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S. K. Böhm, K. McConalogue, and W. Kong are in the Dept. of Surgery, and N. W. Bunnett is in the Dept. of Surgery and Physiology, University of California San Francisco, San Francisco, CA 94143–0660, USA.
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Abstract |
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Introduction |
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 Top Introduction A family of protease-activated...Mechanisms of receptor...Activation by other proteasesPhysiological functions of PARsTurning off the signal:...Conclusions and future...References |
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), which also includes receptors for peptide and nonpeptide hormones and neurotransmitters, lipid mediators, odorant and taste molecules, and photons and ions. Although the PARs are only a small component of this large family, they are functionally important because they regulate inflammation, responses to injury, growth, and development and are thus attractive targets for therapy.
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A family of protease-activated receptors |
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TopIntroductionA family of protease-activated...Mechanisms of receptor...Activation by other proteasesPhysiological functions of PARsTurning off the signal:...Conclusions and future...References |
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-helices spanning the membrane. This arrangement results in an extracellular NH2 terminus of 94 residues, three extracellular loops, four intracellular loops, and an intracellular COOH-terminal tail (Fig. 1A
). Although there are subfamilies of most G protein-coupled receptors, PAR-1 remained a solitary receptor until the serendipitous discovery of the second family member, PAR-2 (9). PAR-2 shares 28% identity to PAR-1 (in the mouse) and is activated by pancreatic trypsin rather than thrombin.
Evidence for the existence of the third member of this receptor family was obtained by the finding that thrombin activated platelets but not fibroblasts from PAR-1 knockout mice (3). Thus PAR-1 accounts for response of fibroblasts to thrombin, but additional receptors must mediate the effects of thrombin on platelets. PAR-3 was subsequently cloned from rat platelets and found to have ~27% amino acid homology to PAR-1 and PAR-2 (5; Fig. 1A). It is highly expressed by megakaryocytes in mouse bone marrow and spleen and widely expressed in human tissues. Thrombin also activated platelets from PAR-3 knockout mice, albeit with reduced efficacy, suggesting the existence of yet another receptor. PAR-4 was recently cloned and found to be 30% identical to PAR-3 (6; Fig. 1A). The roles of these multiple thrombin receptors remain to be determined. Given the differences in potency with which thrombin activates PAR-1 and PAR-3 (high potency) compared with PAR-4 (low potency), multiple receptors may permit cells to respond in a graded manner to a wide range of thrombin concentrations. Additional proteases may also regulate platelets because trypsin activates PAR-4.
Analysis of the structure and chromosomal location of genes encoding PARs provides indirect evidence for a larger family of these receptors. PAR-1, PAR-2, and PAR-3 genes colocalize at chromosomal band 5q13 and span a PAR gene cluster of only ~100 kilobases (kb) (Fig. 1B). All PAR genes have the same structure with two exons separated by an intron. The first exon is small, and the second contains most of the coding sequence and the protease cleavage site. This remarkably similar gene organization suggests that these receptors evolved from a common ancestral gene and supports the existence of an extended gene family.
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Mechanisms of receptor activation by proteolytic cleavage |
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TopIntroductionA family of protease-activated...Mechanisms of receptor...Activation by other proteasesPhysiological functions of PARsTurning off the signal:...Conclusions and future...References |
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). First, thrombin physically interacts with PAR-1 at two sites. The initial interaction is probably between the anion binding site of thrombin, a sequence of basic residues comprising a positively charged patch, with a stretch of negatively charged acidic residues (DKYEPF) in the extracellular NH2 terminus of the receptor. This binding permits interaction of the active site of thrombin with the LDPR sequence of the receptor. Second, thrombin cleaves the receptor at LDPR
SFLL, to expose a new NH2 terminus beginning SFLLRN. Mutation of Arg-Ser to Arg-Pro, which is not cleaved by thrombin, renders the receptor unresponsive to thrombin. Replacement of the thrombin cleavage site with one that is recognized by enterokinase converts the thrombin receptor to an enterokinase receptor. These observations indicate that cleavage at the Arg-Ser bond is necessary and sufficient for receptor activation. Third, the new NH2 terminus acts as a tethered ligand by binding to the cleaved receptor. Synthetic fragments of the receptor that correspond to the tethered ligand domain activate the receptor directly without the need for receptor cleavage. These activating peptides are valuable reagents for investigating receptor functions without the use of proteases, which may cleave other receptors and thus have nonspecific effects. Bioassays of analogs of the tethered ligand sequence, coupled with site-directed mutagenesis within this domain, indicate that Phe2, Leu4, and Arg5 are important for interactions with binding domains of the receptor. Analysis of chimeras of human and Xenopus PAR-1, which have strikingly different tethered ligand domains, shows that the tethered ligand interacts with the second extracellular loop to activate the receptor.
Thrombin also cleaves and activates PAR-3 (5) and PAR-4 (6). As previously described for PAR-1, thrombin initially interacts with extracellular domains of PAR-3 and then cleaves the receptor at LPIKTFRG, exposing a new NH2 terminus beginning TFRGAP (Fig. 1A). This may function as a tethered ligand that binds and activates the cleaved receptor because mutation of this domain abolishes signaling. In contrast to PAR-1 and PAR-2, peptides corresponding to the tethered ligand sequence do not activate PAR-3. Thrombin and trypsin cleave and trigger PAR-4 (6).
Far less is known about the mechanism by which trypsin activates PAR-2 (Fig. 1A). Pancreatic trypsin cleaves PAR-2 at SKGRSLIGK because mutation of Arg-Ser to Arg-Pro, which is not cleaved by trypsin, renders the receptor unresponsive (9). Furthermore, peptides corresponding to the tethered ligand (SLIGK) activate PAR-2, whereas peptides with NH2-terminal extensions of Arg or Gly-Arg are inactive. Cleavage of the receptor by trypsin is evidenced by a loss of binding of antibodies that recognize domains proximal to the cleavage site (1). Substitution of the extracellular face of PAR-2 with the NH2-terminal domain and the extracellular loops of PAR-1 forms a chimeric receptor with PAR-1-like agonist specificity. Substitution of individual domains reveals that the primary determinant of agonist specificity is extracellular loop 2. However, the NH2-terminal domain and extracellular loop 3 also participate in agonist recognition.
PAR-2 responds to both PAR-1 and PAR-2 agonist peptides with similar efficiencies and potencies, whereas PAR-1 is activated only by its own peptide. This situation is reminiscent of the neuropeptide receptor families for somatostatin or tachykinins, in which one peptide recognizes multiple receptors or several peptides bind to the same receptor with different affinities. Whether the tethered ligand of PAR-1 could bind to and activate PAR-2 in vivo is unclear. However, the tethered ligand of a cleaved PAR-1 molecule can bind to an uncleaved receptor, providing evidence for intermolecular interactions. In endothelial cells expressing both PAR-1 and PAR-2, evidence for cross talk between the two receptors exists, supporting the possibility of intermolecular interactions between PAR-1 and PAR-2.
Proteases cleave PARs to expose tethered ligand domains, which bind to and activate the cleaved receptors. Thus PARs may be viewed as specialized peptide receptors; ones in which the peptide ligand is covalently linked to the receptor molecule but only exposed by specific proteolysis. There are informative similarities and differences between the mechanisms that initiate and terminate signaling by proteases and neuropeptides. A comparison of these mechanisms provides insights into signaling by these agonists and G protein-coupled receptors in general (Fig. 2). Proteolysis is important for the initiation of signaling by proteases and peptides. Thus cleavage of PARs is the first step of receptor activation, and posttranslational processing and postsecretory processing of peptide hormones and neurotransmitters are required to generate biologically active forms. Most peptides are synthesized as large, inactive precursors that are processed within cells by a family of prohormone convertases to the biologically active, secreted molecules. Some proteases, exemplified by angiotensin-converting enzyme, convert peptides to their principal biological forms in the extracellular fluid and thereby initiate signaling. Proteolysis is also important for terminating signaling by proteases and peptides. Once cleaved, PARs can no longer be cleaved again and are inactive. Cell-surface proteases, for example, neutral endopeptidase, degrade and inactivate neuropeptides, such as substance P, in the extracellular fluid and terminate their biological actions in a manner that is comparable to the role of acetylcholinesterase in the neuromuscular junction.
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Activation by other proteases |
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TopIntroductionA family of protease-activated...Mechanisms of receptor...Activation by other proteasesPhysiological functions of PARsTurning off the signal:...Conclusions and future...References |
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The presence in PAR-1 of an anion-binding site for thrombin suggests that thrombin is the main activating protease. However, other proteases also cleave PAR-1. Granzyme A, a serine protease secreted by cytolytic T lymphocytes and natural killer cells, cleaves PAR-1 expressed on a mouse neuronal cell at the thrombin cleavage site to cause neurite retraction. This has led to speculation that PAR-1 activation by granzyme A may have pathophysiological implications for autoimmune diseases of the central nervous system involving cytotoxic T lymphocytes. Trypsin and plasmin also cleave PAR-1 at the thrombin site and thus activate the receptor, although they are considerably less potent than thrombin. Cathepsin G activates PAR-1 by cleaving at the thrombin site. It inactivates the receptor by removing the tethered ligand, whereas chymotrypsin only inactivates the receptor. The physiological relevance of proteolytic inactivation of PAR-1 is unknown but could be related to mechanisms of receptor desensitization and downregulation. Trypsin also cleaves and activates PAR-4 (6).
Although nanomolar concentrations of trypsin cleave and activate PAR-2, the widespread distribution of PAR-2 compared with the relatively limited distribution of pancreatic trypsin suggests that other trypsinlike enzymes activate PAR-2 in some locations. PAR-2 is expressed in the gastrointestinal tract, pancreas, kidney, liver, airway, prostate, ovary, and eye and is found in epithelial and endothelial cell lines, smooth muscle, T cell lines, and certain tumor cell lines (2, 9). Pancreatic trypsin may activate PAR-2 in some tissues under physiological and pathophysiological conditions. In the small intestine, PAR-2 is highly expressed by enterocytes. Its presence at the apical membrane places it in a strategic location to be activated by trypsin in the intestinal lumen (Fig. 3) (7). PAR-2 is also highly expressed in the pancreas. Although trypsin in the pancreas is mostly present as trypsinogen, an inactive zymogen, it is prematurely activated in the inflamed pancreas. Thus trypsin may activate PAR-2 during pancreatitis. Elsewhere, other trypsinlike enzymes probably cleave and activate PAR-2. One candidate is mast cell tryptase, which is found in almost all human mast cells, where it comprises up to 25% of the soluble protein. It is released from mast cells during degranulation and could thus cleave PAR-2 in inflamed tissues. Indeed, mast cell tryptase cleaves and activates PAR-2 in transfected cell lines and in cells that naturally express this receptor (8). Experiments with synthetic peptides indicate that tryptase and trypsin cleave PAR-2 at the same site.
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Physiological functions of PARs |
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TopIntroductionA family of protease-activated...Mechanisms of receptor...Activation by other proteasesPhysiological functions of PARsTurning off the signal:...Conclusions and future...References |
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More is known about the signaling mechanisms that are activated by thrombin than its physiological effects (Table 1). Probably all signaling events originate in activation of G and Gß
subunits of heterotrimeric G proteins (4). Cellular responses elicited by thrombin are dependent on the cell type and the complement of G proteins and effectors present in the particular cell type. Thus thrombin activates the ß form of phospholipase C by at least two potential routes: one involving Gß derived from Gi, the other involving G derived of a member of the Gq family. In platelets and HEL cells, the predominant link appears to be Gi, since thrombin-induced phosphoinositide hydrolysis is inhibited by pertussis toxin. In endothelial cells and fibroblasts, the link appears to be mediated by members of the Gq family, since phosphoinositide hydrolysis is largely unaffected by pertussis toxin. The finding that Gß subunits promote activation of the small G protein Ras through the adaptor molecules shc and the Grb2/Sos1 complex indicates that signaling pathways of G protein-coupled receptors and receptor tyrosine kinases, which were previously thought to be distinct, are coupled. Thus, in most cells, thrombin stimulates phospholipases C, A2, and D, inhibits adenylyl cyclase, and activates protein kinase C, mitogen-activated protein kinases, and tyrosine kinases.
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) (7). Trypsin, at concentrations that are present in the intestinal lumen after feeding, cleaves and activates PAR-2 in enterocytes, resulting in activation of phospholipase C-ß, formation of inositol trisphosphate and mobilization of Ca2+, and release of arachidonic acid and prostaglandins (Fig. 4). Eicosanoids act in a paracrine and autocrine manner in the intestine to regulate epithelial transport, intestinal motility, growth, and blood flow and act to mediate inflammation and cytoprotection. Thus, in addition to its role as a digestive enzyme, trypsin may act as an intestinal signaling molecule that regulates the epithelium by specifically cleaving and activating PAR-2.
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and interleukin-1, which orchestrate the acute inflammatory responses by regulating transcription of a large number of genes in responding cells (10). Tryptase is mitogenic for epithelial cells, fibroblasts, and smooth muscle cells and stimulates intracellular adhesion molecule expression by epithelial cells, but the receptor that mediates these effects has not been identified. Although PAR-2 is expressed by several cell types that respond to tryptase, including epithelial, endothelial, and smooth muscle cells, proof that PAR-2 mediates the effects of tryptase will require use of selective agonists and antagonists. PAR-2 and PAR-1 have several common functions. Both receptors contribute to growth regulation and mitogenesis. For example, activation of PAR-1 and PAR-2 stimulates proliferation of endothelial cells (7). Both receptors also induce endothelium-dependent relaxation of coronary artery and aortic smooth muscle, resulting in hypotension. In the gastrointestinal tract, PAR-2 may influence motility, since its activation results in contraction of gastric muscle. Of considerable interest is the extremely high expression of PAR-2 by certain tumor cell lines derived from the lung, colon, and pancreas. Activation of PAR-2 in a lung adenocarcinoma cell line inhibits colony formation (2). The proteases that activate PAR-2 in tumor cells have not been identified. However, some tumors express pancreatic trypsin, raising the possibility that tumor trypsins may regulate cells in an autocrine fashion.
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Turning off the signal: mechanisms of signal attenuation |
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TopIntroductionA family of protease-activated...Mechanisms of receptor...Activation by other proteasesPhysiological functions of PARsTurning off the signal:...Conclusions and future...References |
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). At the level of the agonist, extracellular degradation and inactivation of peptides and proteases attenuate the signal. Degradation of neuropeptides by cell surface proteases such as neutral endopeptidase is one of the earliest mechanisms for quenching a signal. It is not known whether extracellular degradation of thrombin and trypsin contributes to signal attenuation, although the availability of active protease and the presence of protease inhibitors would obviously affect the potential of an enzyme to activate its receptor.
Once G protein-coupled receptors interact with agonists and adopt an active conformation, they transduce signals that are often rapidly (seconds to minutes) quenched, a process known as receptor desensitization. Resensitization is the more gradual (minutes to hours) recovery, which allows tissues to maintain their ability to respond to agonists with time. Once again, there are both similarities and dissimilarities in the mechanisms of desensitization and resensitization of receptors for proteases and classical ligands. One of the first steps of desensitization is receptor phosphorylation by G protein receptor kinases (GRKs) and second messenger kinases (protein kinases A and C) (Fig. 2). Thus isoproterenol and substance P induce phosphorylation of the ß2-adrenergic and neurokinin-1 receptors, respectively, by GRK-2 and -3 (or ß-adrenergic receptor kinases 1 and 2) and second messenger kinases (Fig. 2B). The GRK-phosphorylated receptors interact with ß-arrestins, which interdicts interaction with G proteins and thereby terminates the signal. Similar mechanisms may exist to desensitize signaling by PARs (Fig. 2A). Once a protease cleaves its receptor, the receptor cannot respond again to a protease and is, from this perspective, desensitized. However, the tethered ligand is always exposed and the receptor molecule would be irreversibly activated unless efficient mechanisms exist to quench the signal. Indeed, the formation of second messengers such as Ca2+ and inositol 1,4,5-trisphosphate that follows activation of PAR-1 and PAR-2 is rapidly desensitized. Both GRKs and second messenger kinases contribute to this desensitization. Thrombin stimulates rapid phosphorylation of PAR-1, and GRKs and protein kinase C participate in desensitization of PAR-1 and PAR-2 (1, 4).
Many receptors efficiently internalize after interaction with agonists. Thrombin and trypsin stimulate endocytosis of PAR-1 and PAR-2, respectively, in the same way that isoproterenol and substance P trigger internalization of their receptors (Fig. 2). However, the fate of internalized receptors for proteases and for classical ligands is quite different. Substance P stimulates endocytosis of the neurokinin-1 receptor into early endosomes, and then the receptor recycles to the plasma membrane (Fig. 2B). Proteases similarly cause internalization of PAR-1 and PAR-2 into early endosomes, but the PARs rapidly traverse this compartment and are sorted to lysosomes (1, 4) (Fig. 2A). Little is known about domains that specify intracellular targeting. Recycling may be a default pathway, since certain receptors and lipids recycle at similar rates, but lysosomal targeting probably requires interaction of distinct receptor domains with sorting proteins. Comparisons of protease receptors and neuropeptide receptors may help to identify lysosomal targeting domains. The function of internalization and trafficking of protease receptors and receptors for classical agonists is quite different. Internalization depletes the plasma membrane of receptors and could thus contribute to desensitization of both types of receptors. However, desensitization of the ß2-adrenergic and neurokinin-1 receptors still occurs after endocytosis has been suppressed, indicating that internalization is not the principal mechanism of desensitization. For the protease receptors, internalization and lysosomal degradation would irreversibly terminate the signal and could be a final component of desensitization. Endocytosis is important for resensitization of the ß2-adrenergic or neurokinin-1 receptors, since resensitization is blocked by inhibition of endocytosis and recycling. These findings imply that processing of the internalized receptor, which may include dissociation of the ligand and ß-arrestins, dephosphorylation of the receptor, and receptor recycling, is necessary for resensitization. (Fig. 2B). An alternate mechanism is responsible for resensitization of PAR-1 and PAR-2. Because these receptors are degraded after internalization, resensitization of responses to proteases requires mobilization of intracellular pools and synthesis of new receptors (Fig. 2A). Indeed, large stores of both PAR-1 and PAR-2 exist in the Golgi apparatus. Disruption of the Golgi apparatus with brefeldin A and by inhibition of new receptor synthesis with cycloheximide inhibits resensitization of responses to proteases (1, 4).
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Conclusions and future directions |
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TopIntroductionA family of protease-activated...Mechanisms of receptor...Activation by other proteasesPhysiological functions of PARsTurning off the signal:...Conclusions and future...References |
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PARs could be considered specialized peptide receptors because proteolysis reveals a tethered peptide that binds and activates the receptor. There are similarities between the mechanisms that initiate and terminate signaling by PARs and peptide receptors. Proteases trigger PARs and also generate biologically active peptides. A cleaved PAR cannot be reactivated by proteolysis, and proteases also inactivate peptides. Signaling by both types of receptors is initiated by interaction of specific residues of the ligand, be it tethered to the receptor or soluble within the extracellular fluid, with extracellular domains of the receptor. GRKs phophorylate PARs and peptide receptors, which allows binding of ß-arrestins to uncouple receptors from G proteins and to terminate signaling. There are also informative differences between these types of receptors. Cells expressing PARs detect graded concentrations of a protease by the rate of PAR cleavage, whereas cells expressing peptide receptors sense graded concentrations of peptide by the extent of receptor occupancy. Although both PARs and peptide receptors internalize after agonist binding, PARs are mostly degraded in lysosomes and peptide receptors recycle. Recovery of responses to proteases requires synthesis or mobilization of new receptors, whereas recovery of responses to peptides usually requires recycling of receptors.
The existence of other PARs is almost certain given the recent discovery of PAR-3 and PAR-4 (5, 6). There is also pharmacological evidence for the existence of new PARs. Peptide analogs of the tethered ligand of PAR-2 stimulate short-circuit current in the rat jejunum with potencies that are quite different from their ability to trigger cloned rat PAR-2, which suggests that they activate distinct receptors in the intestine (11). Additional agonists of existing PARs will probably be discovered. The widespread distribution of PAR-2, compared with the more restricted expression of trypsin and tryptase, suggests the existence of other agonists. Although the importance of PAR-1 is firmly established, the physiological and pathophysiological roles of PAR-2, PAR-3, and PAR-4 are still emerging. Understanding the functions of these receptors will require studies of knockout and transgenic animals and the development of selective agonists and antagonists.
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Acknowledgments |
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Research in the author's laboratory is funded by the National Institutes of Health.
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TopIntroductionA family of protease-activated...Mechanisms of receptor...Activation by other proteasesPhysiological functions of PARsTurning off the signal:...Conclusions and future...References |
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