Proteinase-Activated Receptors (PARs)
PAR 4 : Coagulation factor II (thrombin) receptor-like 3; Coagulation factor II receptor-like 3; F2RL3; F2R-like thrombin/trypsin receptor 3; PAR4; PAR-4; Proteinase-activated receptor 4; Thrombin receptor-like 3
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
Structural Features and Activation Mechanism
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).
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 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.
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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