Edited by: Alain Couvineau, Institut National de la Santé et de la Recherche Médicale (INSERM), France
Reviewed by: David Poyner, Aston University, United Kingdom; Rachel Bar-Shavit, Hadassah Medical Center, Israel
This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology
†These authors have contributed equally to this work
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Protease-activated receptors (PARs) belong to the G protein-coupled receptor (GPCR) family. Compared to other GPCRs, the specificity of the four PARs is the lack of physiologically soluble ligands able to induce their activation. Indeed, PARs are physiologically activated after proteolytic cleavage of their N-terminal domain by proteases. The resulting N-terminal end becomes a tethered activation ligand that interact with the extracellular loop 2 domain and thus induce PAR signal. PARs expression is ubiquitous and these receptors have been largely described in chronic inflammatory diseases and cancer. In this review, after describing their discovery, structure, mechanisms of activation, we then focus on the roles of PARs in the intestine and the two main diseases affecting the organ, namely inflammatory bowel diseases and cancer.
Protease-activated receptors (PARs) belong to the family of G-protein coupled receptors (GPCRs). Their activation results from the specific cleavage, by proteases, of the amino terminal sequence that exposes a new N-terminal sequence as a tethered ligand, which then binds intramolecularly to activate the receptor. PARs are ubiquitous throughout the organism, although predominantly expressed in vascular, immune, intestinal epithelial cells and the nervous system. Thus, their activations regulate a set of crucial biological processes involved in physiology and diseases (
In the intestine, cleavage and activation of PARs have been largely described in the modulation of pain (
In polarized intestinal epithelial cells, these receptors are expressed at both apical and basolateral sides, suggesting that luminal, circulating and secreted proteases can reach and activate them (
Four PARs have been identified, PAR1, PAR2, PAR3, and PAR4, according to their cloning order (
The first receptor of this family to be cloned was PAR1, in 1991. Originally, the authors wanted to identify the receptors involved in the mechanisms of action of thrombin in inflammation and haemostasis (
The PARs cloning followed in 1994 with PAR2. After the isolation of a DNA sequence coding for a G-protein-coupled receptor from a mouse genomic library, the predicted protein displayed a structure similar to PAR1 and activated by a similar mechanism. This receptor was first described activated by trypsin, but not by thrombin. In addition, the authors described that an exogenous agonist peptide allowed the receptor activation, suggesting the importance of the proteolytic cleavage in this process. This receptor was indeed named PAR2 (
A similar approach allowed PAR3 discovery in 1997. This thrombin-activated receptor seems to be a PAR4 cofactor (
Finally PAR4 was discovered shortly afterwards. Both trypsin and thrombin can activate this receptor (
The genes encoding the PARs consist in two exons. The exon 1 generates a larger N-terminal sequence than most GPCRs that includes the site of cleavage. The exon 2 codes for the receptor itself (
Protease-activated receptors structure. These receptors present several domains within their structures: the signal (italics lettering) and pro-peptide (green lettering) domains, a NH2-terminal domain (NTD), three extracellular loops (ECL1-3), three intracellular loops (ICL1-3), and a COOH-terminal domain (CTD). Within each receptor, the sequence of their specific tethered ligand are underlined. The blue lettering represents PAR1 and PAR3 Hirudin-like domains. Pink Cysteines are the ones forming a disulfide linkage between the transmembrane domain 3 and ECL2. PARs also present several post-translational modifications sites (N-glycosylation-red N, PAR1 and PAR2 putative palmitoylation sites–Orange lettering, Shadowed and bold lettering, respectively, represent ubiquitination and phosphorylation sites). Finally within PAR1 CTD, the YKKL motif is involved in the regulation of its trafficking.
Under the action of a protease, the proteolytic cleavage of the receptor activator ligand within the canonical N-terminal domain site is irreversible (
Mechanisms regulating protease-activated receptors activation.
PAR1 is cleaved (↓) at its canonical site, LDPR41 ↓ S42FLLRN, by thrombin (
PAR2 is activated at its canonical cleavage site, SKGR34 ↓ S35LIGKV, by trypsin (
PAR3 presents a putative cleavage site for thrombin, LPIK38 ↓ T39FRGAP (
PAR4 can be cleaved by thrombin and trypsin, at similar doses, at this canonical site: PAPR47 ↓ G48YPGQV (
PARs expression sites, activating proteases, tethered peptides, and main effects in the intestine.
Sites of expression in the gut | Entero- and colonocytes, intestinal epithelial primitive cells, myenteric and submucosal neurons, fibroblats, smooth muscles, mast cells, immunes cells, endothelium, human colon epithelial cancer cells | Entero- and colonocytes, intestinal epithelial stem/progenitor cells, myenteric and submucosal neurons, fibroblats, smooth muscles, mast cells, immunes cells, endothelium, human colon epithelial cancer cells | Detected in non-identified cells in the small intestine | Entero- and colonocytes, enteric neurons, immune cells, endothelium, submucosa |
Activating proteases | Thrombin, Factor VIIa, Factor Xa, Trypsin, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, MMP-13, MMP-14, Neutrophil elastase, Proteinase-3, Plasmin, Kallikrein-4,-5,-6, Kallikrein-14, Granzyme A, B, K, Calpain-1, Gingipain, cathepsin G | Trypsin, trypsin-2, trypsin-3, trypsin VI, mast cell tryptase, tissue factor, matriptase/membrane-type serine protease I, Factor Xa, Factor VIIa, gingipain, acrosin, elastase, Thrombin, Tryptase, Cathepsin G, Cathepsin S, Neutrophil elastase, Proteinase-3, Plasmin, Testisin, Kallikrein-4, Kallikrein-5,-6,-14, Calpain-2 | Thrombin, trypsin, Factor Xa | Thrombin, trypsin, cathepsin G, Trypsin VI, Factor Xa, Factor VIIa, gingipain, Kallikrein 14 |
Tethered peptide sequences (human) | SFLLRN | SLIGKV | TFRGAP | GYPGQV |
Effects in the gut | Apoptosis, cell proliferation, motility, increased permeability, ion secretion, smooth muscle contraction and relaxation, inflammation, prostaglandin release | Apoptosis, cell proliferation, motility, increased permeability, ion secretion, ion channel activation, smooth muscle contraction and relaxation, inflammation, prostaglandin and eicosanoid release, neuropeptide release, amilase secretion, neuronal hyperexcitability, visceral hypersensitivity, motor functions | Motor functions, colon cancer cell proliferation |
Then, the notion of biased activation, characterized by a stimulation of the receptor on different sites than the canonical cleavage site, called “non-canonical sites,” appeared. This biased activation causes incomplete or different signaling compared to the ones observed after canonical activation. Proteases can activate PARs in a biased way (
Biased activation was first described for PAR1 signaling (
Regarding PAR2, a study demonstrates the role of neutrophil elastase in MAPK signaling through biased activation of PAR2 (
Thus, considering the diversity of elements able to cleave and activate the PARs, it has not been easy to decipher for each individual receptor its own mechanisms of activation. For example, thrombin can activate PAR1, PAR3, and PAR4. Deciphering the specific signaling triggered by PAR1 via thrombin is in consequence difficult. In that context, using synthetic peptide sequences or agonist peptides of 5–6 amino acids is paramount (
Several peptide sequences, with a different number of amino acids, additional hydrophilic residues or amino acid substitutions relative to the PAR1 activator ligand sequence, have been developed to activate PAR1. The most efficient one is in fact similar to PAR1 activator ligand sequence, TFLLR (
Regarding PAR2, here again, depending on the peptide tested, the results are not identical. Indeed, PAR2 activation via the SLAAAA agonist peptide results in intracellular calcium release, MAPK pathway signaling and receptor internalization (
No PAR3 specific agonist peptides have been generated. Indeed, the peptides designed with that aim, such as TFRGAP-NH2, seem actually to activate PAR4. An explanation could be a PAR3 and PAR4 dimerization as described in response to thrombin (
Regarding PAR4, the agonist peptide GYPGQV-NH2 specifically activates the receptor, causing contractility of the aorta and longitudinal gastric muscles in the rat (
PARs activation can be inhibited by disarming the receptor. Indeed, some proteases can prevent the canonical proteolytic cleavage by a proteolytic cleavage upstream of the activator ligand sequence of the receptor (
PARs can also be activated through co-activation or transactivation. Indeed, the hirudin-like domain present on the PAR1 and PAR3 sequences allows increasing the affinity of these receptors for thrombin, helping in turns to activate PAR4, which does not have such a domain (
PAR4 co-activation by PAR3.
PAR1 can also be activated by a mechanism called transactivation (
PAR2 transactivation via PAR1. PAR1 is clived and activated by thrombin. In turns, its activating ligand links PAR2 ECL2 leading to the receptor and downstream signaling activation.
In addition, this transactivation leads to ERK1/2 signaling (Extracellular signal-Regulated Kinases 1/2) and appears to be more prevalent during pathological events, such as sepsis in endothelial cells, chronic inflammation and carcinogenesis (
Proteases, also known as proteinases and peptidases, are degradative enzymes for protein catabolism that hydrolyse a peptide bond to generate amino acids (
MMPs are a group of zinc- dependent endopeptidases known to degrade and remodel the components of the extracellular matrix. Depending on their substrate specificities, MMPs are subdivided into six groups: Collagenases, gelatinases, stromelysins, matrilysins, membrane-types MMPs, and non-classified MMPs. Besides the extracellular matrix turn over, MMPs are involved in other tissue maintenance functions, such as wound healing, and regulation of a broad range of molecules, such as chemokines, cytokines, growth factors, cytoskeleton, and junctional proteins (
Theses proteases are enzymes that hydrolyze peptide bonds within the protein sequence, in which serine serves as nucleophilic amino acid at the active site. Serine proteases, the most abundant group of proteases, are widely distributed in nature and present in the three domains of life (archaea, bacteria, and eukaryotes) as well as in viral genomes (
Most are found intracellularly. Besides their fundamental functions of catabolism and protein processing, cysteine proteases mediate other signaling pathways involved in programmed cell death, inflammation and intestinal mucosa integrity (epithelium turnover and homeostasis) (
As mentioned above, the colon is highly exposed to proteases, whether pancreatic, bacterial proteases from resident colon cells or proteases produced and secreted by epithelial cells (
In the colon, smooth muscle cells, endothelial cells, enteric neurons, fibroblasts and immune cells, such as neutrophils, lymphocytes, macrophages, express PAR1. Originally, PAR1 expression was only reported in cancerous colon epithelial cells, but not in normal epithelial cells (
One study demonstrated that PAR1 activation on intact cultured monolayers of intestinal epithelial cells in Using chambers resulted in chloride ion release, whereas in intact tissues, PAR1 activation would not result in this release (
Another colon function is to allow the transit of chime. This involves a regulation of the intestinal motility. The role of PAR1 and PAR2 in this process was demonstrated by the stimulation of circular and longitudinal rat colonic muscle layers with either thrombin, trypsin or agonist peptides. This study showed that both PAR1 and PAR2 activations result in muscle contraction and relaxation (
PAR1 and PAR2 play a role in nociception. Thus, several studies have reported that colorectal distension performed by intracolonic administration of trypsin and PAR2 agonist peptide in rats caused visceral pain (
Activation of PARs present in epithelial cells leads to changes in paracellular permeability. Activation of PAR1, in response an agonist peptide, increases the intestinal permeability via the apoptotic process through caspase 3, and an alteration of ZO1 expression (
PAR1 and PAR2 have been both described involved in the stimulation of colorectal cancer cell proliferation (
The IBD, Crohn's disease (CD) and ulcerative colitis (UC), are chronic diseases causing inflammation of the gut (
In the gut, PARs are stimulated by endogenous proteases, such as pancreas trypsin, cells of the intestinal mucosa (immune cells including mast cells, epithelial cells including goblet, neuroendocrine, and enterocyte cells), or gut microbiota. Moreover, PARs expressions in the intestinal epithelium are different between IBD patients and healthy individuals. Colonic biopsies from UC and CD patients exhibited increased expression PAR1, while PAR2 and PAR4 are just upregulated in UC conditions. Enhanced levels of these receptors is linked to its activation-internalization-degradation signaling induced by proteases released from eukaryotic host cells but also from gut micro-organisms. Indeed, on neutrophil cells,
PAR1 stimulation triggers apoptosis of the epithelial cells within the gut mucosa through a mechanism involving caspase-3 activation. This excessive apoptosis is associated to a disruption of the intestinal barrier function, promoting then the development and/or severity of the colitis (
PAR2 receptor is localized at the apical and basolateral membranes (
Altogether, these studies report the role for PAR2 in IBD pathophysiology. However, the most important source of the proteases activating PAR2 to promote IBD is largely unclear.
The biological importance of PAR3 is not fully demonstrated. Thus, PAR3 does not exhibit, as the other PARs, a C-terminal intracytoplasmic tail. However, as described previously, PAR3 could play a role as co-factor or co-receptor for PARs and/or other receptors. Although PAR3 mRNA has been evidenced in the gut, no study has reported its involvement in intestinal inflammation (
PAR4 expression has been detected in the gut (
Worldwide, colorectal cancer is the second and the third most commonly occurring cancer in women and men, respectively. There were over 1.8 million new cases detected in 2018 (GLOBOCAN database 2018,
Since a long time now, proteases have been associated with tumor progression mainly due to their ability to degrade the extracellular matrix (ECM), favoring thus tumor cell invasion and metastasis process (
Evidence that thrombin potentiated the tumor growth and the metastatic process
Patients with IBD are 10–20 times more likely to develop CRC (
Regarding the role of PAR2, several human colon cancer cell lines, namely T84, Caco-2, HT-29, and C1.19A, produce and secrete trypsin at concentrations compatible with PAR2 activation, supporting the idea of a possible autocrine/paracrine regulation of PAR2 activity by trypsin in colon cancer cells (
More recently, we reported in three-dimensional cultures of murine colorectal crypt and in Caco-2 cells, that PAR2 activation decreases the numbers and the size of normal or cancerous spheroids. Spheroids deficient for display an increased proliferation, suggesting that cell proliferation is repressed by PAR2. However, in the same study, PAR2-stimulated normal cells are more resistant to stress, suggesting PAR2 pro-survival roles. Indeed, PAR2-deficient normal spheroids display an increase of active caspase-3. Moreover, we showed that PAR2, but not PAR1, was able to trigger GSK3β activation in normal and tumor cells. The PAR2-triggered GSK3β activation involves an arrestin/PP2A/GSK3β complex that is dependent on the activity of the Rho kinase. Finally, the survival of PAR2-stimulated cultures can be pharmacologically inhibited using a GSK3 inhibitor. This study highlighted the PAR2/GSK3β pathway as a novel critical player in the regulation of stem/progenitor cell survival and proliferation in normal colon crypts and colon cancer (
To date, no clear role of PAR3 in CRC has been reported.
Regarding PAR4, its expression is increased in colorectal cancer tissues compared to the associated normal tissues. This overexpression seems to promote colorectal cancer cell proliferation, survival and metastasis, making PAR4 a potential therapeutic target in CRC (
PAR4 mediates thrombin effects on human colon cancer cells. AP4, a specific PAR4 agonist, mimics the effects of thrombin on cell proliferation. Its effects on calcium mobilization in CHO-PAR4-expressing cells are similar to the one observed in HT-29 cells, while HT-29 cells treatment with a reverse peptide has no effect on calcium mobilization. Finally, AP4 promotes colon cancer cell proliferation. More recently, PAR4 overexpression in LoVo cells has been shown, via activation of the ERK1/2 pathway, to increase their proliferation and migration and tumorigenesis capacities, while its knock-down in HT29 results in opposite effect (
Since their discovery in the early nineties, PARs have been shown to be largely involved in the regulation of the intestine physiological processes, but also in the two main diseases affecting the organ, namely inflammatory bowel diseases and colorectal cancer. Consequently, these G-protein coupled receptors represent attractive targets for therapeutic drug development. More than aiming to target their ligands, efforts to develop specific receptor inhibitors are currently regarded as a priority although to date, the development of effective PAR antagonist yet remains in its early stages.
MS, NS-T, FB, and AF wrote the manuscript. EM did a careful reading and review of the manuscript before its submission.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.