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Departments of Surgery and Physiology, University of California, San Francisco, California
ABSTRACT I. INTRODUCTION II. PROTEASE-ACTIVATED RECEPTORS: MECHANISMS OF ACTIVATION BY CELL-SURFACE PROTEOLYSIS A. Discovery and Structure 1. PAR1 2. PAR2 3. PAR3 4. PAR4 B. Proteases Cleave PARs to Expose a Tethered Ligand Domain 1. PAR1 2. PAR2 3. PAR3 4. PAR4 C. Protease Binding to PARs Facilitates Cleavage and Activation D. There Are Functional Interactions Between PARs 1. Dual receptor systems 2. PAR3 as a cofactor for PAR4 3. Intermolecular PAR signaling E. Protease Binding to Other Membrane Proteins Can Facilitate PAR Cleavage and Activation F. Multiple Proteases Activate PARs 1. Coagulation factors 2. Pancreatic and extrapancreatic trypsins 3. Mast cell proteases 4. Leukocyte proteases 5. Cell-surface proteases 6. Nonmammalian proteases 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 1. Concentration-response relationships 2. PAR1 signaling 3. PAR2 signaling IV. PROTEASE-ACTIVATED RECEPTORS: TERMINATION OF THE SIGNAL A. Cell-Surface Proteolysis Can Disable PARs B. PAR Signaling Is Attenuated by Receptor Desensitization 1. PAR1 2. PAR2 3. PAR3 and PAR4 C. PARs Are Downregulated by Intracellular Proteases 1. PAR1 2. PAR2 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 1. Proteases 2. PARs 3. PAR signaling to circulating cells 4. PAR signaling to the vessels B. Protease Signaling to the Nervous System 1. Proteases 2. PARs 3. PAR signaling in the nervous system 4. Morphology 5. Survival and proliferation 7. Neurogenic inflammation and pain C. Protease Signaling in the Gastrointestinal System 1. Proteases 2. PARs 3. Ion transport 4. Motility 5. Exocrine secretion 6. Neurotransmission 7. Intestinal inflammation D. Protease Signaling in the Airway 1. Proteases 2. PARs 3. Airway resistance 4. Electrolyte transport 5. Inflammation and fibrosis E. Protease Signaling in the Skin 1. Proteases 2. PARs 3. Pigmentation 4. Proliferation and wound healing 5. Inflammation VI. PROTEASE-ACTIVATED RECEPTORS: MECHANISMS OF DISEASE AND TARGETS FOR THERAPY A. Cardiovascular Disease B. Inflammatory Disease C. Cancer VII. CONCLUSIONS AND FUTURE PERSPECTIVES
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I. INTRODUCTION |
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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 cell-surface 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 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.
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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).
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II. PROTEASE-ACTIVATED RECEPTORS: MECHANISMS OF ACTIVATION BY CELL-SURFACE PROTEOLYSIS |
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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 (LDPR41 
S42FLLRN, where denotes cleavage) (Fig. 2).
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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 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 SKGR34 S35LIGKV (Fig. 2).
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 LPIK38 T39FRGAP (Fig. 2).
Two laboratories identified PAR-like sequences by searching expressed sequence tag (EST) libraries, and both 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: PAPR47 G48YPGQV (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.
The mechanism by which thrombin activates PAR1 has been investigated in detail. Thrombin cleaves PAR1 at R41 S42FLLRN 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).
Similar observations indicate that trypsin cleaves PAR2 at R34 S35 LIGKV 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).
Thrombin cleaves PAR3 at K38 T39FRGAP, 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).
Thrombin and trypsin cleave PAR4 at R47 G48YPGQV, 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 100-fold 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.
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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.
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 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 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.
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 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 PAR1-mediated 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 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 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.
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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 circulating anticoagulant protein by degrading FVa and FVIIa. However, when retained at the cell surface, the EPCR-APC 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.
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.
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 µM) 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 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.
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 dramatically increased by mutation Asn30, by enzymatic deglycosylation, or by expression of PAR2 in glycosylation-defective 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).
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 desensitization of PAR2, suggesting that proteinase 3 is a PAR2 activator (292).
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.
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 PAR1- and 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 proteases 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.
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III. PROTEASE-ACTIVATED RECEPTORS: TRANSDUCTION OF THE SIGNAL |
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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 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
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 µM 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.
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 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.
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 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.
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PAR1 also couples to G12 and G13 in platelets and 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 thrombin-induced 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), 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 observ
