The classic ligand-initiated paradigm of G protein coupled receptor (GPCR) signaling was challenged with the discovery of protease-activated receptors (PARs). Unlike canonical GPCR activation, which requires a soluble ligand, PARs are enzymatically cleaved at their N-terminus to free a tethered ligand, which subsequently activates the receptor and downstream signaling.1 The elucidation of this unique activation mechanism has transformed how we think about GPCR signaling, and more importantly, emphasizes the critical role of proteases in influencing cellular behavior. This text focuses on the contributions of protease-activated receptors -1 and -2 (PAR1 and PAR2) to cellular injury and repair, with a special focus on matrix metalloprotease-PAR1 signaling and cellular differentiation in cardiovascular and hepatic pathophysiology. The following introductory reading will summarize the current literature on PARs and matrix metalloproteases (MMPs), and bring the reader up to date on PAR1 and PAR2 signaling in the cardiovascular and hepatic systems, respectively.
Proteases comprise approximately 2% of the human genome and contribute to cell signaling through zymogen/ligand activation, degradation/inactivation of soluble ligands, and activation/inactivation of cell-surface receptors. PARs are a subfamily of GPCRs that are cleaved and activated by an array of proteases including those associated with coagulation, inflammatory cells, the digestive tract, and matrix remodeling. PARs signal through heterotrimeric G proteins to activate a multitude of signaling outputs that influence cell behavior and function. Aberrant PAR signaling has been implicated in thrombosis, in-stent restenosis, heart failure, sepsis, arthritis, inflammation, wound-healing, and cancer among others. This section will summarize the cloning, receptor activation, signal termination, and physiological roles PARs, with a focus on PAR1 and PAR2.
Fig. 1 Mechanism of PAR activation. PARs are activated through proteolytic cleavage of the N-terminus, which liberates a tethered ligand that is then capable of interacting with extracellular loop 2, resulting in conformational changes in the transmembrane regions that activate G-protein signaling. Activation of the receptor results in the exchange of GDP for GTP in the Gα subunit, resulting in dissociation of the α and βγ subunits. Both the α and βγ subunits are capable of activating downstream signaling cascades that are cell type- and context-specific.
PARs exert their effects through activation of various G protein constituents. Specifically, PAR1 has been shown to activate the Gi, Gq, and G12/13 families of G proteins. The exact nature of each interaction and the functional consequences that result from G protein activation are cell type- and environment-specific. Most studies attempting to identify specific interactions have relied on G protein-blocking antibodies or have utilized cells derived from G protein knock-out animals. Therefore, although there are general guidelines to PAR1-dependent G protein signaling, it is by no means comprehensive. A recent study attempted to ectopically express both PAR1 and PAR2 in the PAR-null Cos7 cell line to elucidate how PARs couple to G proteins under similar cellular conditions. The researchers found that PAR1 and PAR2 both signal through Gq and G12, whereas only PAR1 signaled through Gi. Although these results are intriguing, Cos7 cells are not human derived and the receptors are ectopically expressed, which are important limitations to the study.
One of the more prominent PAR1-coupled pathways involves Gq activation and subsequent PLC-β signaling. Fibroblasts, cancer cells, and platelets utilize this signaling cascade to mobilize intracellular calcium and stimulate calcium-dependent pathways. Activation of this signaling axis promotes proliferation, migration, and secretion, among others. PAR1 is also capable of coupling to Gi dependent pathways in certain cell lines and cell types. In Chinese hamster ovary (CHO) cells, PAR1 signaling is almost entirely Gi-dependent, and stimulates PLC-β activation and arachidonic acid release through the βγ subunits. Gi signaling is sensitive to inhibition by pertussis toxin, which is a useful reagent when interrogating Gi -dependent pathways. G12/13 pathways are important for cytoskeletal reorganization and cellular shape change. PAR1 couples to G12/13 pathways in both platelets and endothelial cells, which results in platelet activation and endothelial barrier disruption, respectively. PAR1 has consistently been shown to activate MAP- kinase (MAPK) signaling pathways, although it is unclear whether this occurs through a specific G protein pathway, or is ubiquitous to all G proteins.
Although PAR2 G protein signaling has not been as thoroughly studied as PAR1, some similarities have been identified. PAR2 is known to couple to inositol triphosphate (IP3)-calcium-dependent pathways in a number of cell types, suggesting that PAR2 probably couples to Gq. Studies have not demonstrated pertussis toxin-sensitive PAR2 signaling, indicating that Gi is not a prominent pathway for PAR2. Similar to PAR1, PAR2 strongly activates MAPK signaling, particularly the ERK1/2 pathway; the exact mechanism of MAPK activation is elusive, but arrestins are thought to play an important scaffolding role for PAR2.