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1 Proteinase-Activated Receptor: Activation, Signal Transduction and Desensitisation

1 Proteinase-Activated Receptor: Activation, Signal Transduction and Desensitisation

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The Role of Proteases in Pain


Table 1 Common PAR-cleaving enzymes, activating peptides and antagonists

Proteinase-activated receptor











Granzyme A





Factor VIIa

Factor Xa



Granzyme A














Cathepsin G


PAR antagonist
















Pepducin P4pal10



and other signalling molecules to the intracellular domains of the receptors. PAR3

is a distinct receptor in that it acts as a cofactor for thrombin-mediated activation of

the PAR4 receptor (Nakanishi-Matsui et al. 2000). For practical purposes, short

synthetic peptides derived from the sequence of the unmasked tethered ligand have

been developed which can also activate PARs in the absence of receptor proteolysis

(Fig. 1). These experimental tools allow for the pharmacological study of PAR

processes in vitro and in vivo without the confounding multifactorial effects of

proteolytic activity. Hence, these short activating peptides have been used in

studying the role of PARs in different physiological and pathophysiological

situations (Russell and McDougall 2009). In addition to protease activation, some

proteases can also disarm PAR signalling by cleaving the N-terminus downstream

of the receptor-activating site, thereby detaching the tethered ligand making it

unavailable for receptor activation (Fig. 1). A recent report confirmed that serine

proteinase enzymes such as neutrophil elastase can disarm trypsin-mediated PAR2

signalling and at the same time activate PAR2 signalling selectively via a mitogen-


J.J. McDougall and M.M. Muley

Cleavage site





Inactive Receptor



Activated Receptor




Disarmed Receptor



Antagonist Blockade

Fig. 1 Schematic representation of proteinase-activated receptor (PAR) modulation. (a) Inactive

PARs can be cleaved by a protease revealing a tethered ligand (orange box) which binds to the

active domain of the same receptor leading to cell signalling. (b) The PAR binding site can be

blocked with a small molecule antagonist (red box). (c) Alternative proteases can remove a

segment of the N-terminal including the binding domain leading to disarmament of the receptor

activated protein kinase (MAPK) pathway, without triggering an elevation in

intracellular calcium levels (Ramachandran et al. 2011).




PARs couple with G proteins and activate multiple pathways and hence they can

regulate various cellular functions. PAR1 interacts with several α-subunits particularly G1α, G12/13α and Gq11α. G1α leading to inhibition of adenylyl cyclase (AC) to

reduce cyclic adenosine monophosphate (cAMP) (Benka et al. 1995). Gq11α

activates phospholipase Cβ (PLCβ) to generate inositol trisphosphate (InsP3),

which mobilises Ca2+, and diacylglycerol (DAG), which activates protein kinase

C (PKC). G12/13α couples to guanine nucleotide exchange factors (GEF), resulting

in activation of Rho, Rho-kinase (ROK) and serum response elements (SRE)

(Ossovskaya and Bunnett 2004). Recent studies suggest that the cleaved

N-terminal domain of PAR1 is released and exhibits biological activity in certain

settings and has been termed ‘parstatin’ (Duncan and Kalluri 2009). PAR1 can

activate the MAPK cascade by transactivation of the EGF receptor, through activation of PKC, Phosphoinositide 3-kinase (PI3K), Pyk2 and other mechanisms

(Coughlin 2000).

The Role of Proteases in Pain



PAR2 couples to Gqα and stimulates the generation of InsP3 and mobilisation of

Ca2+ in PAR2-transfected cell lines (Bohm et al. 1996). In the case of enterocytes

and transfected epithelial cells, activation of PAR2 leads to arachidonic acid release

and the generation of prostaglandins E2 and F1α (Kong et al. 1997). This suggests

that PAR2 cleavage involves the activation of phospholipase A2 and

cyclooxygenase-1 (Kong et al. 1997). Also, it has been reported that PAR2 activates

MAP kinases ERK1/2 and weakly stimulates MAP kinase p38, although c-Jun

amino-terminal kinase is not activated (Belham et al. 1996; DeFea et al. 2000).

PAR3 and PAR4

PAR3 does not signal autonomously and is only considered as a cofactor for PAR4

activation by thrombin (Nakanishi-Matsui et al. 2000). However, in a recent report,

it was observed that PAR3 can elicit Rho- and Ca2+-dependent release of ATP from

lung epithelial A549 cells and PAR4 couples to Gq and G12/13 signalling effectors in

order to signal through G proteins (Seminario-Vidal et al. 2009).

1.1.3 Desensitisation

The desensitisation mechanisms for the PARs are distinct but poorly understood.

Phosphorylation of activated PAR1 takes place to uncouple it from G proteins and

G protein-coupled receptor kinase 3 or 5 (GRK 3 or 5) which enhances PAR1

phosphorylation. Moreover, the β-arrestin 1 mechanism also contributes to

desensitisation of PAR1. In the case of PAR2, the main mechanism of uncoupling

is phosphorylation by PKC and other kinases, by binding both β-arrestin 1 and

β-arrestin 2 leading to a rapid uncoupling from G protein signalling at the cell

surface. The mechanisms contributing to the desensitisation of PAR3 and PAR4 are

unknown; however, it has been suggested that receptor internalisation may contribute to the termination of PAR4 signalling (Soh et al. 2010).


Proteinase-Activated Receptor: Role in Physiology

and Disease

1.2.1 Cardiovascular System

Protease signalling through PARs contributes to both normal homeostasis and

various cardiovascular disease states such as thrombosis and atherosclerosis. Evidence that PARs are involved in cardiovascular homeostasis comes from the

observation that the receptors are expressed on platelets, endothelial cells and

smooth muscle cells (Nelken et al. 1992; Takada et al. 1998). PAR1, but not

PAR2, is expressed on rat cardiac fibroblast (Steinberg 2005). A study, however,

showed the presence of PAR2 in rat cardiac fibroblast (Murray et al. 2012). PAR1

and PAR4 mainly coordinate thrombin-mediated platelet aggregation. After activation, PAR1 rapidly transmits a signal across the plasma membrane to G proteins,

which results in the formation of platelet–platelet aggregates. It also causes stimulation of Gq proteins which culminates in a rapid rise in intracellular calcium and


J.J. McDougall and M.M. Muley

activation of the GP IIb/IIIa (αIIbβ3) fibrinogen receptor. PAR4 is cleaved and

signals more slowly but, despite its slower response, generates the majority of the

intracellular calcium flux and does not require additional input from ADP receptor

to form stable platelet clumping. Blockade of thrombin binding to mouse PAR3 or

knockout of the PAR3 gene inhibited mouse platelet aggregation indicating the

importance of PAR3 for thrombin signalling in mouse platelets. However, when

mouse PAR3 was overexpressed in mouse platelets, it did not trigger thrombin

signalling. Mouse platelets express both PAR3 and PAR4, so Matsui et al. carried

out a series of experiments and showed that PAR3 and PAR4 interact with each

other and PAR3 functions as a cofactor in cleavage and activation of PAR4 by

thrombin (Nakanishi-Matsui et al. 2000). PAR2 is expressed by numerous cell

types within the cardiovascular system. Functional PAR2 expression has been

demonstrated on vascular endothelium, smooth muscle cells and cardiomyocytes

(Steinberg 2005; Sabri et al. 2000). Armed with this information, a group of

researchers carried out an investigation which involved evaluation of the role of

PAR2 in a cardiac ischaemia/reperfusion injury model (Antoniak et al. 2010). It

was demonstrated that PAR2 deficiency reduced myocardial infarction and heart

remodelling after ischaemia/reperfusion injury. In another study, it has been

reported that PAR2 contributes to the pathogenesis of heart hypertrophy and failure.

In a similar study, it was demonstrated that cardiomyocyte-specific overexpression

of PAR2 led to pathological heart hypertrophy associated with cardiac fibrosis

(Antoniak et al. 2013). Pathological remodelling of the heart in αMHC-PAR2

mice was accompanied by increased ANP: Atrial natriuretic peptide, BNP:

B-type natriuretic peptide and βMHC expression and decreased MHC expression

(Antoniak et al. 2013).

1.2.2 Nervous System

Various reports available in the literature show that thrombin changes the morphology of neurones and astrocytes, induces glial cell proliferation and even exerts,

depending on the concentration applied, either cytoprotective or cytotoxic effects

on neurones (Wang and Reiser 2003). Thrombin induces various neuronal changes

such as neurite retraction, cell rounding, NMDA receptor potentiation and protection from cell death which are all mediated by PAR1 (Jalink and Moolenaar 1992;

Turnell et al. 1995; Gingrich et al. 2000). PAR1 agonists also stimulate proliferation and shape changes in astrocytes, which results in the release of endothelin-1

and nerve growth factor and to inhibit the expression of glutamate receptors

(Beecher et al. 1994). When thrombin was infused into the brain, it has been

shown to reproduce inflammatory signs observed after injury in the CNS (Suidan

et al. 1996). Although expression of the other two thrombin receptors (PAR3 and

PAR4) in the brain has also been detected by several studies, the physiological role

of PAR3 and PAR4 in neuronal differentiation is presently unknown (Wang and

Reiser 2003). In a recent study, animals subjected to transient middle cerebral

artery occlusion followed by reperfusion showed an increase in PAR2 expression.

Also, there was significant decrease in the neuronal expression of phosphorylated

extracellular signal-regulated kinase (p-ERK) in PAR2 KO mice (Jin et al. 2005).

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