In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain, and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an emerging therapeutic target for major diseases. Most information about PAR activation and function derives from studies of a few proteases, for example thrombin in the case of PAR 1, PAR 3, and PAR 4, and trypsin in the case of PAR 2 and PAR 4. These proteases cleave PARs at established sites with the extracellular N-terminal domains, and expose tethered ligands that stabilize conformations of the cleaved receptors that activate the canonical pathways of G protein- and/or β-arrestin-dependent signaling. However, a growing number of proteases have been identified that cleave PARs at divergent sites to activate distinct patterns of receptor signaling and trafficking. The capacity of these proteases to trigger distinct signaling pathways is referred to as biased signaling, and can lead to unique patho-physiological outcomes. Given that a different repertoire of proteases are activated in various patho-physiological conditions that may activate PARs by different mechanisms, signaling bias may account for the divergent actions of proteases and PARs. Moreover, therapies that target disease-relevant biased signaling pathways may be more effective and selective approaches for the treatment of protease- and PAR-driven diseases. Thus, rather than mediating the actions of a few proteases, PARs may integrate the biological actions of a wide spectrum of proteases in different patho-physiological conditions.
With over 800 members in mammals, G protein-coupled receptors (GPCRs) are the largest family of cell-surface signaling proteins. They are receptors for an extraordinary range of structurally diverse agonists in the extracellular fluid, including endogenous hormones, neurotransmitters, and paracrine regulators, as well as multiple exogenous ligands. Due to their critical importance in the control of most patho-physiological processes, GPCRs are the primary target for over 30% of the clinically used drugs. The established mechanism of GPCR activation is that agonist binding results in conformational changes in the receptor that activate the Gα subunits of heterotrimeric G proteins, leading to the dissociation of Gβγ dimers from Gα. Activated Gα and Gβγ then initiate downstream signaling processes. To control the duration and magnitude of this signaling, activated receptors are phosphorylated by G protein-coupled receptor kinases (GRKs) or other kinases, and then interact with β-arrestins, which mediate receptor desensitization and endocytosis. Depending on the receptor and the agonist, internalized receptors are then sorted to lysosomes for degradation, or move to the plasma membrane for another cycle of activation. However, a common feature of GPCRs is that a single receptor can interact with multiple endogenous and exogenous ligands, each of which may activate the receptor in different ways. For example, a large number of endogenous opioid neuropeptides as well as many different opiate drugs interact with opioid receptors, and different opioids and opiates result in divergent processes of receptor activation and regulation. Thus, the simplistic view of receptor activation and regulation has been revised by the appreciation that different agonists of the same receptor can result in distinct patterns of signaling and regulation.
The early two-state model of receptor function suggested that a receptor adopts active conformation upon ligand binding. This model considered only one active state, leading to a single functional readout. However, increased understanding of receptor signaling has revealed that different ligands can initiate distinct signaling events through the same GPCR. The heterogeneity of signaling events by a single GPCR can include different maximum responses from a single pathway (i.e., full or partial agonism) or activation of distinctly different signaling pathways by different agonists. The capacity of different agonists to initiate signaling of the same GPCR by distinct mechanisms is referred to as biased agonism or signaling, and has been described for many GPCRs, including opioid receptors, angiotensin receptors, and glutamate receptors. This phenomenon of signaling bias is not surprising because GPCRs are flexible proteins that interact with multiple ligands and regulatory proteins, all of which may influence the capacity of the receptor to signal by particular mechanisms. Indeed, recent advances in our understanding of the structure of GPCRs in various activation states has revealed that a single GPCR can exist in multiple active conformations that may favor coupling to different signaling pathways.
This review focuses on the capacity of different proteases and synthetic ligands to induce biased signaling of protease-activated receptors (PARs). The PARs are a family of four GPCRs (PAR 1–4) that belong to group A rhodopsin-like GPCR subfamily. The first family member, PAR 1, was identified as a receptor for thrombin, a serine protease coagulation factor. PAR 2 was subsequently identified as a receptor for the serine protease trypsin, followed by PAR 3, another thrombin receptor, and PAR 4, a receptor for both thrombin and trypsin. PARs are expressed in many tissues and cell types, where they regulate multiple patho-physiological processes, including hemostasis, inflammation, pain, cellular proliferation, and healing. However, in addition to thrombin and trypsin, a large number of proteases have been identified that can cleave PARs. In some cases, these proteases cleave at the same sites as thrombin or trypsin and thereby initiate common signaling events. However, in other cases, proteases cleave PARs at distinct sites, and either activate distinct signals (biased agonism), or disarm the receptor by removing or destroying tethered ligand domains (receptor antagonism). We will review mechanisms by which various proteases and synthetic agonists activate PARs, and will discuss the implications of protease-biased signaling of PARs for patho-physiological control and therapeutic targeting.
Unlike other GPCRs, the endogenous ligands for PARs reside within the extracellular N-terminus of the receptors. Receptor cleavage at the defined sites within the N-terminus by proteases such as thrombin and trypsin reveals these tethered ligands that, once exposed, can bind to regions in the second extracellular loops of the cleaved receptors, initiating conformational changes in the receptors that activate downstream signals. This is the canonical mechanism of PAR activation (Figure 1A). There are subtle differences in the mechanisms by which different proteases initiate the canonical pathways of receptor activation, which depend on the protease and PAR in question. For example, thrombin first binds to PAR 1 and PAR 3; this action facilitates receptor cleavage and exposure of the tethered ligand sequence. Mutation of the binding site reduces the efficacy with which thrombin activates these receptors, and mutation of the cleavage site prevents receptor activation. On the other hand, trypsin activates PAR 2 directly, without first binding to the receptor. Accessory proteins can also influence the capacity of proteases to activate PARs. In particular, proteins that anchor proteases to the plasma membrane can enhance proteolytic activation. For example, during tissue damage and inflammation, tissue factor (TF) binds coagulation factors (F) VIIa, which in turn activates FX to FXa. FXa and its co-factor FVa promote conversion of prothrombin to thrombin, and subsequent PAR 1 activation. Besides promoting thrombin activation, FVIIa and FXa both can signal directly through PAR 1 and PAR 2, although the efficiency and potency of receptor activation is substantially enhanced when they are coupled with TF. Similarly, the proteolytic activity of the anticoagulant activated protein C (APC) toward PARs is largely regulated by its association with the endothelial protein C receptor (EPCR) at the surface of endothelial cells.
Support for the tethered ligand mechanism of PAR activation is provided by the observation that synthetic peptides, referred to as activating peptides (APs), that mimic the tethered ligand domain can also activate certain PARs directly, without the requirement for proteolysis (Figure 1C). Peptides mimicking the tethered ligands of PAR 1, PAR 2, and PAR 4 can directly activate these receptors, although with a considerably lower potency than the activating proteases, especially in the case of PAR 4. The higher EC 50 values of APs compare to those of proteases possibly reflect the differences between a tethered ligand and an untethered ligand in solution. PAR 3 is not activated by tethered ligand-derived peptides, and appears to be unable to signal directly, but rather to serve as a co-factor for other PARs, such as PAR 1 and PAR 4.
Activating peptides have been considered to mimic the effects of proteases and have been widely used to probe the functions of PARs without the use of proteases, which can cleave multiple other proteins that may influence outcomes. However, this is not always the case because in some circumstances proteases and APs agonists can exert different effects. For example, in human brain microvascular endothelial cells, thrombin activation of PAR 1 triggers endothelial barrier permeability, whereas PAR1-AP (SFLLRN-NH 2) has no significant effect. In addition, the signaling properties of a PAR 2 mutant with substitutions within the trypsin-revealed tethered ligand domain differ from those of APs with the same substitutions, suggesting distinct activation modes by tethered versus soluble peptides. The divergent signaling effects of proteases and APs provide evidence for biased signaling of PARs.
In addition to the diversity of signals that can originate from the same receptor after activation by proteases or synthetic agonists (i.e., biased signaling), many other factors also affect patho-physiological outcome of PAR activation. The
