Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Proteinase-Activated Receptors (PARs)

  • Roland Kaufmann
  • Utz Settmacher
  • Hendrik Ungefroren
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101885

Synonyms

Historical Background

Since about 50 years, serine proteinases like chymotrypsin and pepsin have been known to cause hormone-like effects in target tissues. In addition, in the 1970, thrombin and trypsin have been demonstrated to stimulate mitogenesis by acting at the cell surface. However, the mechanisms responsible for the growth factor-like action of these proteolytic enzymes remained undefined for a long time. It was in 1991 when the search for the mechanisms of thrombin-induced platelet activation and fibroblast mitogenesis led to the discovery of the proteinase-activated receptor (PAR) family of G-protein-coupled receptors. Extensive research in this field over the last decades provided evidence for a fundamental role of PARs in mediating cellular responses to proteinases.

Proteinase-Activated Receptors (PARs): A Family of G-Protein-Coupled Receptors

PARs belong to the class A G-protein-coupled receptor (GPCR) family with currently four members, PAR1, PAR2, PAR3, and PAR4, that all mediate cellular effects of proteinases (for reviews, see Adams et al. 2011; Ossovskaya and Bunnett 2004; Ramachandran and Hollenberg 2008; Ramachandran et al. 2012; Steinhoff et al. 2005). PAR1, PAR3, and PAR4 are activated by the coagulation enzyme thrombin, though numerous other proteinases have been shown to cleave and activate PAR1 including factor Xa, plasmin, kallikreins, activated protein C (APC), matrix metalloproteinase-1 (MMP1), neutrophil elastase (NE), and neutrophil proteinase-3 (PR3). PAR2, like PAR1, is also targeted by many serine proteinases including trypsin, neutrophil elastase, neutrophil proteinase 3, mast cell tryptase, tissue factor/factor VIIa/factor Xa, human kallikrein-related peptidases (KLKs), and membrane-tethered serine proteinase-1 (MT-SP1)/matriptase 1 as well as by parasite cysteine proteinase and bacterial gingipains. Although generally regarded as a targeted for trypsin but not for thrombin signaling (Adams et al. 2011), recent results suggest that under certain conditions, e.g., at sites of acute injury or inflammation, or in the tumor microenvironment, PAR2 may be also be activated by thrombin (see Fig. 1).
Proteinase-Activated Receptors (PARs), Fig. 1

Activating proteinases of PAR1–4. APC activated protein C, EPCR endothelial protein C receptor, MASP-1 mannan-binding lectin serine protease 1 also known as mannose-associated serine protease 1, MT-SP1 membrane-tethered serine proteinase-1, MMP-1 matrix metalloproteinase-1, TF tissue factor

Structural Features and Activation Mechanism

The PARs are encoded by genes that map to either a gene cluster on chromosome 5q13 (F2R encoding PAR1, F2RL1 encoding PAR2, and F2RL2 encoding PAR3) or chromosome 19p12 (F2RL3 encoding PAR4). Each gene spans two exons, with the first exon encoding the signal peptide and the second the mature protein. As shown in Fig. 2, the PARs contain seven transmembrane helices (TM1–7), an extracellular amino-terminal domain encompassing a signal peptide of between 17 and 26 residues and a propeptide domain of between 11 and 30 amino acids, three intracellular loops (ICL1–3), three extracellular loops (ECL1–3), and an intracellular C-terminal domain varying between 13 and 51 amino acids. As well as connecting TM4 and TM5, ECL2 also makes a disulfide bond with TM3 that is conserved among GPCRs and contributes to receptor structural stability (Fig. 2). To facilitate thrombin binding and proteolysis, PAR1 and PAR3 each contain a hirudin-like binding domain within the N-terminus (Vu et al. 1991; Macfarlane et al. 2001; Fig. 2). In contrast, another thrombin-responsive receptor, PAR4, lacks a functional hirudin-like binding domain. Instead it contains a motif, P44APR, that provides a low-affinity thrombin-binding site and an anionic cluster, D57, D59, E62, and D65, which slows the rate of thrombin dissociation (Fig. 2).
Proteinase-Activated Receptors (PARs), Fig. 2

PAR key structural features. Sequence alignments of the signal peptide and propeptide regions; the mature amino terminus after cleavage at the PAR canonical site; extracellular loop (ECL) domains 1, 2, and 3; intracellular loop (ICL) domains 1, 2, and 3; and the C-terminal sequence and amino acid modifications by N-linked glycosylation and disulfide bond of the four human PAR family members are shown. The hirudin-like domains in the mature amino terminus are highlighted in magenta color. The numbers in brackets indicate the amino acid position in the PAR protein sequence [sequence data from: UniProtKB – P25116 (PAR1_HUMAN), UniProtKB – P55085 (PAR2_HUMAN), UniProtKB – O00254 (PAR3_HUMAN), UniProtKB - Q96RI0 (PAR4_HUMAN)]

Although the PAR family members share in common basic structural features of all GPCRs, including a central core domain composed of seven transmembrane helices (TM1–7) connected by three intracellular (ICL1–3) and three extracellular loops (ECL1–3), they exhibit a unique mechanism of proteolytic activation. Most GPCRs are activated in a reversible fashion by small hydrophilic molecules to elicit a cellular response; however, PAR activation by endogenous proteinases involves the unmasking of an N-terminal “tethered ligand” (TL) that remains attached to the receptor and cannot diffuse away (Adams et al. 2011; Alexander et al. 2008; Hollenberg and Compton 2002; Ossovskaya and Bunnett 2004; Ramachandran and Hollenberg 2008; Ramachandran et al. 2012; Steinhoff et al. 2005). Serine proteinases, such as thrombin or trypsin, are able to cleave PARs 1, 2, and 4 at specific recognition sites in the extracellular N-terminus (see Fig. 3 for PAR1 activation). The unmasked amino terminus functions as a tethered ligand (curved arrow, Fig. 3, left) and binds to the extracellular receptor domains to trigger conformational changes and signaling.
Proteinase-Activated Receptors (PARs), Fig. 3

Model for activation of PAR1. The scheme illustrates activation of the intact receptor by either proteolysis (left) or binding of the synthetic PAR1-activating peptide TFLLRN-NH2 (right)

Analogous cleavage of the N-terminus of PAR3 also exposes a potential “tethered ligand,” but the ability of the cleaved receptor to signal on its own is unclear. Rather, it appears that PAR3 acts as a cofactor for PAR4 activation by thrombin (Nakanishi-Matsui et al. 2000), although “autonomous” signaling by PAR3 has been reported in select circumstances. Alternatively, PARs can be activated via proteinases by a “noncanonical” mechanism involving cleavage at a site distinct from the “canonical tethered ligand” motif (Fig. 3, left). For example, matrix-metalloproteinase 1 (MMP1) and activated protein C (APC) can cleave the N-terminal domain of PAR1 to unmask a “noncanonical” tethered activating sequence different from the one revealed by serine proteinases (SFLLRNPNDK…, Fig. 3, left). As illustrated explicitly in Fig. 3, PAR1 can also be cleaved by the neutrophil enzymes, proteinase-3 (PR3), and elastase (NE) to reveal receptor-activating sequences that differ not only from each other but also from those resulting from MMP1 and APC. Of importance, these “noncanonical” tethered ligands interact with the receptor to drive distinct biased signaling pathways (e.g., via mitogen-activated protein kinase (MAPK) but not calcium). Neutrophil elastase (NE) has recently been shown to activate PAR2 signaling in a so-called “biased” manner, by exposing yet another “noncanonical” PAR2 tethered ligand sequence that selectively stimulates a MAPK pathway without triggering an elevation in intracellular calcium levels as is caused by a “canonical” trypsin-exposed PAR2 tethered ligand (Ramachandran et al. 2011). Furthermore, when PAR1 was cloned (Rasmussen et al. 1991; Vu et al. 1991), it was established that, in addition to proteinase-triggered PAR activation, short synthetic peptides derived from the proteolytically exposed “tethered ligand” sequences are able to activate PARs without receptor proteolysis (Scarborough et al. 1992; Vu et al. 1991, shown for PAR1 in Fig. 3, right). All of the PARs harbor in their N-terminal sequences a principal serine proteinase-targeted arginine at which enzymatic cleavage exposes a distinct tethered ligand for each PAR subtype. Unmasking of such TLs in PAR1 and PAR4 by thrombin and in PAR2 by trypsin induces cell signaling that involves a number of G-proteins (Gq, Gi, G12/13). Further, the synthetic peptides based on the revealed TL sequences can also stimulate comparable signaling via the “partner” G-proteins. Of importance, it has been possible to synthesize PAR-selective activating peptides to evaluate the effects of signaling by PARs 1, 2, and 4 in a variety of cultured cells and under in vivo conditions. PAR3 appears to be the exception, where synthetic peptides corresponding to its thrombin-revealed sequence do not seem to cause PAR3 signaling (Nakanishi-Matsui et al. 2000) and instead are able to activate PAR1 and PAR2. These so-called PAR-activating peptides (PAR-APs) have proved to be useful tools to study the function of PARs especially in settings in which more than one PAR subtype is expressed and stimulated by the same proteolytic enzyme (Macfarlane et al. 2001, Ramachandran and Hollenberg 2008). Moreover, synthetic peptides derived from the “noncanonical” cleavage of PAR1 (e.g., TLDPRSF-NH2 for a PR3 tethered ligand derived-activating peptide; or RNPNDKYEPF-NH2 for a NE tethered ligand-derived activating peptide) can serve as “biased” agonists of PAR1 to activate MAPK but not calcium signaling. These “biased signaling” pathways that are selective for either G-protein-coupled responses or for arrestin-mediated processes may lead to distinct signaling events and cellular responses (see below).

Signaling

Currently, PAR signaling is known to activate several major signal pathways: (I) the “classical pathway” in which receptor activation causes signaling via heterotrimeric G-proteins and downstream targets, (II) a β-arrestin pathway of signaling involving ligand-regulated scaffolds, and (III) by the transactivation of a variety of receptors and other signaling proteins. Transactivation can include (Adams et al. 2011) the rapid cellular release of agonists-like prostaglandins or EGF receptor (EGFR) ligands that can trigger non-PAR receptors by an autocrine or paracrine mechanism, (Alexander et al. 2008) an intracellular kinase pathway (e.g., Src-family tyrosine kinase) that targets and activates another cell surface receptor like the EGFR in an agonist-independent way and (Cunningham et al. 2016) a direct or indirect impact of the PARs on other signal mediators, either via GPCR-dimer formation or via transactivation of cell signaling components like ion channels, toll-like receptors (TLRs) or Nod-like receptors (NLRs) (see Fig. 4). Thus, the “transactivation” mechanisms in which the PARs participate can involve not only growth factor receptors and G-protein-coupled receptors (GPCRs) but also a diversity of other “signal generators” (Fig. 4) (reviewed in Gieseler et al. 2013). Given the complexity of the intracellular signaling networks (for review see Adams et al. 2011), the ability of PARs to generate a “biased signal” adds yet another layer of complexity to PAR regulation of cell behavior.
Proteinase-Activated Receptors (PARs), Fig. 4

PAR-mediated signaling mechanisms. GPCR G-protein-coupled receptor, NLR Nod-like receptor, RTK receptor tyrosine kinase, RSTK receptor serine/threonine kinase, TLRs toll-like receptors

Biased Signaling

Functional selectivity or biased agonism describes agonists that preferentially activate specific pathways downstream of the receptor. In keeping with the established floating or “mobile” receptor hypothesis, GPCRs couple to multiple effectors via their intracellular loop domains. PARs, like other GPCRs, can exhibit biased signaling. For example, the peptide agonist-induced activation of PAR1 can trigger signaling preferentially via Gαq, whereas thrombin-triggered PAR1 signaling is preferentially coupled to Gα12 or Gα13. Furthermore, signaling via PAR1 and PAR2 can differ depending on the location of the receptor. For example, sequestration of PAR1 in a membrane microdomain favors its signaling via activated protein C (APC) over that of thrombin, thus triggering the activation of RAC1 rather than RHOA51. Lipid raft integrity appears to be important for the activation of PAR2 by the tissue factor-factor VIIa complex (see also references that specifically focus on biased signaling from PARs in detail: Hollenberg et al. 2014; Russo et al. 2009).

Signal Termination and Intracellular Trafficking

As demonstrated for various other GPCRs, PAR signaling is rapidly terminated by multiple mechanisms (for review, see, e.g., Ramachandran et al. 2012). Upon activation, PAR1 is phosphorylated on sites within its C-terminus by several GPCR kinases (GRKs), including GRK3 and GRK5. There is evidence to indicate that PAR1 signaling is desensitized by its interaction with β-arrestin 1, but this interaction seems not to play a role in PAR1 trafficking. Rather, PAR1 internalization and degradation involves deubiquitylation of the receptor and interaction with the adaptor protein AP2 and epsin1. This leads to clathrin- and dynamin-dependent processes that specify the lysosomal localization of the receptors. In addition, regions within the C-terminus of PAR1 are crucial for specifying lysosomal targeting, and sorting nexin 1 (SNX1), a membrane-associated lysosomal sorting protein, is necessary for the transport of PAR1 from early endosomes to lysosomes. Finally, bicaudal D homolog 1 (BIC D1) has been identified as an important adaptor protein involved in the transfer of PAR1 from the plasma membrane to endocytic vesicles (Fig. 5).
Proteinase-Activated Receptors (PARs), Fig. 5

Signal termination and intracellular trafficking of PAR1, PAR2, PAR3 and PAR4.AP2 clathrin adaptor protein complex, ASMH deubiquitylating protease-associated molecule with the SH3 domain of STAM, BIC D1 bicaudal D homolog1, DUB deubiquitylating enzyme, GRKs GPCR kinases, SNX1 sorting nexin 1, UBPY ubiquitin isopeptidase Y

As illustrated in Fig. 5, PAR2 activation leads to rapid phosphorylation by GRKs, a process that is considered to be important for the recruitment of β-arrestins to the receptor. In contrast to PAR1, the interactions of PAR2 with β-arrestin 1 and β-arrestin 2 are mandatory for the desensitization of signaling as well as for receptor internalization and lysosomal targeting. The PAR2-β-arrestin complex is targeted to Rab5 early endosomes and finally to late endosomes and lysosomes. This process involves deubiquitylation of PAR2 via a complex formed by ubiquitin isopeptidase Y (UBPY) and the adaptor protein deubiquitylating proteinase-associated molecule with the SH3 domain of STAM (AMSH). In addition, PAR2 lysosomal targeting depends on deubiquitylation of the receptor and interaction with the lysosomal sorting protein hepatocyte growth factor-regulated tyrosine kinase substrate (HGS).

The mechanisms regulating the trafficking of PAR3 and PAR4 remain largely unknown (Fig. 5). A recent study has provided evidence for the interaction of β-arrestin 2 with PAR4 and its regulation of PAR4-dependent AKT signaling. The involvement of β-arrestins in regulating PAR4 desensitization and internalization is, however, still unclear.

Pathophysiological Roles

Beside a function of/in various physiological responses especially regarding blood coagulation and vascular integrity, PARs are involved in various disease processes. As demonstrated in Fig. 6, PARs play critical roles in cardiovascular disease, neurodegenerative disorders, infection, inflammation and pain, renal ischemia/reperfusion (IR) injury, arthritis, and cancer (for review, see Adams et al. 2011). Once it was established that PARs regulate a number of cellular responses, it became clear that antagonists of these receptors could potentially be useful in clinical settings. However, although a number of PAR1 antagonists have been evaluated (for review, see Cunningham et al. 2016; Franchi and Angiolillo 2015), to date no PAR-targeting drug has yet found its way into routine use in the clinic.
Proteinase-Activated Receptors (PARs), Fig. 6

PARs are involved in different diseases

Summary

Proteinase-activated receptors (PARs) are a subfamily of GPCRs encompassing four members, PAR1, PAR2, PAR3, and PAR4. This receptor class is characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular N-terminus and the exposure of N-terminal “tethered ligand“ domains that intramolecularly bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex “biased” intracellular signaling via classical G-protein-mediated pathways and β-arrestin signaling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological and pathophysiological effects. Although PARs are potential targets for the therapy of various diseases, at present no PAR antagonist has found its way into the clinic.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Roland Kaufmann
    • 1
  • Utz Settmacher
    • 1
  • Hendrik Ungefroren
    • 2
  1. 1.Department of General, Visceral and Vascular SurgeryJena University HospitalJenaGermany
  2. 2.University of LübeckLübeckGermany