is also expressed by extrinsic neurons that innervate the intestine. Thus the administration of PAR2 activators into the lumen of the rat intestine induces a hyperalgesia to distension of the intestine with a balloon, consistent with a visceral hyperalgesia (60). However, in contrast to the somatic hyperalgesia that depends on the spinal release of SP, PAR2-dependent visceral hyperalgesia involves peripheral release of SP. The widespread expression of PARs in the intrinsic and extrinsic nervous systems of the gut may be of considerable interest during inflammation, where proteases from the circulation, inflammatory cells, and the intestinal lumen could alter neuronal excitability and induce alterations in motility, secretion, and pain perception. 607 608 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT 4. Electrolyte transport PAR2 also regulates ion transport in the airway epithelium (78, 171). PAR2 activators, added to the basolateral surface of bronchial epithelial cells and airway tissue, cause a transient Ca2⫹-dependent increase in short-circuit current, followed by a sustained inhibition of amilioridePhysiol Rev • VOL sensitive current. This mechanism may control fluid volume and composition at the airway surface. 5. Inflammation and fibrosis There has been considerable interest in the role of tryptase in airway inflammation and asthma. Hyperplasia of smooth muscle contributes to remodeling of the airways that accompanies asthma, and tryptase induces proliferation of airway smooth muscle cells, fibroblasts, and epithelial cells (29, 33, 244). Tryptase also induces hyperresponsiveness in the airway (17), and when injected into the skin tryptase causes plasma extravasation and neutrophil infiltration (119, 120). In a sheep model, inhaled tryptase causes bronchial hyperresponsiveness, whereas a tryptase inhibitor reduces early and late phase bronchoconstriction and hyperresponsiveness to allergen challenge (55). Although it is not established that these effects of tryptase are mediated by PAR2, PAR2 AP does have similar effects. For example, PAR2 AP also induces proliferation of airway smooth muscle cells and lung fibroblasts, and thus PAR2 may mediate the proliferation effects of tryptase on lung fibroblasts and myocytes (2, 18). PAR2 may have both proinflammatory and protective effects in the airway. Airway epithelial cells express PAR1, PAR2, and PAR4, and activation of these receptors triggers the release of cytokines such as interleukin-6, interleukin-8, and prostaglandin E2 that can regulate inflammation (12). Observations on PAR2 knockout mice and animals overexpressing human PAR2 suggest that this receptor contributes to allergic inflammation (255). In an ovalbumin-induced allergic inflammation of the airway, both the infiltration of eosinophils into the airway lumen and methacholine-induced airway hyperreactivity are markedly diminished in PAR2-deficient animals, and exacerbated in mice overexpressing PAR2. PAR2 deletion also reduces IgE levels to ovalbumin sensitization by fourfold compared with wild-type animals. These results, together with the observation of upregulation of PAR2 in airway epithelium of asthmatics (164), suggest that PAR2 contributes to the development of immunity and to allergic inflammation of the airway. However, PAR2 can also be protective against airway inflammation. Intranasal delivery of PAR2 AP in mice does not cause any inflammation in the airways but inhibits the marked immune cell inflammatory response, characterized by a recruitment of polymorphonuclear leukocytes, triggered by administration of lipopolysaccharide (195). Although less is known about PAR1 in the airway, there are reports of a role for thrombin and PAR1 in fibrotic diseases. Thrombin and PAR1 AP are mitogenic for human airway smooth muscle cells (280). Thrombin also stimulates the release of the proinflammatory and fibrogenic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) from airway smooth muscle 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 carbachol (49, 56, 58, 172). This relaxation requires the presence of the epithelium and is mediated by prostanoids but not by NO. Surprisingly, in the rat, PAR2 AP but not trypsin causes relaxation, possibly due to the existence of trypsin inhibitors in that tissue (49). PAR2-induced relaxation of the isolated mouse trachea is unaffected by acute damage to the epithelium caused either by mechanical rubbing or infection with influenza A virus (173). However, the effect of PAR2 activation in the airway may depend on the tissue chosen for study and the species. For example, whereas PAR2 activation causes relaxation of isolated guinea pig trachea and main bronchi by a mechanism that is dependent on the presence of the epithelium and that is mediated by NO and prostanoids, in smaller intrapulmonary bronchi the sole effect of trypsin and PAR2 AP is to trigger smooth muscle contraction (237). PAR2 activators also stimulate contraction of lobar or segmental bronchial rings from humans that is mediated in part by a direct activation of PAR2 on smooth muscle cells (256). Since smaller bronchioles are the principal site of airway resistance, contraction would be expected to increase total airway resistance, which would be detrimental. In addition to the aforementioned indirect effects of PAR2, which often require the presence of the epithelium, PAR2 activators, including tryptase, can directly signal to isolated smooth muscle cells from the human airway by mobilizing intracellular Ca2⫹ (19, 256). Studies in intact animals also show a protective or detrimental effect of PAR2 activation. In support of a protective role, aerosol administration of PAR2 AP to intact rats inhibits serotonin-induced bronchoconstriction (56, 58). Similarly, intravenous administration of PAR2 AP inhibits histamine-induced increase in airway resistance in guinea pigs by a mechanism independent of the release of prostaglandins, NO, or the effect of circulating epinephrine (51). In contrast, intravenous and intratracheal administration PAR2 activators causes bronchoconstriction in intact guinea pigs, mediated in part by prostanoids and involving the release of tachykinins from sensory nerve endings and activation of neurokinin-1 and -2 receptors (237). Thus PAR2 may be protective or detrimental in the airways, depending on the experimental model and species. It will be of great interest to examine the contribution of proteases and PAR2 to airway disease by using specific antagonists and genetically modified animals. PROTEASE-ACTIVATED RECEPTORS E. Protease Signaling in the Skin The role of proteases in cutaneous biology and disease has recently been reviewed (234). 1. Proteases Cells in the epidermis and dermis could be regulated by proteases of the coagulation cascade. Inflammation of the skin is associated with an influx of inflammatory cells that release proteases that activate PAR2. Thus whereas in normal skin mast cells are found in the dermis, in atopic dermatitis and psoriasis there is an influx of tryptase-containing mast cells in the dermal and epidermal junction, as well as the epidermis (268). In addition, epidermal cells express proteases, some of which could activate PARs. Potential activating enzymes include stratum corneum tryptic enzyme and chymotryptic enzyme (96, 275). However, it remains to be determined if proteases such as these activate PARs. Exogenous proteases from mites, bacteria, and fungi could also signal to epidermal cells. PAR2 (126, 268). The expression of PAR2 has been evaluated in some skin diseases (268). Thus PAR2 immunoreactivity is strong in the hyperproliferative granular layer of skin from patients with lichen planus, and in atopic dermatitis there is strong staining of the whole epidermis, as well as in blood vessels and dendritic-like cells. 3. Pigmentation There is considerable interest in the role of PAR2 in pigmentation. The interaction between keratinocytes and neighboring melanocytes is essential for skin pigmentation. Melanocytes produce secretory granules, melanosomes, which produce melanin. Melanin-containing melanosomes are transferred to dendrites of melanocytes and are then taken up by keratinocytes by phagocytosis, resulting in skin darkening. Although melanocytes do not express PAR2, PAR2 activators regulate the phagocytosis of melanosomes by keratinocytes and thereby control pigmentation (259, 264). Thus trypsin and PAR2 AP stimulate pigmentation of cocultures of keratinocytes and melanocytes. Remarkably, the topical application of PAR2 AP induces pigmentation of human skin transplanted onto mice. Conversely, a serine protease inhibitor causes lightening of the skin (224). The mechanism of these effects of PAR2 on pigmentation depends on the stimulation of phagocytosis in keratinocytes. Thus activation of PAR2 increases the ingestion of particles and bacteria by keratinocytes (264). Phagocytosis by keratinocytes not only plays a role in pigmentation but also enables “nonimmune” cells such as these to participate in protection of the skin, with implications for wound healing and inflammation. Of particular interest, the increased phagocytic response of keratinocytes correlates with increased activity of soluble serine proteases. Serine protease inhibitors downregulated both constitutive and PAR2 stimulated phagocytosis, suggesting that these proteases also activate PAR2 and thereby amplify the response. PAR2 is upregulated in keratinocytes after ultraviolet exposure, with implications for pigmentation (257). 4. Proliferation and wound healing 2. PARs Immunoreactive PAR1 is found in the basal and spinous layers of the human epidermis but not in the granular layer, and human basal keratinocytes and a keratinocytes cell line express PAR1 mRNA and protein (76). PAR2 immunoreactivity is predominantly localized to the basal and suprabasal spinous layers, although the stratum corneum is unstained (76, 268). PAR2 is also expressed in the inner root sheath of hair follicles and in myoepithelial cells of sweat glands. Keratinocytes in culture also express PAR2 mRNA and protein (251). PAR2 mRNA is also moderately expressed in cultured human microvasculature endothelial cells, but melanocytes do not express Physiol Rev • VOL Several findings suggest that PAR1 could play a role in wound healing. Activation of PAR1 stimulates proliferation of keratinocytes (5, 251) and fibroblasts (45), and also induce angiogenesis (285). Plasmin, thrombin, and PAR1 AP induce expression of cysteine-rich angiogenic protein Cyr-61, a growth factor-like gene implicated in angiogenesis and wound healing, in fibroblasts from wildtype but not PAR1-deficient mice (227). Moreover, the topical application of thrombin and PAR1 AP to incisional wounds in rats improves wound strength and increases angiogenesis, thereby promoting wound healing (36). The role of PAR1 in wound healing has been examined in PAR1-deficient mice (65). Although fibroblasts from these 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 cells. Although this effect is blocked by pertussis toxin, and thus likely mediated by a GPCR, it is not mimicked by activators of PAR1 and PAR4, and may thus involve a novel receptor. However, PAR1 may have a role in fibrotic diseases of the lungs. Instillation of bleomycin into the airway induces pulmonary fibrosis in rats, which is associated with elevated expression of thrombin and PAR1 in macrophages and fibroblasts (127). Administration of a thrombin inhibitor UK-156406 attenuates bleomycin-stimulated deposition of collagen in the lung by 40%, suggesting an important role of coagulation proteases in fibrogenesis in the lung. Moreover, thrombin and FXa stimulate expression of connective tissue growth factor from fibroblasts by activation PAR1, which may contribute to normal repair and fibrosis (42). Inhibitors of coagulation proteases and antagonists of PAR1 could be novel therapies for pulmonary fibrosis. 609 610 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT animals do not respond to thrombin, the time to closure of open skin wounds and the incision strength of healed wounds is unaffected by deletion of PAR1. The discrepancy between this observation and the finding that PAR1 AP promotes healing in rats is difficult to explain, but could be related to differences in species and the wound model chosen. 5. Inflammation VI. PROTEASE-ACTIVATED RECEPTORS: MECHANISMS OF DISEASE AND TARGETS FOR THERAPY Proteases and PARs make important contributions to disease and are targets for therapies. First, PARs regulate many biological processes that are critical in disease, including coagulation, proliferation and survival, inflammation, neurotransmission, and pain. Second, proteases that activate PARs are generated during diseases, for Physiol Rev • VOL A. Cardiovascular Disease The inappropriate aggregation of platelets makes an important contribution to occlusive vascular disorders such as stroke, angina, and myocardial infarction, where accumulation of atherosclerotic plaques promotes platelet-mediated thrombus formation. Thrombin is a major mediator of platelet aggregation and fibrin deposition, and thrombin inhibitors are useful antithrombotic agents. One drawback of such inhibitors is their disruption of the normal hemostatic mechanism, and a selective suppression of the effects of thrombin on platelets may be advantageous. A difficulty of studying the role of PARs in thrombus formation is that platelets express several thrombin receptors, which vary among species. However, despite the redundancy and interspecies differences, recent observations in cynomologus monkeys, whose platelets, like those of humans, express PAR1 and PAR4, suggest that PAR1 antagonists may be useful therapeutic agents in humans (88). Thus the PAR1 antagonist RWJ-58259 markedly inhibits thrombus formation and vessel occlusion in a model of electrolytic injury of the carotid artery in cynomologus monkeys. Despite the fact that platelets from these animals possess the dual PAR1/PAR4 system, antagonism of a single receptor is effective, raising the possibility that antagonism of a PAR1 may be of use for treatment of thrombosis disorders in humans. In guinea pigs, whose platelets express three thrombin receptors (PAR1, PAR3, and PAR4), RWJ-58259 is only marginally effective in arterial thrombosis, which emphasizes the 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 Activation of PAR1 and PAR2 induces expression of interleukin-6 and -8 as well as GM-CSF in cultured human keratinocytes, indicating that serine proteases modulate cytokine expression in the epidermis (126, 307). GM-CSF mediates Langerhans cell maturation, activates tissue macrophages, and stimulates proliferation of keratinocytes. PAR2 agonists also activate stress-activated protein kinases (c-Jun amino-terminal kinase or JNK and p38) and the NFB pathways in the keratinocyte cell line NCTC2544 (145). The NFB family of transcription factors is implicated in regulation of a number of proinflammatory genes in these cells. The proinflammatory effects of PAR2 have also been examined in the intact animal. PAR2-deficient mice exhibit diminished ear swelling and infiltration of inflammatory cells in a model of allergic dermatitis, suggesting that PAR2 mediates allergic dermatitis (159). PAR2 activators also signal to primary spinal afferent neurons that innervate the skin, resulting in the release of the neuropeptides SP and CGRP, which together induce neurogenic inflammation (270) (Fig. 8). This signaling also triggers a prolonged mechanical and thermal somatic hyperalgesia, which is due to the release of SP from central projections of these neurons within the dorsal horn of the spinal cord (296). Protease signaling to sensory nerves in the skin has recently been investigated in humans (234). Atopic dermatitis is associated with an elevated release of tryptase in the skin and an upregulation of PAR2 on nerve cells. Cutaneous administration of PAR2 AP to these patients results in prolonged itch, even in the presence of antihistamine drugs (269). These results suggest that tryptase could act on PAR2-expressing sensory nerves in human skin to produce itch, which may explain the insensitivity of these patients to antihistamines. example, in trauma, hemostasis, inflammation, and tumor formation. Third, the expression and function of PARs are altered in disease. Thus PAR2 is upregulated during inflammation (216), and a polymorphic variant of PAR2 has been identified with markedly abnormal sensitivity to PAR agonists (61). Finally, studies of genetically modified animals and using PAR antagonists and protease inhibitors suggest a role of PARs in models of diseases. However, there are formidable obstacles toward understanding of the role of proteases and PARs in disease. A major problem is the lack of widely available antagonists; although PAR-deficient mice are available, mice are not always the optimal species to model human diseases. Another problem is the apparent redundancy of the PAR system. Cells express several PARs for the same protease, and several proteases can activate the same receptor. Effective blockade of protease signaling may require antagonism of several PARs or proteases. Finally, the roles of PARs have been specifically studied in few animal models of disease. Moreover, little is known about the expression of proteases and PARs in diseased human tissues. With these caveats in mind, the role of PARs will be briefly discussed in several diseases. PROTEASE-ACTIVATED RECEPTORS B. Inflammatory Disease There is considerable interest in understanding the role of PARs, in particular PAR2, in inflammatory diseases, and this topic has been thoroughly reviewed (54, 57, 69). Observations on PAR2-deficient mice suggest a role for this receptor in inflammation of the airway, joints, and kidney. In allergic inflammation of the airway induced by immunization and challenge with ovalbumin, there is diminished infiltration of eosinophils and reduced hyperreactivity to methacholine in PAR2-deficient animals, whereas these responses are exacerbated in mice overexpressing PAR2 (255). Rheumatoid arthritis is a chronic inflammatory condition that is associated with inflammation of the synovium and hyperplasia. There is also elevated expression of TF and thrombin in the synovium and the deposition of fibrin in the inflamed joint, indicating activation of the coagulation system. Immunization and subsequent challenge of mice with chicken collagen results in arthritis, and administration of the thrombin inhibitor hirudin reduces the severity of the inflammation, assessed by clinical scoring and measurement of expression of proinflammatory cytokines, providing direct evidence for the involvement of thrombin in the inflammation (186). Evidence for a role of PAR2 in arthritis has been provided from observations of PAR2-deficient animals, which are protected against arthritis induced by Physiol Rev • VOL intra-articular and periarticular injection of adjuvant (101). This inflammation is accompanied by an upregulation of PAR2 expression, which is normally confined to the vasculature of the joints. The protease that activates PAR2 in the inflamed joint remains to be determined, although it is tempting to speculate that FVIIa and FXa could be involved in light of the established activation of the coagulation cascade in arthritis. Proteases and PARs may also contribute to renal inflammation. Cresentic glomerulonephritis is a severe and progressive form of renal inflammation that is a common cause of end-stage kidney disease. It is associated with fibrin deposition, suggesting activation of the coagulation proteases. Immunization with sheep globulin followed by challenge with sheepanti-mouse glomerular basement membrane induces glomerulonephritis in mice that is characterized by fibrin deposition, infiltration of leukocytes, and reduced glomerular filtration (75). This inflammation is markedly diminished by treatment with the thrombin inhibitor hirudin, and inflammation is reduced in PAR1-deficient mice, indicating involvement of coagulation proteases and PAR1. The observation that inflammation is diminished in PAR-deficient animals suggests that PAR antagonists and protease inhibitors may be useful anti-inflammatory agents. Indeed, tryptase inhibitors have been used to treat asthma and inflammatory bowel disease in humans (170, 284). However, it must be emphasized that PARs can exert anti-inflammatory and protective effects in certain conditions. Thus administration of recombinant APC blocks apoptosis in human brain endothelial cells and is neuroprotective in an animal model of ischemic brain injury (46). Moreover, APC reduces mortality of patients with sepsis (20). These protective actions of APC on endothelial cells are dependent on PAR1 (46, 238). In addition, activation of PAR2 induces bronchodilatation (56), inhibits lipopolysaccharide-induced pulmonary neutrophilia (195), diminishes formation of gastric ulcers (153) and experimental colitis (103), and protects against ischemia-perfusion injury in the heart (203). Thus agonists of PAR1 and PAR2 could be useful therapies for certain conditions. C. Cancer The microenvironment of tumors is replete with proteases that can activate PARs, and tumor cells themselves express PARs. Malignant cells secrete thrombin and trypsin, which can affect proliferation and mediate metastatic processes such as cellular invasion, extracellular matrix degradation, angiogenesis, and tissue remodeling. PAR1 and PAR2 are expressed by a wide range of tumor cells (24, 80, 82, 208, 316). In breast tumor tissues, PAR1 and PAR2 are expressed in the tumor cells, mast cells, macrophages, endothelial cells, and vascular 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 importance of selecting the appropriate species to model human disease (8). PAR antagonists may also be of interest in treating vascular injury. In addition to its role in thrombus formation, thrombin is also implicated in restenosis of the blood vessels after vascular injury. Thus a PAR1 blocking antibody or a PAR1 antagonist attenuates thickening of the neointima of the rat carotid artery rat, initiated by damage to the endothelium by balloon angioplasty (8). Similarly, vascular injury is attenuated in PAR1-deficient mice (301). The protective effect of PAR1 antagonism or deletion occurs even though rat and mouse platelets do not express PAR1 and thus exhibit normal aggregation in the absence of functional PAR1. These effects of thrombin on restenosis are therefore dependent on the proliferative or proinflammatory effects of PAR1 on vascular smooth muscle. PAR2 may contribute to injury that follows ischemia and reperfusion of tissues (203). Ischemia and reperfusion of the heart induces injury that is characterized by generalized inflammation and necrosis. PAR2 is upregulated in a model of coronary ischemia and reperfusion injury in rats, and administration of a PAR2 AP markedly protects the heart from injury. PAR2 activation also improves efficiency of ischemic preconditioning and reduces cardiac inflammation in the rat heart (204). 611 612 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT VII. CONCLUSIONS AND FUTURE PERSPECTIVES There have been substantive advances in our understanding of protease signaling. Four PARs have been cloned and characterized, and the mechanisms by which proteases cleave and activate PARs are understood in considerable detail. A number of mammalian and nonmammalian proteases have been identified that can activate PARs, providing new insights into protease signaling. The realization that anchoring proteins, including PARs themselves or other nonreceptor proteins, play a critical Physiol Rev • VOL role as cofactors for protease signaling has led to the identification of new activating enzymes. PARs have been used as model receptors to investigate mechanisms of desensitization and downregulation of GPCRs in general. Tools have been developed to examine receptor functions, including specific agonists and antagonists and both knockout and transgenic mice. With the use of these tools, the contribution of PARs to disease has been investigated in experimental animals. Finally, protease inhibitors, for example, tryptase inhibitors, and proteases themselves, such as APC, have been used to successfully treat human diseases. The effectiveness of these therapies may well be attributable to effects on signaling by PARs, raising the possibility that antagonists and agonists of PARs may be useful drugs. Despite this impressive progress, there is much to learn about proteases and their receptors. First, the proteases that activate PARs are not fully characterized. Given the extremely widespread distribution of PARs, it is almost certain that new activating enzymes await discovery. This omission is particularly true for PAR2; although trypsin is a very potent agonist of this receptor, almost nothing is known about the function and regulation of extrapancreatic trypsins, which are likely to activate PAR2 in certain tissues. Second, there is good evidence for the existence of other PARs. Many proteases have biological actions that appear to be receptor mediated but which cannot be explained by the known PARs. Moreover, differences in the rank order of potencies of PAR agonists in bioassays point to the existence of receptor subtypes. Whether these novel receptors are PAR-like GPCRs or different types of receptor remains to be determined. Third, the mechanisms of PAR signaling are not fully explored. Thus the process by which proteolytic cleavage of an extracellular portion of the receptor induces the interaction of intracellular domains with heterotrimeric G proteins is not understood. Furthermore, the molecular events that terminate this irreversible mechanism of activation are not completely defined. For example, little is known about the postendocytic sorting mechanisms that target PARs for degradation. Fourth, and most important, from the standpoint of physiology and medicine, the optimal tools to study the function of PARs, receptor antagonists, are generally lacking. Although antagonists have been reported for certain PARs, they are not widely available. Finally, information about the role of proteases and PARs in many experimental systems is simply lacking. It will be of great interest to investigate the contributions of proteases, protease inhibitors, cofactors, and PARs in experimental models of human disease using genetically modified animals and available pharmacological reagents. We thank members of our laboratory for valuable discussion and advice and for critically reading this manuscript. 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 smooth muscle cells of the metastatic tumor microenvironment (80). In particular, there is an upregulation of PAR1 and PAR2 in proliferating stromal fibroblasts surrounding the carcinoma cells. In pulmonary tumor alveolar walls, the expression of PAR1 and PAR2 mRNA is increased by 10- and 16-fold, respectively, compared with normal alveolar tissues (140). Expression of trypsin is also detected in this tumor tissue. The metastasis of tumor cells requires cell detachment from the expanding tumor mass, degradation of the stromal tissue and basement membrane, and increased cell motility. There is considerable evidence that proteases and PAR1 may contribute to these processes. The level of expression of PAR1 on tumor cells directly correlates with metastatic potential in both primary breast carcinoma and established cancer cell lines (97). Introduction of PAR1 antisense cDNA inhibits the invasion of metastatic breast carcinoma cells in culture through a reconstituted basement membrane, indicating an important role for PAR1 in tumor cell migration. In support of this observation, thrombin and PAR1 AP promote the invasion of a highly aggressive breast cancer cell line expressing PAR1 in an in vitro assay (122). Exposure of certain tumor cells to thrombin and PAR1 AP also enhances their adhesion to platelets, fibronectin, and von Willebrand factor in vitro and promotes pulmonary metastasis when cells are administered to mice in vivo (209, 210). Moreover, overexpression of PAR1 in certain tumor cells can enhance their metastatic potential in animal models. In colon cancer cell lines, activation of PAR1 induces a marked mitogenic response, which is dependent on activation of MAP kinase ERK1/2, and also stimulate motility of wounded cells (81). Together, these results suggest that antagonists of PAR1 may be useful treatments for proliferation and metastases of certain tumors. However, PAR1 AP has also reported to inhibit migration and invasion of breast cancer cell lines when applied as a concentration gradient in the direction of movement (144). PAR2 may also contribute to tumor formation and metastasis since PAR2 AP stimulates proliferation of colon tumor cell lines (82). PROTEASE-ACTIVATED RECEPTORS This review is not exhaustive; some papers have been omitted due to space limitations. Research in the authors’ laboratory in this area is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-43207, DK-57489, and DK-39957 and by the RW Johnson Focused Giving Program (to N. W. Bunnett). Address for reprint requests and other correspondence: N. W. Bunnett, Room C317, UCSF, 513 Parnassus Ave., San Francisco, CA 94143-0660 (E-mail: [email protected]). 16. 17. REFERENCES Physiol Rev • VOL 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. Wadsworth RM, Gould GW, and Plevin R. Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases. Biochem J 320: 939 – 946, 1996. Benka ML, Lee M, Wang GR, Buckman S, Burlacu A, Cole L, DePina A, Dias P, Granger A, Grant B, Hayward-Lester A, Karki S, Mann S, Marcu O, Nussenzweig A, Piepenhagen P, Raje M, Roegiers F, Rybak S, Salic A, Smith-Hall J, Waters J, Yamamoto N, Yanowitz J, Yeow K, Busa WB, and Mendelsohn ME. The thrombin receptor in human platelets is coupled to a GTP binding protein of the G alpha q family. FEBS Lett 363: 49 –52, 1995. Berger P, Compton SJ, Molimard M, Walls AF, N’Guyen C, Marthan R, and Tunon-De-Lara JM. Mast cell tryptase as a mediator of hyperresponsiveness in human isolated bronchi. Clin Exp Allergy 29: 804 – 812, 1999. Berger P, Perng DW, Thabrew H, Compton SJ, Cairns JA, McEuen AR, Marthan R, Tunon De Lara JM, and Walls AF. Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells. J Appl Physiol 91: 1372–1379, 2001. Berger P, Tunon-De-Lara JM, Savineau JP, and Marthan R. Selected contribution: tryptase-induced PAR-2-mediated Ca2⫹ signaling in human airway smooth muscle cells. J Appl Physiol 91: 995–1003, 2001. Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, Steingrub JS, Garber GE, Helterbrand JD, Ely EW, and Fisher CJ Jr. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 344: 699 –709, 2001. Blackhart BD, Emilsson K, Nguyen D, Teng W, Martelli AJ, Nystedt S, Sundelin J, and Scarborough RM. Ligand crossreactivity within the protease-activated receptor family. J Biol Chem 271: 16466 –16471, 1996. Bohm SK, Grady EF, and Bunnett NW. Regulatory mechanisms that modulate signalling by G-protein-coupled receptors. Biochem J 322: 1–18, 1997. Bohm SK, Khitin LM, Grady EF, Aponte G, Payan DG, and Bunnett NW. Mechanisms of desensitization and resensitization of proteinase-activated receptor-2. J Biol Chem 271: 22003–22016, 1996. Bohm SK, Kong W, Bromme D, Smeekens SP, Anderson DC, Connolly A, Kahn M, Nelken NA, Coughlin SR, Payan DG, and Bunnett NW. Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2. Biochem J 314: 1009 –1016, 1996. Bouton MC, Jandrot-Perrus M, Moog S, Cazenave JP, Guillin MC, and Lanza F. Thrombin interaction with a recombinant Nterminal extracellular domain of the thrombin receptor in an acellular system. Biochem J 305: 635– 641, 1995. Brass LF, Pizarro S, Ahuja M, Belmonte E, Blanchard N, Stadel JM, and Hoxie JA. Changes in the structure and function of the human thrombin receptor during receptor activation, internalization, and recycling. J Biol Chem 269: 2943–2952, 1994. Bretschneider E, Kaufmann R, Braun M, Nowak G, Glusa E, and Schror K. Evidence for functionally active protease-activated receptor-4 (PAR-4) in human vascular smooth muscle cells. Br J Pharmacol 132: 1441–1446, 2001. Bretschneider E, Kaufmann R, Braun M, Wittpoth M, Glusa E, Nowak G, and Schror K. Evidence for proteinase-activated receptor-2 (PAR-2)-mediated mitogenesis in coronary artery smooth muscle cells. Br J Pharmacol 126: 1735–1740, 1999. Brown JK, Tyler CL, Jones CA, Ruoss SJ, Hartmann T, and Caughey GH. Tryptase, the dominant secretory granular protein in human mast cells, is a potent mitogen for cultured dog tracheal smooth muscle cells. J Clin Invest 88: 493– 499, 1991. Buresi MC, Buret AG, Hollenberg MD, and MacNaughton WK. Activation of proteinase-activated receptor 1 stimulates epithelial chloride secretion through a unique MAP kinase- and cyclo-oxygenase-dependent pathway. FASEB J 16: 1515–1525, 2002. Buresi MC, Schleihauf E, Vergnolle N, Buret A, Wallace JL, Hollenberg MD, and MacNaughton WK. Protease-activated receptor-1 stimulates Ca2⫹-dependent Cl⫺ secretion in human intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 281: G323–G332, 2001. 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 1. Abraham LA, Chinni C, Jenkins AL, Lourbakos A, Ally N, Pike RN, and Mackie EJ. Expression of protease-activated receptor-2 by osteoblasts. Bone 26: 7–14, 2000. 2. Akers IA, Parsons M, Hill MR, Hollenberg MD, Sanjar S, Laurent GJ, and McAnulty RJ. Mast cell tryptase stimulates human lung fibroblast proliferation via protease-activated receptor-2. Am J Physiol Lung Cell Mol Physiol 278: L193–L201, 2000. 3. Al-Ani B, Saifeddine M, and Hollenberg MD. Detection of functional receptors for the proteinase-activated-receptor-2-activating polypeptide, SLIGRL-NH2, in rat vascular and gastric smooth muscle. Can J Phys Pharmacol 73: 1203–1207, 1995. 4. Al-Ani B, Saifeddine M, Kawabata A, and Hollenberg MD. Proteinase activated receptor 2: role of extracellular loop 2 for ligand-mediated activation. Br J Pharmacol 128: 1105–1113, 1999. 5. Algermissen B, Sitzmann J, Nurnberg W, Laubscher JC, Henz BM, and Bauer F. Distribution and potential biologic function of the thrombin receptor PAR-1 on human keratinocytes. Arch Dermatol Res 292: 488 – 495, 2000. 6. Alm AK, Gagnemo-Persson R, Sorsa T, and Sundelin J. Extrapancreatic trypsin-2 cleaves proteinase-activated receptor-2. Biochem Biophys Res Commun 275: 77– 83, 2000. 7. Andersen H, Greenberg DL, Fujikawa K, Xu W, Chung DW, and Davie EW. Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity. Proc Natl Acad Sci USA 96: 11189 –11193, 1999. 8. Andrade-Gordon P, Derian CK, Maryanoff BE, Zhang HC, Addo MF, Cheung W, Damiano BP, D’Andrea MR, Darrow AL, de Garavilla L, Eckardt AJ, Giardino EC, Haertlein BJ, and McComsey DF. Administration of a potent antagonist of proteaseactivated receptor-1 (PAR-1) attenuates vascular restenosis following balloon angioplasty in rats. J Pharmacol Exp Ther 298: 34 – 42, 2001. 9. Andrade-Gordon P, Maryanoff BE, Derian CK, Zhang HC, Addo MF, Darrow AL, Eckardt AJ, Hoekstra WJ, McComsey DF, Oksenberg D, Reynolds EE, Santulli RJ, Scarborough RM, Smith CE, and White KB. Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tetheredligand receptor. Proc Natl Acad Sci USA 96: 12257–12262, 1999. 10. Aragay AM, Collins LR, Post GR, Watson AJ, Feramisco JR, Brown JH, and Simon MI. G12 requirement for thrombin-stimulated gene expression and DNA synthesis in 1321N1 astrocytoma cells. J Biol Chem 270: 20073–20077, 1995. 11. Asfaha S, Brussee V, Chapman K, Zochodne DW, and Vergnolle N. Proteinase-activated receptor-1 agonists attenuate nociception in response to noxious stimuli. Br J Pharmacol 135: 1101–1106, 2002. 12. Asokananthan N, Graham PT, Fink J, Knight DA, Bakker AJ, McWilliam AS, Thompson PJ, and Stewart GA. Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells. J Immunol 168: 3577–3585, 2002. 13. Baffy G, Yang L, Raj S, Manning DR, and Williamson JR. G protein coupling to the thrombin receptor in Chinese hamster lung fibroblasts. J Biol Chem 269: 8483– 8487, 1994. 14. Beecher KL, Andersen TT, Fenton JWN, and Festoff BW. Thrombin receptor peptides induce shape change in neonatal murine astrocytes in culture. J Neurosci Res 37: 108 –115, 1994. 15. Belham CM, Tate RJ, Scott PH, Pemberton AD, Miller HR, 613 614 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT Physiol Rev • VOL 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. bin receptor activating peptide in guinea-pigs in vivo. Br J Pharmacol 126: 478 – 484, 1999. Cicala C, Morello S, Santagada V, Caliendo G, Sorrentino L, and Cirino G. Pharmacological dissection of vascular effects caused by activation of protease-activated receptors 1 and 2 in anesthetized rats. FASEB J 15: 1433–1435, 2001. Cicala C, Pinto A, Bucci M, Sorrentino R, Walker B, Harriot P, Cruchley A, Kapas S, Howells GL, and Cirino G. Proteaseactivated receptor-2 involvement in hypotension in normal and endotoxemic rats in vivo. Circulation 99: 2590 –2597, 1999. Cirino G, Bucci M, Cicala C, and Napoli C. Inflammation-coagulation network: are serine protease receptors the knot? Trends Pharmacol Sci 21: 170 –172, 2000. Clark JM, Abraham WM, Fishman CE, Forteza R, Ahmed A, Cortes A, Warne RL, Moore WR, and Tanaka RD. Tryptase inhibitors block allergen-induced airway and inflammatory responses in allergic sheep. Am J Respir Crit Care Med 152: 2076 – 2083, 1995. Cocks TM, Fong B, Chow JM, Anderson GP, Frauman AG, Goldie RG, Henry PJ, Carr MJ, Hamilton JR, and Moffatt JD. A protective role for protease-activated receptors in the airways. Nature 398: 156 –160, 1999. Cocks TM and Moffatt JD. Protease-activated receptors: sentries for inflammation? Trends Pharmacol Sci 21: 103–108, 2000. Cocks TM and Moffatt JD. Protease-activated receptor-2 (PAR2) in the airways. Pulm Pharmacol Ther 14: 183–191, 2001. Cocks TM, Sozzi V, Moffatt JD, and Selemidis S. Proteaseactivated receptors mediate apamin-sensitive relaxation of mouse and guinea pig gastrointestinal smooth muscle. Gastroenterology 116: 586 –592, 1999. Coelho AM, Vergnolle N, Guiard B, Fioramonti J, and Bueno L. Proteinases and proteinase-activated receptor 2: a possible role to promote visceral hyperalgesia in rats. Gastroenterology 122: 1035–1047, 2002. Compton SJ, Cairns JA, Palmer KJ, Al-Ani B, Hollenberg MD, and Walls AF. A polymorphic protease-activated receptor 2 (PAR2) displaying reduced sensitivity to trypsin and differential responses to PAR agonists. J Biol Chem 275: 39207–39212, 2000. Compton SJ, Renaux B, Wijesuriya SJ, and Hollenberg MD. Glycosylation and the activation of proteinase-activated receptor 2 [PAR(2)] by human mast cell tryptase. Br J Pharmacol 134: 705– 718, 2001. Compton SJ, Sandhu S, Wijesuriya SJ, and Hollenberg MD. Glycosylation of human proteinase-activated receptor-2 (hPAR2): role in cell surface expression and signalling. Biochem J 368: 495–505, 2002. Connolly AJ, Ishihara H, Kahn ML, Farese RV Jr, and Coughlin SR. Role of the thrombin receptor in development and evidence for a second receptor. Nature 381: 516 –519, 1996. Connolly AJ, Suh DY, Hunt TK, and Coughlin SR. Mice lacking the thrombin receptor, PAR1, have normal skin wound healing. Am J Pathol 151: 1199 –1204, 1997. Corvera CU, Dery O, McConalogue K, Bohm SK, Khitin LM, Caughey GH, Payan DG, and Bunnett NW. Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2. J Clin Invest 100: 1383–1393, 1997. Corvera CU, Dery O, McConalogue K, Gamp P, Thoma M, Al-Ani B, Caughey GH, Hollenberg MD, and Bunnett NW. Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and -2. J Physiol 517: 741–756, 1999. Coughlin SR. Thrombin signalling and protease-activated receptors. Nature 407: 258 –264, 2000. Coughlin SR and Camerer E. PARticipation in inflammation. J Clin Invest 111: 25–27, 2003. Covic L, Gresser AL, and Kuliopulos A. Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets. Biochemistry 39: 5458 –5467, 2000. Covic L, Gresser AL, Talavera J, Swift S, and Kuliopulos A. Activation and inhibition of G protein-coupled receptors by cellpenetrating membrane-tethered peptides. Proc Natl Acad Sci USA 99: 643– 648, 2002. Covic L, Misra M, Badar J, Singh C, and Kuliopulos A. Pepdu- 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 32. Bustos D, Negri G, De Paula JA, Di Carlo M, Yapur V, Facente A, and De Paula A. Colonic proteinases: increased activity in patients with ulcerative colitis. Medicina 58: 262–264, 1998. 33. Cairns JA and Walls AF. Mast cell tryptase is a mitogen for epithelial cells. Stimulation of IL-8 production and intercellular adhesion molecule-1 expression. J Immunol 156: 275–283, 1996. 34. Camerer E, Huang W, and Coughlin SR. Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proc Natl Acad Sci USA 97: 5255–5260, 2000. 35. Camerer E, Kataoka H, Kahn M, Lease K, and Coughlin SR. Genetic evidence that protease-activated receptors mediate factor Xa signaling in endothelial cells. J Biol Chem 277: 16081–16087, 2002. 36. Carney DH, Mann R, Redin WR, Pernia SD, Berry D, Heggers JP, Hayward PG, Robson MC, Christie J, Annable C, Fenton JW II, and Glenn KC. Enhancement of incisional wound healing and neovascularization in normal rats by thrombin and synthetic thrombin receptor-activating peptides. J Clin Invest 89: 1469 –1477, 1992. 37. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, and Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389: 816 – 824, 1997. 38. Caughey GH. Mast cell chymases and tryptases: phylogeny, family relations, and biogenesis. In: Mast Cell Proteases in Immunology and Biology, edited by Caughey GH. New York: Dekker, 1995, p. 305–329. 39. Cavanaugh KP, Gurwitz D, Cunningham DD, and Bradshaw RA. Reciprocal modulation of astrocyte stellation by thrombin and protease nexin-1. J Neurochem 54: 1735–1743, 1990. 40. Cenac N, Coelho AM, Nguyen C, Compton S, Andrade-Gordon P, MacNaughton WK, Wallace JL, Hollenberg MD, Bunnett NW, Garcia-Villar R, Bueno L, and Vergnolle N. Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2. Am J Pathol 161: 1903–1915, 2002. 41. Cenac N, Garcia-Villar R, Ferrier L, Larauche M, Vergnolle N, Bunnett NW, Coelho AM, Fioramonti J, and Bueno L. Proteinase-activated receptor-2-induced colonic inflammation in mice: possible involvement of afferent neurons, nitric oxide, and paracellular permeability. J Immunol 170: 4296 – 4300, 2003. 42. Chambers RC, Leoni P, Blanc-Brude OP, Wembridge DE, and Laurent GJ. Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of proteaseactivated receptor-1. J Biol Chem 275: 35584 –35591, 2000. 43. Chen J, Ishii M, Wang L, Ishii K, and Coughlin SR. Thrombin receptor activation. Confirmation of the intramolecular tethered liganding hypothesis and discovery of an alternative intermolecular liganding mode. J Biol Chem 269: 16041–16045, 1994. 44. Chen X, Earley K, Luo W, Lin SH, and Schilling WP. Functional expression of a human thrombin receptor in Sf9 insect cells: evidence for an active tethered ligand. Biochem J 314: 603– 611, 1996. 45. Chen YH, Pouyssegur J, Courtneidge SA, and Van ObberghenSchilling E. Activation of Src family kinase activity by the G protein-coupled thrombin receptor in growth-responsive fibroblasts. J Biol Chem 269: 27372–27377, 1994. 46. Cheng T, Liu D, Griffin JH, Fernandez JA, Castellino F, Rosen ED, Fukudome K, and Zlokovic BV. Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective. Nat Med 9: 338 –342, 2003. 47. Cheung WM, Andrade-Gordon P, Derian CK, and Damiano BP. Receptor-activating peptides distinguish thrombin receptor (PAR-1) and protease activated receptor 2 (PAR-2) mediated hemodynamic responses in vivo. Can J Physiol Pharmacol 76: 16 –25, 1998. 48. Cheung WM, D’Andrea MR, Andrade-Gordon P, and Damiano BP. Altered vascular injury responses in mice deficient in proteaseactivated receptor-1. Arterioscler Thromb Vasc Biol 19: 3014 –3024, 1999. 49. Chow JM, Moffatt JD, and Cocks TM. Effect of protease-activated receptor (PAR)-1, -2 and -4-activating peptides, thrombin and trypsin in rat isolated airways. Br J Pharmacol 131: 1584–1591, 2000. 50. Cicala C. Protease activated receptor 2 and the cardiovascular system. Br J Pharmacol 135: 14 –20, 2002. 51. Cicala C, Bucci M, De Dominicis G, Harriot P, Sorrentino L, and Cirino G. Bronchoconstrictor effect of thrombin and throm- PROTEASE-ACTIVATED RECEPTORS 73. 74. 75. 76. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. Physiol Rev • VOL 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. ase-activated receptors: novel mechanisms of signaling by serine proteases. Am J Physiol Cell Physiol 274: C1429 –C1452, 1998. Dery O, Thoma MS, Wong H, Grady EF, and Bunnett NW. Trafficking of proteinase-activated receptor-2 and beta-arrestin-1 tagged with green fluorescent protein. beta-Arrestin-dependent endocytosis of a proteinase receptor. J Biol Chem 274: 18524 –18535, 1999. Dihanich M, Kaser M, Reinhard E, Cunningham D, and Monard D. Prothrombin mRNA is expressed by cells of the nervous system. Neuron 6: 575–581, 1991. Donovan FM and Cunningham DD. Signaling pathways involved in thrombin-induced cell protection. J Biol Chem 273: 12746 – 12752, 1998. Donovan FM, Pike CJ, Cotman CW, and Cunningham DD. Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities. J Neurosci 17: 5316 –5326, 1997. Ducroc R, Bontemps C, Marazova K, Devaud H, Darmoul D, and Laburthe M. Trypsin is produced by and activates proteaseactivated receptor-2 in human cancer colon cells: evidence for new autocrine loop. Life Sci 70: 1359 –1367, 2002. Dulon S, Cande C, Bunnett NW, Hollenberg MD, Chignard M, and Pidard D. Proteinase-activated receptor-2 and human lung epithelial cells: disarming by neutrophil serine proteinases. Am J Respir Cell Mol Biol 28: 339 –346, 2003. Ekholm IE, Brattsand M, and Egelrud T. Stratum corneum tryptic enzyme in normal epidermis: a missing link in the desquamation process? J Invest Dermatol 114: 56 – 63, 2000. Even-Ram S, Uziely B, Cohen P, Grisaru-Granovsky S, Maoz M, Ginzburg Y, Reich R, Vlodavsky I, and Bar-Shavit R. Thrombin receptor overexpression in malignant and physiological invasion processes. Nat Med 4: 909 –914, 1998. Faruqi TR, Weiss EJ, Shapiro MJ, Huang W, and Coughlin SR. Structure-function analysis of protease-activated receptor 4 tethered ligand peptides. Determinants of specificity and utility in assays of receptor function. J Biol Chem 275: 19728 –19734, 2000. Fee JA, Monsey JD, Handler RJ, Leonis MA, Mullaney SR, Hope HM, and Silbert DF. A Chinese hamster fibroblast mutant defective in thrombin-induced signaling has a low level of phospholipase C-beta 1. J Biol Chem 269: 21699 –21708, 1994. Feng DM, Veber DF, Connolly TM, Condra C, Tang MJ, and Nutt RF. Development of a potent thrombin receptor ligand. J Med Chem 38: 4125– 4130, 1995. Ferrell WR, Lockhart JC, Kelso EB, Dunning L, Plevin R, Meek SE, Smith AJ, Hunter GD, McLean JS, McGarry F, Ramage R, Jiang L, Kanke T, and Kawagoe J. Essential role for proteinase-activated receptor-2 in arthritis. J Clin Invest 111: 35– 41, 2003. Festoff BW, Smirnova IV, Ma J, and Citron BA. Thrombin, its receptor and protease nexin I, its potent serpin, in the nervous system. Semin Thromb Hemost 22: 267–271, 1996. Fiorucci S, Mencarelli A, Palazzetti B, Distrutti E, Vergnolle N, Hollenberg MD, Wallace JL, Morelli A, and Cirino G. Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis. Proc Natl Acad Sci USA 98: 13936 –13941, 2001. Gao C, Liu S, Hu HZ, Gao N, Kim GY, Xia Y, and Wood JD. Serine proteases excite myenteric neurons through protease-activated receptors in guinea pig small intestine. Gastroenterology 123: 1554 –1564, 2002. Gerszten RE, Chen J, Ishii M, Ishii K, Wang L, Nanevicz T, Turck CW, Vu TK, and Coughlin SR. Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface. Nature 368: 648 – 651, 1994. Gill JS, Pitts K, Rusnak FM, Owen WG, and Windebank AJ. Thrombin induced inhibition of neurite outgrowth from dorsal root ganglion neurons. Brain Res 797: 321–327, 1998. Gingrich MB, Junge CE, Lyuboslavsky P, and Traynelis SF. Potentiation of NMDA receptor function by the serine protease thrombin. J Neurosci 20: 4582– 4595, 2000. Grand RJ, Turnell AS, and Grabham PW. Cellular consequences of thrombin-receptor activation. Biochem J 313: 353–368, 1996. Green BT, Bunnett NW, Kulkarni-Narla A, Steinhoff M, and 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 77. cin-based intervention of thrombin-receptor signaling and systemic platelet activation. Nat Med 8: 1161–1165, 2002. Cuffe JE, Bertog M, Velazquez-Rocha S, Dery O, Bunnett N, and Korbmacher C. Basolateral PAR-2 receptors mediate KCl secretion and inhibition of Na⫹ absorption in the mouse distal colon. J Physiol 539: 209 –222, 2002. Cumashi A, Ansuini H, Celli N, De Blasi A, O’Brien PJ, Brass LF, and Molino M. Neutrophil proteases can inactivate human PAR3 and abolish the co-receptor function of PAR3 on murine platelets. Thromb Haemostasis 85: 533–538, 2001. Cunningham MA, Rondeau E, Chen X, Coughlin SR, Holdsworth SR, and Tipping PG. Protease-activated receptor 1 mediates thrombin-dependent, cell-mediated renal inflammation in crescentic glomerulonephritis. J Exp Med 191: 455– 462, 2000. Daaka Y, Luttrell LM, Ahn S, Della Rocca GJ, Ferguson SS, Caron MG, and Lefkowitz RJ. Essential role for G proteincoupled receptor endocytosis in the activation of mitogen-activated protein kinase. J Biol Chem 273: 685– 688, 1998. Damiano BP, Cheung WM, Santulli RJ, Fung-Leung WP, Ngo K, Ye RD, Darrow AL, Derian CK, de Garavilla L, and Andrade-Gordon P. Cardiovascular responses mediated by proteaseactivated receptor-2 (PAR- 2) and thrombin receptor (PAR-1) are distinguished in mice deficient in PAR-2 or PAR-1. J Pharmacol Exp Ther 288: 671– 678, 1999. Danahay H, Withey L, Poll CT, van de Graaf SF, and Bridges RJ. Protease-activated receptor-2-mediated inhibition of ion transport in human bronchial epithelial cells. Am J Physiol Cell Physiol 280: C1455–C1464, 2001. D’Andrea MR, Derian CK, Leturcq D, Baker SM, Brunmark A, Ling P, Darrow AL, Santulli RJ, Brass LF, and AndradeGordon P. Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues. J Histochem Cytochem 46: 157–164, 1998. D’Andrea MR, Derian CK, Santulli RJ, and Andrade-Gordon P. Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues. Am J Pathol 158: 2031–2041, 2001. Darmoul D, Gratio V, Devaud H, Lehy T, and Laburthe M. Aberrant expression and activation of the thrombin receptor protease-activated receptor-1 induces cell proliferation and motility in human colon cancer cells. Am J Pathol 162: 1503–1513, 2003. Darmoul D, Marie JC, Devaud H, Gratio V, and Laburthe M. Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2. Br J Cancer 85: 772–779, 2001. Darrow AL, Fung-Leung WP, Ye RD, Santulli RJ, Cheung WM, Derian CK, Burns CL, Damiano BP, Zhou L, Keenan CM, Peterson PA, and Andrade-Gordon P. Biological consequences of thrombin receptor deficiency in mice. Thromb Haemostasis 76: 860 – 866, 1996. De Candia E, Hall SW, Rutella S, Landolfi R, Andrews RK, and De Cristofaro R. Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets. J Biol Chem 276: 4692– 4698, 2001. Defea K, Schmidlin F, Dery O, Grady EF, and Bunnett NW. Mechanisms of initiation and termination of signalling by neuropeptide receptors: a comparison with the proteinase-activated receptors. Biochem Soc Trans 28: 419 – 426, 2000. DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins RD, and Bunnett NW. Beta-arrestin-dependent endocytosis of proteinaseactivated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol 148: 1267–1281, 2000. De Garavilla L, Vergnolle N, Young SH, Ennes H, Steinhoff M, Ossovskaya VS, D’Andrea MR, Mayer EA, Wallace JL, Hollenberg MD, Andrade-Gordon P, and Bunnett NW. Agonists of proteinase-activated receptor 1 induce plasma extravasation by a neurogenic mechanism. Br J Pharmacol 133: 975–987, 2001. Derian CK, Damiano BP, Addo MF, Darrow AL, D’Andrea MR, Nedelman M, Zhang HC, Maryanoff BE, and Andrade-Gordon P. Blockade of the thrombin receptor protease-activated receptor-1 with a small-molecule antagonist prevents thrombus formation and vascular occlusion in nonhuman primates. J Pharmacol Exp Ther 304: 855– 861, 2003. Dery O, Corvera CU, Steinhoff M, and Bunnett NW. Protein- 615 616 110. 111. 112. 113. 114. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. Brown DR. Intestinal type 2 proteinase-activated receptors: expression in opioid-sensitive secretomotor neural circuits that mediate epithelial ion transport. J Pharmacol Exp Ther 295: 410 – 416, 2000. Griffin CT, Srinivasan Y, Zheng YW, Huang W, and Coughlin SR. A role for thrombin receptor signaling in endothelial cells during embryonic development. Science 293: 1666 –1670, 2001. Griffin JH, Zlokovic B, and Fernandez JA. Activated protein C: potential therapy for severe sepsis, thrombosis, and stroke. Semin Hematol 39: 197–205, 2002. Gurwitz D and Cunningham DD. Thrombin modulates and reverses neuroblastoma neurite outgrowth. Proc Natl Acad Sci USA 85: 3440 –3444, 1988. Hamilton JR, Chow JM, and Cocks TM. Protease-activated receptor-2 turnover stimulated independently of receptor activation in porcine coronary endothelial cells. Br J Pharmacol 127: 617– 622, 1999. Hamilton JR and Cocks TM. Heterogeneous mechanisms of endothelium-dependent relaxation for thrombin and peptide activators of protease-activated receptor-1 in porcine isolated coronary artery. Br J Pharmacol 130: 181–188, 2000. Hamilton JR, Frauman AG, and Cocks TM. Increased expression of protease-activated receptor-2 (par2) and par4 in human coronary artery by inflammatory stimuli unveils endothelium- dependent relaxations to par2 and par4 agonists. Circ Res 89: 92–98, 2001. Hamilton JR, Moffatt JD, Frauman AG, and Cocks TM. Protease-activated receptor (PAR) 1 but not PAR2 or PAR4 mediates endothelium-dependent relaxation to thrombin and trypsin in human pulmonary arteries. J Cardiovasc Pharmacol 38: 108 –119, 2001. Hamilton JR, Moffatt JD, Tatoulis J, and Cocks TM. Enzymatic activation of endothelial protease-activated receptors is dependent on artery diameter in human and porcine isolated coronary arteries. Br J Pharmacol 136: 492–501, 2002. Hayes KL and Tracy PB. The platelet high affinity binding site for thrombin mimics hirudin, modulates thrombin-induced platelet activation, and is distinct from the glycoprotein Ib-IX-V complex. J Biol Chem 274: 972–980, 1999. He S, Peng Q, and Walls AF. Potent induction of a neutrophil and eosinophil-rich infiltrate in vivo by human mast cell tryptase: selective enhancement of eosinophil recruitment by histamine. J Immunol 159: 6216 – 6225, 1997. He S and Walls AF. Human mast cell tryptase: a stimulus of microvascular leakage and mast cell activation. Eur J Pharmacol 328: 89 –97, 1997. Hein L, Ishii K, Coughlin SR, and Kobilka BK. Intracellular targeting and trafficking of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor. J Biol Chem 269: 27719 –27726, 1994. Henrikson KP, Salazar SL, Fenton JW II, and Pentecost BT. Role of thrombin receptor in breast cancer invasiveness. Br J Cancer 79: 401– 406, 1999. Hollenberg MD, Saifeddine M, al-Ani B, and Kawabata A. Proteinase-activated receptors: structural requirements for activity, receptor cross-reactivity, and receptor selectivity of receptoractivating peptides. Can J Physiol Pharmacol 75: 832– 841, 1997. Hoogerwerf WA, Zou L, Shenoy M, Sun D, Micci MA, LeeHellmich H, Xiao SY, Winston JH, and Pasricha PJ. The proteinase-activated receptor 2 is involved in nociception. J Neurosci 21: 9036 –9042, 2001. Horvat R and Palade GE. The functional thrombin receptor is associated with the plasmalemma and a large endosomal network in cultured human umbilical vein endothelial cells. J Cell Sci 108: 1155–1164, 1995. Hou L, Kapas S, Cruchley AT, Macey MG, Harriott P, Chinni C, Stone SR, and Howells GL. Immunolocalization of proteaseactivated receptor-2 in skin: receptor activation stimulates interleukin-8 secretion by keratinocytes in vitro. Immunology 94: 356 – 362, 1998. Howell DC, Laurent GJ, and Chambers RC. Role of thrombin and its major cellular receptor, protease-activated receptor-1, in pulmonary fibrosis. Biochem Soc Trans 30: 211–216, 2002. Physiol Rev • VOL 128. Howells GL, Macey MG, Chinni C, Hou L, Fox MT, Harriott P, and Stone SR. Proteinase-activated receptor-2: expression by human neutrophils. J Cell Sci 110: 881– 887, 1997. 129. Hoxie JA, Ahuja M, Belmonte E, Pizarro S, Parton R, and Brass LF. Internalization and recycling of activated thrombin receptors. J Biol Chem 268: 13756 –13763, 1993. 130. Huang C, De Sanctis GT, O’Brien PJ, Mizgerd JP, Friend DS, Drazen JM, Brass LF, and Stevens RL. Evaluation of the substrate specificity of human mast cell tryptase beta I and demonstration of its importance in bacterial infections of the lung. J Biol Chem 276: 26276 –26284, 2001. 131. Hung DT, Vu TH, Nelken NA, and Coughlin SR. Thrombininduced events in non-platelet cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor. J Cell Biol 116: 827– 832, 1992. 132. Hung DT, Wong YH, Vu TK, and Coughlin SR. The cloned platelet thrombin receptor couples to at least two distinct effectors to stimulate phosphoinositide hydrolysis and inhibit adenylyl cyclase. J Biol Chem 267: 20831–20834, 1992. 133. Iaccarino G, Rockman HA, Shotwell KF, Tomhave ED, and Koch WJ. Myocardial overexpression of GRK3 in transgenic mice: evidence for in vivo selectivity of GRKs. Am J Physiol Heart Circ Physiol 275: H1298 –H1306, 1998. 134. Idell S, Gonzalez K, Bradford H, MacArthur CK, Fein AM, Maunder RJ, Garcia JG, Griffith DE, Weiland J, and Martin TR. Procoagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome. Contribution of tissue factor associated with factor VII. Am Rev Respir Dis 136: 1466 –1474, 1987. 135. Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, Timmons C, Tram T, and Coughlin SR. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386: 502–506, 1997. 136. Ishii K, Chen J, Ishii M, Koch WJ, Freedman NJ, Lefkowitz RJ, and Coughlin SR. Inhibition of thrombin receptor signaling by a G-protein coupled receptor kinase. Functional specificity among G-protein coupled receptor kinases. J Biol Chem 269: 1125–1130, 1994. 137. Ishii K, Hein L, Kobilka B, and Coughlin SR. Kinetics of thrombin receptor cleavage on intact cells. Relation to signaling. J Biol Chem 268: 9780 –9786, 1993. 138. Jalink K, van Corven EJ, Hengeveld T, Morii N, Narumiya S, and Moolenaar WH. Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho. J Cell Biol 126: 801– 810, 1994. 139. Jarjour NN, Calhoun WJ, Schwartz LB, and Busse WW. Elevated bronchoalveolar lavage fluid histamine levels in allergic asthmatics are associated with increased airway obstruction. Am Rev Respir Dis 144: 83– 87, 1991. 140. Jin E, Fujiwara M, Pan X, Ghazizadeh M, Arai S, Ohaki Y, Kajiwara K, Takemura T, and Kawanami O. Protease-activated receptor (PAR)-1 and PAR-2 participate in the cell growth of alveolar capillary endothelium in primary lung adenocarcinomas. Cancer 97: 703–713, 2003. 141. Julius D and Basbaum AI. Molecular mechanisms of nociception. Nature 413: 203–210, 2001. 142. Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, and Coughlin SR. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 103: 879 – 887, 1999. 143. Kahn ML, Zheng YW, Huang W, Bigornia V, Zeng D, Moff S, Farese RV Jr, Tam C, and Coughlin SR. A dual thrombin receptor system for platelet activation. Nature 394: 690 – 694, 1998. 144. Kamath L, Meydani A, Foss F, and Kuliopulos A. Signaling from protease-activated receptor-1 inhibits migration and invasion of breast cancer cells. Cancer Res 61: 5933–5940, 2001. 145. Kanke T, Macfarlane SR, Seatter MJ, Davenport E, Paul A, McKenzie R, and Plevin R. Proteinase-activated receptor-2-mediated activation of stress-activated protein kinases and inhibitory kappa B kinases in NCTC 2544 keratinocytes. J Biol Chem 18: 18, 2001. 146. Katona G, Berglund GI, Hajdu J, Graf L, and Szilagyi L. 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 115. VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT PROTEASE-ACTIVATED RECEPTORS 147. 148. 149. 150. 151. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. Physiol Rev • VOL 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. airways: upregulation of PAR-2 in respiratory epithelium from patients with asthma. J Allergy Clin Immunol 108: 797– 803, 2001. Koivunen E, Huhtala ML, and Stenman UH. Human ovarian tumor-associated trypsin. Its purification and characterization from mucinous cyst fluid and identification as an activator of pro-urokinase. J Biol Chem 264: 14095–14099, 1989. Koivunen E, Saksela O, Itkonen O, Osman S, Huhtala ML, and Stenman UH. Human colonic carcinoma, fibrosarcoma and leukemia cell lines produce tumor-associated trypsinogen. Int J Cancer 47: 592–596, 1991. Kong W, McConalogue K, Khitin LM, Hollenberg MD, Payan DG, Bohm SK, and Bunnett NW. Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2. Proc Natl Acad Sci USA 94: 8884 – 8889, 1997. Koshikawa N, Hasegawa S, Nagashima Y, Mitsuhashi K, Tsubota Y, Miyata S, Miyagi Y, Yasumitsu H, and Miyazaki K. Expression of trypsin by epithelial cells of various tissues, leukocytes, and neurons in human and mouse. Am J Pathol 153: 937–944, 1998. Koshikawa N, Nagashima Y, Miyagi Y, Mizushima H, Yanoma S, Yasumitsu H, and Miyazaki K. Expression of trypsin in vascular endothelial cells. FEBS Lett 409: 442– 448, 1997. Krishna MT, Chauhan A, Little L, Sampson K, Hawksworth R, Mant T, Djukanovic R, Lee T, and Holgate S. Inhibition of mast cell tryptase by inhaled APC 366 attenuates allergen-induced latephase airway obstruction in asthma. J Allergy Clin Immunol 107: 1039 –1045, 2001. Kunzelmann K, Schreiber R, Konig J, and Mall M. Ion transport induced by proteinase-activated receptors (PAR2) in colon and airways. Cell Biochem Biophys 36: 209 –214, 2002. Lan RS, Knight DA, Stewart GA, and Henry PJ. Role of PGE(2) in protease-activated receptor-1, -2 and -4 mediated relaxation in the mouse isolated trachea. Br J Pharmacol 132: 93–100, 2001. Lan RS, Stewart GA, and Henry PJ. Modulation of airway smooth muscle tone by protease activated receptor- 1,-2,-3 and -4 in trachea isolated from influenza A virus-infected mice. Br J Pharmacol 129: 63–70, 2000. Laposata M, Dovnarsky DK, and Shin HS. Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro. Blood 62: 549 –556, 1983. Lerner DJ, Chen M, Tram T, and Coughlin SR. Agonist recognition by proteinase-activated receptor 2 and thrombin receptor. Importance of extracellular loop interactions for receptor function. J Biol Chem 271: 13943–13947, 1996. Linden DR, Manning BP, Bunnett NW, and Mawe GM. Agonists of proteinase-activated receptor 2 excite guinea pig ileal myenteric neurons. Eur J Pharmacol 431: 311–314, 2001. Lindner JR, Kahn ML, Coughlin SR, Sambrano GR, Schauble E, Bernstein D, Foy D, Hafezi-Moghadam A, and Ley K. Delayed onset of inflammation in protease-activated receptor-2-deficient mice. J Immunol 165: 6504 – 6510, 2000. Lourbakos A, Chinni C, Thompson P, Potempa J, Travis J, Mackie EJ, and Pike RN. Cleavage and activation of proteinaseactivated receptor-2 on human neutrophils by gingipain-R from Porphyromonas gingivalis. FEBS Lett 435: 45– 48, 1998. Lourbakos A, Potempa J, Travis J, D’Andrea MR, AndradeGordon P, Santulli R, Mackie EJ, and Pike RN. Arginine-specific protease from Porphyromonas gingivalis activates proteaseactivated receptors on human oral epithelial cells and induces interleukin-6 secretion. Infect Immun 69: 5121–5130., 2001. Lourbakos A, Yuan YP, Jenkins AL, Travis J, Andrade-Gordon P, Santulli R, Potempa J, and Pike RN. Activation of protease-activated receptors by gingipains from Porphyromonas gingivalis leads to platelet aggregation: a new trait in microbial pathogenicity. Blood 97: 3790 –3797, 2001. Luttrell LM and Lefkowitz RJ. The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J Cell Sci 115: 455– 465, 2002. Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, and Plevin R. Proteinase-activated receptors. Pharmacol Rev 53: 245–282, 2001. Madamanchi NR, Li S, Patterson C, and Runge MS. Thrombin regulates vascular smooth muscle cell growth and heat shock 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 152. Crystal structure reveals basis for the inhibitor resistance of human brain trypsin. J Mol Biol 315: 1209 –1218, 2002. Kauffman HF, Tomee JF, van de Riet MA, Timmerman AJ, and Borger P. Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production. J Allergy Clin Immunol 105: 1185–1193, 2000. Kaufmann R, Patt S, Zieger M, Kraft R, Tausch S, Henklein P, and Nowak G. The two-receptor system PAR-1/PAR-4 mediates alpha-thrombin-induced [Ca2⫹]i mobilization in human astrocytoma cells. J Cancer Res Clin Oncol 126: 91–94, 2000. Kawabata A, Kawao N, Kuroda R, Itoh H, and Nishikawa H. Specific expression of spinal Fos after PAR-2 stimulation in mast cell-depleted rats. Neuroreport 13: 511–514, 2002. Kawabata A, Kawao N, Kuroda R, Tanaka A, Itoh H, and Nishikawa H. Peripheral PAR-2 triggers thermal hyperalgesia and nociceptive responses in rats. Neuroreport 12: 715–719, 2001. Kawabata A, Kawao N, Kuroda R, Tanaka A, and Shimada C. The PAR-1-activating peptide attenuates carrageenan-induced hyperalgesia in rats. Peptides 23: 1181–1183, 2002. Kawabata A, Kinoshita M, Kuroda R, and Kakehi K. Capsazepine partially inhibits neurally mediated gastric mucus secretion following activation of protease-activated receptor 2. Clin Exp Pharmacol Physiol 29: 360 –361, 2002. Kawabata A, Kinoshita M, Nishikawa H, Kuroda R, Nishida M, Araki H, Arizono N, Oda Y, and Kakehi K. The protease-activated receptor-2 agonist induces gastric mucus secretion and mucosal cytoprotection. J Clin Invest 107: 1443–1450, 2001. Kawabata A, Kuroda R, Kuroki N, Nishikawa H, and Kawai K. Dual modulation by thrombin of the motility of rat oesophageal muscularis mucosae via two distinct protease-activated receptors (PARs): a novel role for PAR-4 as opposed to PAR-1. Br J Pharmacol 131: 578 –584, 2000. Kawabata A, Kuroda R, Nagata N, Kawao N, Masuko T, Nishikawa H, and Kawai K. In vivo evidence that protease-activated receptors 1 and 2 modulate gastrointestinal transit in the mouse. Br J Pharmacol 133: 1213–1218, 2001. Kawabata A, Kuroda R, Nishida M, Nagata N, Sakaguchi Y, Kawao N, Nishikawa H, Arizono N, and Kawai K. Proteaseactivated receptor-2 (PAR-2) in the pancreas and parotid gland: immunolocalization and involvement of nitric oxide in the evoked amylase secretion. Life Sci 71: 2435–2446, 2002. Kawabata A, Morimoto N, Nishikawa H, Kuroda R, Oda Y, and Kakehi K. Activation of protease-activated receptor-2 (PAR-2) triggers mucin secretion in the rat sublingual gland. Biochem Biophys Res Commun 270: 298 –302, 2000. Kawabata A, Nishikawa H, Kuroda R, Kawai K, and Hollenberg MD. Proteinase-activated receptor-2 (PAR-2): regulation of salivary and pancreatic exocrine secretion in vivo in rats and mice. Br J Pharmacol 129: 1808 –1814, 2000. Kawagoe J, Takizawa T, Matsumoto J, Tamiya M, Meek SE, Smith AJ, Hunter GD, Plevin R, Saito N, Kanke T, Fujii M, and Wada Y. Effect of protease-activated receptor-2 deficiency on allergic dermatitis in the mouse ear. Jpn J Pharmacol 88: 77– 84, 2002. Kawao N, Sakaguchi Y, Tagome A, Kuroda R, Nishida S, Irimajiri K, Nishikawa H, Kawai K, Hollenberg MD, and Kawabata A. Protease-activated receptor-2 (PAR-2) in the rat gastric mucosa: immunolocalization and facilitation of pepsin/pepsinogen secretion. Br J Pharmacol 135: 1292–1296, 2002. Keogh RJ, Houliston RA, and Wheeler-Jones CP. Thrombinstimulated Pyk2 phosphorylation in human endothelium is dependent on intracellular calcium and independent of protein kinase C and Src kinases. Biochem Biophys Res Commun 294: 1001–1008, 2002. King C, Brennan S, Thompson PJ, and Stewart GA. Dust mite proteolytic allergens induce cytokine release from cultured airway epithelium. J Immunol 161: 3645–3651, 1998. Klages B, Brandt U, Simon MI, Schultz G, and Offermanns S. Activation of G12/G13 results in shape change and Rho/Rho-kinasemediated myosin light chain phosphorylation in mouse platelets. J Cell Biol 144: 745–754, 1999. Knight DA, Lim S, Scaffidi AK, Roche N, Chung KF, Stewart GA, and Thompson PJ. Protease-activated receptors in human 617 618 184. 185. 186. 187. 188. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. proteins via the JAK-STAT pathway. J Biol Chem 276: 18915–18924, 2001. Magazine HI, King JM, and Srivastava KD. Protease activated receptors modulate aortic vascular tone. Int J Cardiol 53 Suppl: S75–S80, 1996. Marchese A and Benovic JL. Agonist-promoted ubiquitination of the G protein-coupled receptor CXCR4 mediates lysosomal sorting. J Biol Chem 276: 45509 – 45512, 2001. Marty I, Peclat V, Kirdaite G, Salvi R, So A, and Busso N. Amelioration of collagen-induced arthritis by thrombin inhibition. J Clin Invest 107: 631– 640, 2001. Maryanoff BE, Santulli RJ, McComsey DF, Hoekstra WJ, Hoey K, Smith CE, Addo M, Darrow AL, and Andrade-Gordon P. Protease-activated receptor-2 (PAR-2): structure-function study of receptor activation by diverse peptides related to tethered-ligand epitopes. Arch Biochem Biophys 386: 195–204, 2001. McEuen AR, He S, Brander ML, and Walls AF. Guinea pig lung tryptase. Localisation to mast cells and characterisation of the partially purified enzyme. Biochem Pharmacol 52: 331–340, 1996. McNamara CA, Sarembock IJ, Gimple LW, Fenton JW, Coughlin SR, and Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 91: 94 –98, 1993. Metcalfe DD, Baram D, and Mekori YA. Mast cells. Physiol Rev 77: 1033–1079, 1997. Milia AF, Salis MB, Stacca T, Pinna A, Madeddu P, Trevisani M, Geppetti P, and Emanueli C. Protease-activated receptor-2 stimulates angiogenesis and accelerates hemodynamic recovery in a mouse model of hindlimb ischemia. Circ Res 91: 346 –352, 2002. Miotto D, Hollenberg MD, Bunnett NW, Papi A, Braccioni F, Boschetto P, Rea F, Zuin A, Geppetti P, Saetta M, Maestrelli P, Fabbri LM, and Mapp CE. Expression of protease activated receptor-2 (PAR-2) in central airways of smokers and non-smokers. Thorax 57: 146 –151, 2002. Mirza H, Schmidt VA, Derian CK, Jesty J, and Bahou WF. Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell alpha- or beta-tryptases. Blood 90: 3914 –3922, 1997. Mirza H, Yatsula V, and Bahou WF. The proteinase activated receptor-2 (PAR-2) mediates mitogenic responses in human vascular endothelial cells. J Clin Invest 97: 1705–1714, 1996. Moffatt JD, Jeffrey KL, and Cocks TM. Protease-activated receptor-2 activating peptide SLIGRL inhibits bacterial lipopolysaccharide-induced recruitment of polymorphonuclear leukocytes into the airways of mice. Am J Respir Cell Mol Biol 26: 680 – 684, 2002. Molino M, Barnathan ES, Numerof R, Clark J, Dreyer M, Cumashi A, Hoxie JA, Schechter N, Woolkalis M, and Brass LF. Interactions of mast cell tryptase with thrombin receptors and PAR-2. J Biol Chem 272: 4043– 4049, 1997. Molino M, Blanchard N, Belmonte E, Tarver AP, Abrams C, Hoxie JA, Cerletti C, and Brass LF. Proteolysis of the human platelet and endothelial cell thrombin receptor by neutrophil-derived cathepsin G. J Biol Chem 270: 11168 –11175, 1995. Mule F, Baffi MC, and Cerra MC. Dual effect mediated by protease-activated receptors on the mechanical activity of rat colon. Br J Pharmacol 136: 367–374, 2002. Nakanishi-Matsui M, Zheng YW, Sulciner DJ, Weiss EJ, Ludeman MJ, and Coughlin SR. PAR3 is a cofactor for PAR4 activation by thrombin. Nature 404: 609 – 613, 2000. Nakayama T, Hirano K, Shintani Y, Nishimura J, Nakatsuka A, Kuga H, Takahashi S, and Kanaide H. Unproductive cleavage and the inactivation of protease-activated receptor-1 by trypsin in vascular endothelial cells. Br J Pharmacol 138: 121–130, 2003. Nanevicz T, Ishii M, Wang L, Chen M, Chen J, Turck CW, Cohen FE, and Coughlin SR. Mechanisms of thrombin receptor agonist specificity. Chimeric receptors and complementary mutations identify an agonist recognition site. J Biol Chem 270: 21619 – 21625, 1995. Nanevicz T, Wang L, Chen M, Ishii M, and Coughlin SR. Thrombin receptor activating mutations. Alteration of an extracellular agonist recognition domain causes constitutive signaling. J Biol Chem 271: 702–706, 1996. Physiol Rev • VOL 203. Napoli C, Cicala C, Wallace JL, de Nigris F, Santagada V, Caliendo G, Franconi F, Ignarro LJ, and Cirino G. Proteaseactivated receptor-2 modulates myocardial ischemia-reperfusion injury in the rat heart. Proc Natl Acad Sci USA 97: 3678 –3683, 2000. 204. Napoli C, De Nigris F, Cicala C, Wallace JL, Caliendo G, Condorelli M, Santagada V, and Cirino G. Protease-activated receptor-2 activation improves efficiency of experimental ischemic preconditioning. Am J Physiol Heart Circ Physiol 282: H2004 – H2010, 2002. 205. Nguyen TD, Moody MW, Steinhoff M, Okolo C, Koh DS, and Bunnett NW. Trypsin activates pancreatic duct epithelial cell ion channels through proteinase-activated receptor-2. J Clin Invest 103: 261–269, 1999. 206. Niclou S, Suidan HS, Brown-Luedi M, and Monard D. Expression of the thrombin receptor mRNA in rat brain. Cell Mol Biol 40: 421– 428, 1994. 207. Niclou SP, Suidan HS, Pavlik A, Vejsada R, and Monard D. Changes in the expression of protease-activated receptor 1 and protease nexin-1 mRNA during rat nervous system development and after nerve lesion. Eur J Neurosci 10: 1590 –1607, 1998. 208. Nierodzik ML, Bain RM, Liu LX, Shivji M, Takeshita K, and Karpatkin S. Presence of the seven transmembrane thrombin receptor on human tumour cells: effect of activation on tumour adhesion to platelets and tumor tyrosine phosphorylation. Br J Haematol 92: 452– 457, 1996. 209. Nierodzik ML, Chen K, Takeshita K, Li JJ, Huang YQ, Feng XS, D’Andrea MR, Andrade-Gordon P, and Karpatkin S. Protease-activated receptor 1 (PAR-1) is required and rate-limiting for thrombin-enhanced experimental pulmonary metastasis. Blood 92: 3694 –3700, 1998. 210. Nierodzik ML, Kajumo F, and Karpatkin S. Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo. Cancer Res 52: 3267–3272, 1992. 211. Nishikawa H, Kawai K, Nishimura S, Tanaka S, Araki H, Al-Ani B, Hollenberg MD, Kuroda R, and Kawabata A. Suppression by protease-activated receptor-2 activation of gastric acid secretion in rats. Eur J Pharmacol 447: 87–90, 2002. 212. Norton KJ, Scarborough RM, Kutok JL, Escobedo MA, Nannizzi L, and Coller BS. Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment. Blood 82: 2125–2136, 1993. 213. Nystedt S, Emilsson K, Larsson AK, Strombeck B, and Sundelin J. Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. Eur J Biochem 232: 84 – 89, 1995. 214. Nystedt S, Emilsson K, Wahlestedt C, and Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 91: 9208 –9212, 1994. 215. Nystedt S, Larsson AK, Aberg H, and Sundelin J. The mouse proteinase-activated receptor-2 cDNA and gene. Molecular cloning and functional expression. J Biol Chem 270: 5950 –5955, 1995. 216. Nystedt S, Ramakrishnan V, and Sundelin J. The proteinaseactivated receptor 2 is induced by inflammatory mediators in human endothelial cells. Comparison with the thrombin receptor. J Biol Chem 271: 14910 –14915, 1996. 217. O’Brien PJ, Molino M, Kahn M, and Brass LF. Protease activated receptors: theme and variations. Oncogene 20: 1570 –1581, 2001. 218. O’Brien PJ, Prevost N, Molino M, Hollinger MK, Woolkalis MJ, Woulfe DS, and Brass LF. Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1. J Biol Chem 275: 13502–13509, 2000. 219. Offermanns S. In vivo functions of heterotrimeric G-proteins: studies in Galpha-deficient mice. Oncogene 20: 1635–1642, 2001. 220. Offermanns S, Laugwitz KL, Spicher K, and Schultz G. G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets. Proc Natl Acad Sci USA 91: 504 –508, 1994. 221. Offermanns S, Mancino V, Revel JP, and Simon MI. Vascular 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 189. VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT PROTEASE-ACTIVATED RECEPTORS 222. 223. 224. 225. 226. 228. 229. 230. 231. 232. 233. 234. 235. 236. 237. 238. 239. Physiol Rev • VOL 240. 241. 242. 243. 244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254. 255. 256. and initiation of coagulation by tissue factor. Proc Natl Acad Sci USA 98: 7742–7747, 2001. Riewald M and Ruf W. Science review: role of coagulation protease cascades in sepsis. Crit Care 7: 123–129, 2003. Robin J, Kharbanda R, McLean P, Campbell R, and Vallance P. Protease-activated receptor 2-mediated vasodilatation in humans in vivo: role of nitric oxide and prostanoids. Circulation 107: 954 –959, 2003. Roosterman D, Schmidlin F, and Bunnett NW. Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2. Am J Physiol Cell Physiol 284: C1319 –C1329, 2003. Roy SS, Saifeddine M, Loutzenhiser R, Triggle CR, and Hollenberg MD. Dual endothelium-dependent vascular activities of proteinase-activated receptor-2-activating peptides: evidence for receptor heterogeneity. Br J Pharmacol 123: 1434 –1440, 1998. Ruoss S, Hartmann T, and Caughey G. Mast cell tryptase is a mitogen for cultured fibroblasts. J Clin Invest 88: 493– 499, 1991. Sabri A, Alcott SG, Elouardighi H, Pak E, Derian C, AndradeGordon P, Kinnally K, and Steinberg SF. Neutrophil cathepsin G promotes detachment-induced cardiomyocyte apoptosis via a protease-activated receptor-independent mechanism. J Biol Chem. In press. Sabri A, Short J, Guo J, and Steinberg SF. Protease-activated receptor-1-mediated DNA synthesis in cardiac fibroblast is via epidermal growth factor receptor transactivation: distinct PAR-1 signaling pathways in cardiac fibroblasts and cardiomyocytes. Circ Res 91: 532–539, 2002. Saifeddine M, al-Ani B, Cheng CH, Wang L, and Hollenberg MD. Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue. Br J Pharmacol 118: 521–530, 1996. Saifeddine M, Roy SS, Al-Ani B, Triggle CR, and Hollenberg MD. Endothelium-dependent contractile actions of proteinase-activated receptor-2-activating peptides in human umbilical vein: release of a contracting factor via a novel receptor. Br J Pharmacol 125: 1445–1454, 1998. Sambrano GR, Huang W, Faruqi T, Mahrus S, Craik C, and Coughlin SR. Cathepsin G activates protease-activated receptor-4 in human platelets. J Biol Chem 275: 6819 – 6823, 2000. Santos J, Bayarri C, Saperas E, Nogueiras C, Antolin M, Mourelle M, Cadahia A, and Malagelada JR. Characterisation of immune mediator release during the immediate response to segmental mucosal challenge in the jejunum of patients with food allergy. Gut 45: 553–558, 1999. Santulli RJ, Derian CK, Darrow AL, Tomko KA, Eckardt AJ, Seiberg M, Scarborough RM, and Andrade-Gordon P. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes. Proc Natl Acad Sci USA 92: 9151–9155, 1995. Sawada K, Nishibori M, Nakaya N, Wang Z, and Saeki K. Purification and characterization of a trypsin-like serine proteinase from rat brain slices that degrades laminin and type IV collagen and stimulates protease-activated receptor-2. J Neurochem 74: 1731– 1738, 2000. Scarborough RM, Naughton MA, Teng W, Hung DT, Rose J, Vu TK, Wheaton VI, Turck CW, and Coughlin SR. Tethered ligand agonist peptides. Structural requirements for thrombin receptor activation reveal mechanism of proteolytic unmasking of agonist function. J Biol Chem 267: 13146 –13149, 1992. Schechter NM, Brass LF, Lavker RM, and Jensen PJ. Reaction of mast cell proteases tryptase and chymase with protease activated receptors (PARs) on keratinocytes and fibroblasts. J Cell Physiol 176: 365–373, 1998. Schmidlin F, Amadesi S, Dabbagh K, Lewis DE, Knott P, Bunnett NW, Gater PR, Geppetti P, Bertrand C, and Stevens ME. Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway. J Immunol 169: 5315–5321, 2002. Schmidlin F, Amadesi S, Vidil R, Trevisani M, Martinet N, Caughey G, Tognetto M, Cavallesco G, Mapp C, Geppetti P, and Bunnett NW. Expression and function of proteinase-activated receptor 2 in human bronchial smooth muscle. Am J Respir Crit Care Med 164: 1276 –1281, 2001. 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 227. system defects and impaired cell chemokinesis as a result of Galpha13 deficiency. Science 275: 533–536, 1997. Offermanns S, Toombs CF, Hu YH, and Simon MI. Defective platelet activation in G alpha(q)-deficient mice. Nature 389: 183– 186, 1997. Ofosu FA, Freedman J, Dewar L, Song Y, and Fenton JW II. A trypsin-like platelet protease propagates protease-activated receptor-1 cleavage and platelet activation. Biochem J 336: 283–285, 1998. Paine C, Sharlow E, Liebel F, Eisinger M, Shapiro S, and Seiberg M. An alternative approach to depigmentation by soybean extracts via inhibition of the PAR-2 pathway. J Invest Dermatol 116: 587–595, 2001. Paing MM, Stutts AB, Kohout TA, Lefkowitz RJ, and Trejo J. beta-Arrestins regulate protease-activated receptor-1 desensitization but not internalization or down-regulation. J Biol Chem 277: 1292–1300, 2002. Patterson C, Stouffer GA, Madamanchi N, and Runge MS. New tricks for old dogs: nonthrombotic effects of thrombin in vessel wall biology. Circ Res 88: 987–997, 2001. Pendurthi UR, Ngyuen M, Andrade-Gordon P, Petersen LC, and Rao LV. Plasmin induces Cyr61 gene expression in fibroblasts via protease-activated receptor-1 and p44/42 mitogen-activated protein kinase-dependent signaling pathway. Arterioscler Thromb Vasc Biol 22: 1421–1426, 2002. Pereira PJ, Bergner A, Macedo-Ribeiro S, Huber R, Matschiner G, Fritz H, Sommerhoff CP, and Bode W. Human beta-tryptase is a ring-like tetramer with active sites facing a central pore. Nature 392: 306 –311, 1998. Perraud F, Besnard F, Sensenbrenner M, and Labourdette G. Thrombin is a potent mitogen for rat astroblasts but not for oligodendroblasts and neuroblasts in primary culture. Int J Dev Neurosci 5: 181–188, 1987. Pindon A, Berry M, and Hantai D. Thrombomodulin as a new marker of lesion-induced astrogliosis: involvement of thrombin through the G-protein-coupled protease-activated receptor-1. J Neurosci 20: 2543–2550, 2000. Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C, and Ullrich A. EGF receptor transactivation by G-proteincoupled receptors requires metalloproteinase cleavage of proHBEGF. Nature 402: 884 – 888, 1999. Raithel M, Winterkamp S, Pacurar A, Ulrich P, Hochberger J, and Hahn EG. Release of mast cell tryptase from human colorectal mucosa in inflammatory bowel disease. Scand J Gastroenterol 36: 174 –179, 2001. Rasmussen UB, Vouret-Craviari V, Jallat S, Schlesinger Y, Pages G, Pavirani A, Lecocq JP, Pouyssegur J, and Van Obberghen-Schilling E. cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2⫹ mobilization. FEBS Lett 288: 123–128, 1991. Rattenholl A and Steinhoff M. Role of proteinase-activated receptors in cutaneous biology and disease. In: Drug Development Research, edited by M. Hollenberg and N. Vergrolle. New York: Wiley-Liss, 2003, vol. 59, p. 408 – 417. Reed DE, Barajas-Lopez C, Cottrell G, Velazquez-Rocha S, Dery O, Grady EF, Bunnett NW, and Vanner SJ. Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons. J Physiol 547: 531–542, 2003. Renesto P, Si-Tahar M, Moniatte M, Balloy V, Van Dorsselaer A, Pidard D, and Chignard M. Specific inhibition of thrombininduced cell activation by the neutrophil proteinases elastase, cathepsin G, and proteinase 3: evidence for distinct cleavage sites within the aminoterminal domain of the thrombin receptor. Blood 89: 1944 –1953, 1997. Ricciardolo FL, Steinhoff M, Amadesi S, Guerrini R, Tognetto M, Trevisani M, Creminon C, Bertrand C, Bunnett NW, Fabbri LM, Salvadori S, and Geppetti P. Presence and bronchomotor activity of protease-activated receptor-2 in guinea pig airways. Am J Respir Crit Care Med 161: 1672–1680, 2000. Riewald M, Petrovan RJ, Donner A, Mueller BM, and Ruf W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 296: 1880 –1882, 2002. Riewald M and Ruf W. Mechanistic coupling of protease signaling 619 620 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT Physiol Rev • VOL 275. Suzuki Y, Nomura J, Hori J, Koyama J, Takahashi M, and Horii I. Detection and characterization of endogeneous protease associated with desquamation of stratum corneum. Arch Dermatol Res 285: 372–377, 1993. 276. Takeuchi T, Harris JL, Huang W, Yan KW, Coughlin SR, and Craik CS. Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and singlechain urokinase-type plasminogen activator as substrates. J Biol Chem 275: 26333–26342, 2000. 277. Tay-Uyboco J, Poon MC, Ahmad S, and Hollenberg MD. Contractile actions of thrombin receptor-derived polypeptides in human umbilical and placental vasculature: evidence for distinct receptor systems. Br J Pharmacol 115: 569 –578, 1995. 278. Tiruppathi C, Yan W, Sandoval R, Naqvi T, Pronin AN, Benovic JL, and Malik AB. G protein-coupled receptor kinase-5 regulates thrombin-activated signaling in endothelial cells. Proc Natl Acad Sci USA 97: 7440 –7445, 2000. 279. Toyoda N, Gabazza EC, Inoue H, Araki K, Nakashima S, Oka S, Taguchi Y, Nakamura M, Suzuki Y, Taguchi O, Imoto I, Suzuki K, and Adachi Y. Expression and cytoprotective effect of protease-activated receptor-1 in gastric epithelial cells. Scand J Gastroenterol 38: 253–259, 2003. 280. Tran T and Stewart AG. Protease-activated receptor (PAR)-independent growth and pro-inflammatory actions of thrombin on human cultured airway smooth muscle. Br J Pharmacol 138: 865– 875, 2003. 281. Trejo J, Altschuler Y, Fu HW, Mostov KE, and Coughlin SR. Protease-activated receptor-1 down-regulation: a mutant HeLa cell line suggests novel requirements for PAR1 phosphorylation and recruitment to clathrin-coated pits. J Biol Chem 275: 31255–31265, 2000. 282. Trejo J and Coughlin SR. The cytoplasmic tails of proteaseactivated receptor-1 and substance P receptor specify sorting to lysosomes versus recycling. J Biol Chem 274: 2216 –2224, 1999. 283. Trejo J, Hammes SR, and Coughlin SR. Termination of signaling by protease-activated receptor-1 is linked to lysosomal sorting. Proc Natl Acad Sci USA 95: 13698 –13702, 1998. 284. Tremaine WJ, Brzezinski A, Katz JA, Wolf DC, Fleming TJ, Mordenti J, Strenkoski-Nix LC, and Kurth MC. Treatment of mildly to moderately active ulcerative colitis with a tryptase inhibitor (APC 2059): an open-label pilot study. Aliment Pharmacol Ther 16: 407– 413, 2002. 285. Tsopanoglou NE and Maragoudakis ME. On the mechanism of thrombin-induced angiogenesis. Potentiation of vascular endothelial growth factor activity on endothelial cells by up-regulation of its receptors. J Biol Chem 274: 23969 –23976, 1999. 286. Turgeon VL, Lloyd ED, Wang S, Festoff BW, and Houenou LJ. Thrombin perturbs neurite outgrowth and induces apoptotic cell death in enriched chick spinal motoneuron cultures through caspase activation. J Neurosci 18: 6882– 6891, 1998. 287. Ubl JJ and Reiser G. Activity of protein kinase C is necessary for sustained thrombin-induced [Ca2⫹]i oscillations in rat glioma cells. Pflügers Arch 433: 312–320, 1997. 288. Ubl JJ and Reiser G. Characteristics of thrombin-induced calcium signals in rat astrocytes. Glia 21: 361–369, 1997. 289. Ubl JJ, Sergeeva M, and Reiser G. Desensitisation of proteaseactivated receptor-1 (PAR-1) in rat astrocytes: evidence for a novel mechanism for terminating Ca2⫹ signalling evoked by the tethered ligand. J Physiol 525: 319 –330, 2000. 290. Ubl JJ, Vohringer C, and Reiser G. Co-existence of two types of [Ca2⫹]i-inducing protease-activated receptors (PAR-1 and PAR-2) in rat astrocytes and C6 glioma cells. Neuroscience 86: 597– 609, 1998. 291. Uehara A, Muramoto K, Takada H, and Sugawara S. Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through protease-activated receptor 2. J Immunol 170: 5690 –5696, 2003. 292. Uehara A, Sugawara S, Muramoto K, and Takada H. Activation of human oral epithelial cells by neutrophil proteinase 3 through protease-activated receptor-2. J Immunol 169: 4594 – 4603, 2002. 293. Vaughan PJ, Pike CJ, Cotman CW, and Cunningham DD. Thrombin receptor activation protects neurons and astrocytes 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 257. Scott G, Deng A, Rodriguez-Burford C, Seiberg M, Han R, Babiarz L, Grizzle W, Bell W, and Pentland A. Protease-activated receptor 2, a receptor involved in melanosome transfer, is upregulated in human skin by ultraviolet irradiation. J Invest Dermatol 117: 1412–1420, 2001. 258. Scudamore CL, Thornton EM, McMillan L, Newlands GF, and Miller HR. Release of the mucosal mast cell granule chymase, rat mast cell protease-II, during anaphylaxis is associated with the rapid development of paracellular permeability to macromolecules in rat jejunum. J Exp Med 182: 1871–1881, 1995. 259. Seiberg M, Paine C, Sharlow E, Andrade-Gordon P, Costanzo M, Eisinger M, and Shapiro SS. The protease-activated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions. Exp Cell Res 254: 25–32, 2000. 260. Seiler SM, Peluso M, Michel IM, Goldenberg H, Fenton JWN, Riexinger D, and Natarajan S. Inhibition of thrombin and SFLLR-peptide stimulation of platelet aggregation, phospholipase A2 and Na⫹/H⫹ exchange by a thrombin receptor antagonist. Biochem Pharmacol 49: 519 –528, 1995. 261. Shapiro MJ and Coughlin SR. Separate signals for agonist-independent and agonist-triggered trafficking of protease-activated receptor 1. J Biol Chem 273: 29009 –29014, 1998. 262. Shapiro MJ, Trejo J, Zeng D, and Coughlin SR. Role of the thrombin receptor’s cytoplasmic tail in intracellular trafficking. Distinct determinants for agonist-triggered versus tonic internalization and intracellular localization. J Biol Chem 271: 32874 –32880, 1996. 263. Shapiro MJ, Weiss EJ, Faruqi TR, and Coughlin SR. Proteaseactivated receptors 1 and 4 are shutoff with distinct kinetics after activation by thrombin. J Biol Chem 275: 25216 –25221, 2000. 264. Sharlow ER, Paine CS, Babiarz L, Eisinger M, Shapiro S, and Seiberg M. The protease-activated receptor-2 upregulates keratinocyte phagocytosis. J Cell Sci 113: 3093–3101, 2000. 265. Smith-Swintosky VL, Cheo-Isaacs CT, D’Andrea MR, Santulli RJ, Darrow AL, and Andrade-Gordon P. Protease-activated receptor-2 (PAR-2) is present in the rat hippocampus and is associated with neurodegeneration. J Neurochem 69: 1890 –1896, 1997. 266. Southan C. A genomic perspective on human proteases. FEBS Lett 498: 214 –218, 2001. 267. Stead RH, Tomioka M, Quinonez G, Simon GT, Felten SY, and Bienenstock J. Intestinal mucosal mast cells in normal and nematode-infected rat intestines are in intimate contact with peptidergic nerves. Proc Natl Acad Sci USA 84: 2975–2979, 1987. 268. Steinhoff M, Corvera CU, Thoma MS, Kong W, McAlpine BE, Caughey GH, Ansel JC, and Bunnett NW. Proteinase-activated receptor-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase. Exp Dermatol 8: 282–294, 1999. 269. Steinhoff M, Neisius U, Ikoma A, Fartasch M, Heyer G, Skov PS, Luger TA, and Schmelz M. Proteinase-activated receptor-2 mediates itch: a novel pathway for pruritus in human skin. J Neurosci. In press. 270. Steinhoff M, Vergnolle N, Young SH, Tognetto M, Amadesi S, Ennes HS, Trevisani M, Hollenberg MD, Wallace JL, Caughey GH, Mitchell SE, Williams LM, Geppetti P, Mayer EA, and Bunnett NW. Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism. Nat Med 6: 151–158, 2000. 271. Striggow F, Riek M, Breder J, Henrich-Noack P, Reymann KG, and Reiser G. The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations. Proc Natl Acad Sci USA 97: 2264 –2269, 2000. 272. Suidan HS, Bouvier J, Schaerer E, Stone SR, Monard D, and Tschopp J. Granzyme A released upon stimulation of cytotoxic T lymphocytes activates the thrombin receptor on neuronal cells and astrocytes. Proc Natl Acad Sci USA 91: 8112– 8116, 1994. 273. Sun G, Stacey MA, Schmidt M, Mori L, and Mattoli S. Interaction of mite allergens der p3 and der p9 with protease-activated receptor-2 expressed by lung epithelial cells. J Immunol 167: 1014 – 1021, 2001. 274. Suo Z, Wu M, Citron BA, Gao C, and Festoff BW. Persistent protease-activated receptor 4 signaling mediates thrombin-induced microglial activation. J Biol Chem. In press. PROTEASE-ACTIVATED RECEPTORS 294. 295. 296. 297. 298. 300. 301. 302. 303. 304. 305. 306. Physiol Rev • VOL 307. 308. 309. 310. 311. 312. 313. 314. 315. 316. 317. 318. 319. Domains specifying thrombin-receptor interaction. Nature 353: 674 – 677, 1991. Wakita H, Furukawa F, and MT. Thrombin and trypsin stimulate granulocyte-macrophage colony-stimulating factor and interleukin-6 gene expression in cultured normal human keratinocytes. Proc Assoc Am Phys 109: 190 –207, 1997. Wang H and Reiser G. Thrombin signaling in the brain: the role of protease-activated receptors. J Biol Chem 384: 193–202, 2003. Wang H, Ubl JJ, and Reiser G. Four subtypes of proteaseactivated receptors, co-expressed in rat astrocytes, evoke different physiological signaling. Glia 37: 53– 63, 2002. Wang H, Ubl JJ, Stricker R, and Reiser G. Thrombin (PAR-1)induced proliferation in astrocytes via MAPK involves multiple signaling pathways. Am J Physiol Cell Physiol 283: C1351–C1364, 2002. Wang Y, Zhou Y, Szabo K, Haft CR, and Trejo J. Down-regulation of protease-activated receptor-1 is regulated by sorting nexin 1. Mol Biol Cell 13: 1965–1976, 2002. Weinstein JR, Gold SJ, Cunningham DD, and Gall CM. Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA. J Neurosci 15: 2906 –2919, 1995. Weiss EJ, Hamilton JR, Lease KE, and Coughlin SR. Protection against thrombosis in mice lacking PAR3. Blood 100: 3240 – 3244, 2002. Widmann C, Gibson S, Jarpe MB, and Johnson GL. Mitogenactivated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev 79: 143–180, 1999. Winitz S, Gupta SK, Qian NX, Heasley LE, Nemenoff RA, and Johnson GL. Expression of a mutant Gi2 alpha subunit inhibits ATP and thrombin stimulation of cytoplasmic phospholipase A2mediated arachidonic acid release independent of Ca2⫹ and mitogen-activated protein kinase regulation. J Biol Chem 269: 1889 – 1895, 1994. Wojtukiewicz MZ, Tang DG, Ben-Josef E, Renaud C, Walz DA, and Honn KV. Solid tumor cells express functional “tethered ligand” thrombin receptor. Cancer Res 55: 698 –704, 1995. Xu WF, Andersen H, Whitmore TE, Presnell SR, Yee DP, Ching A, Gilbert T, Davie EW, and Foster DC. Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci USA 95: 6642– 6646, 1998. Yang PC, Berin MC, Yu L, and Perdue MH. Mucosal pathophysiology and inflammatory changes in the late phase of the intestinal allergic reaction in the rat. Am J Pathol 158: 681– 690, 2001. Yu Z, Ahmad S, Schwartz JL, Banville D, and Shen SH. Proteintyrosine phosphatase SHP2 is positively linked to proteinase-activated receptor 2-mediated mitogenic pathway. J Biol Chem 272: 7519 –7524, 1997. 84 • APRIL 2004 • www.prv.org Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017 299. from cell death produced by environmental insults. J Neurosci 15: 5389 –5401, 1995. Vergnolle N. Proteinase-activated receptor-2-activating peptides induce leukocyte rolling, adhesion, and extravasation in vivo. J Immunol 163: 5064 –5069, 1999. Vergnolle N. Review article: proteinase-activated receptors: novel signals for gastrointestinal pathophysiology. Aliment Pharmacol Ther 14: 257–266, 2000. Vergnolle N, Bunnett NW, Sharkey KA, Brussee V, Compton SJ, Grady EF, Cirino G, Gerard N, Basbaum AI, AndradeGordon P, Hollenberg MD, and Wallace JL. Proteinase-activated receptor-2 and hyperalgesia: a novel pain pathway. Nat Med 7: 821– 826, 2001. Vergnolle N, Derian CK, D’Andrea MR, Steinhoff M, and Andrade-Gordon P. Characterization of thrombin-induced leukocyte rolling and adherence: a potential proinflammatory role for proteinase-activated receptor-4. J Immunol 169: 1467–1473, 2002. Vergnolle N, Hollenberg MD, Sharkey KA, and Wallace JL. Characterization of the inflammatory response to proteinase-activated receptor-2 (PAR2)-activating peptides in the rat paw. Br J Pharmacol 127: 1083–1090, 1999. Vergnolle N, Hollenberg MD, and Wallace JL. Pro- and antiinflammatory actions of thrombin: a distinct role for proteinaseactivated receptor-1 (PAR1). Br J Pharmacol 126: 1262–1268, 1999. Vergnolle N, MacNaughton WK, Al-Ani B, Saifeddine M, Wallace JL, and Hollenberg MD. Proteinase-activated receptor 2 (PAR2)-activating peptides: identification of a receptor distinct from PAR2 that regulates intestinal transport. Proc Natl Acad Sci USA 95: 7766 –7771, 1998. Vogel SM, Gao X, Mehta D, Ye RD, John TA, Andrade-Gordon P, Tiruppathi C, and Malik AB. Abrogation of thrombin-induced increase in pulmonary microvascular permeability in PAR-1 knockout mice. Physiol Genomics 4: 137–145, 2000. Vouret-Craviari V, Auberger P, Pouyssegur J, and Van Obberghen-Schilling E. Distinct mechanisms regulate 5-HT2 and thrombin receptor desensitization. J Biol Chem 270: 4813– 4821, 1995. Vouret-Craviari V, Boquet P, Pouyssegur J, and Van Obberghen-Schilling E. Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function. Mol Biol Cell 9: 2639 –2653, 1998. Vouret-Craviari V, Grall D, Chambard JC, Rasmussen UB, Pouyssegur J, and Van Obberghen-Schilling E. Post-translational and activation-dependent modifications of the G proteincoupled thrombin receptor. J Biol Chem 270: 8367– 8372, 1995. Vu TK, Hung DT, Wheaton VI, and Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64: 1057–1068, 1991. Vu TK, Wheaton VI, Hung DT, Charo I, and Coughlin SR. 621
