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First published online March 7, 2007
doi: 10.1242/10.1242/jcs.03409

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Protease-activated receptor signalling, endocytic sorting and dysregulation in cancer

Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365, USA

View larger version (23K): [in this window] [in a new window]   Fig. 1. PAR1 activation and signalling. PAR1 is a seven-transmembrane-span G-protein-coupled receptor that is activated by a unique proteolytic mechanism. Thrombin (-Th), a serine protease, binds to and cleaves the N-terminal exodomain of PAR1. The newly unmasked N-terminus of PAR1 then acts as a tethered ligand and binds intramolecularly to the receptor to trigger transmembrane signalling. Synthetic peptides that mimic the tethered ligand domain can activate PAR1 independently of proteolysis. PAR1 couples to Gq, G12/13, Gi and G to activate a variety of signalling cascades and cellular responses. The G12/13 subunits bind RhoGEFs, which activate small G-proteins such as Rho. Gq activates phospholipase C, triggering phosphoinositide hydrolysis, resulting in inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) production and Ca2+ mobilization, protein kinase C (PKC) and MAP kinase activation. G can activate PI-3-kinase, G-protein-coupled receptor kinases (GRKs) and other effectors. PAR1 is uncoupled from G-protein signalling by rapid phosphorylation and arrestin binding.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Human PAR N-terminal and C-terminal sequences. Residues flanking the cleavage site specify protease recognition. These are defined as unprimed (P) and primed sites (P'), respectively (i.e. P3-P2-P1/P1'-P2'-P3', where / indicates the cleavage site). Only transmembrane domain (TM) 1 and TM7 are shown. The number of N-terminal residues omitted before the cleavage site is shown in parentheses and the sequences of the tethered ligand domains are shown in yellow. The hirudin-like sequence in PAR1 and PAR3 responsible for high-affinity thrombin binding is underlined. The potential sites of PAR phosphorylation are shown in red and the PAR1 distal tyrosine-based motif important for µ2 binding is underlined. Asterisks indicate the end of the PAR sequences.

View larger version (47K): [in this window] [in a new window]   Fig. 3. PAR endocytic sorting. PAR1 displays both constitutive and agonist-induced internalization. Uncleaved PAR1 constitutively cycles between the plasma membrane and an intracellular compartment. The clathrin adaptor AP2, which binds to a tyrosine-based motif in the PAR1 cytoplasmic tail, mediates constitutive internalization. PAR1 is basally ubiquitylated. Agonist-induced internalization of PAR1 requires phosphorylation and ubiquitylation specifies internalization through clathrin-coated pits. The clathrin adaptor that mediates agonist-induced internalization of ubiquitylated PAR1 has not been identified. Internalized PAR1 is sorted from endosomes to lysosomes through a sorting nexin-1 (SNX1)-dependent pathway, which is independent of ubiquitylation and the ESCRT-1 machinery. Arrestins mediate PAR2 internalization. Internalized PAR2 bound to arrestins appear to signal to ERK1/2 from an endocytic compartment. Sorting of PAR2 from endosomes to lysosomes requires ubiquitylation. The sorting mechanism(s) and adaptor proteins that mediate PAR4 internalization and lysosomal sorting are not known, and whether PAR3 redistributes from the cell surface remains to be determined.

View larger version (33K): [in this window] [in a new window]   Fig. 4. PARs function in the vasculature and in tumor invasion and metastasis. PAR function is shown in the context of a blood vessel. Thrombin, the main effector protease of the coagulation cascade, activates PAR1, PAR3 and PAR4 to elicit signalling in a variety of cell types. Thrombin activates PAR1 and PAR4 in human platelets and generates fibrin, which is important for thrombus formation and tumor cell survival and metastasis. FXa can also cleave and activate PAR1. Activated endothelial cells express tissue factor (TF) and are represented by an oval shape. PAR2 is activated by TF-bound FVIIa and FXa but not by thrombin. PAR1 and PAR2 are also cleaved and activated by tumor-generated proteases, which contribute to tumor cell growth, invasion and metastasis.