1. Protease Activated Receptors (PARs) Molecular Cell Biology, 2013 Paul Bauer Jens Berndtsson Eva Darai Adams et al. 2011, Pharmacology & Therapeutics Coughlin. 2000, Nature Member of seven transmembrane helices receptor superfamily (GPCR) Four different types in humans, PAR1-4 Irreversible activated/inactivated by N-terminal peptide cleavage by proteases Adams et al. 2011, Pharmacology & Therapeutics Signaling through both G-protein pathway and β-arrestin pathway PARs activates multiple downstream signaling pathways through these two general ways (e.g. MAPK, NFκB) Precise protease activity determine receptor activity Target sequence differs between PAR1-4 PARs are essential for normal development Misregulation of signal peptide cleavage can result in unwanted receptor activation PAR activity has been implicated in several diseases Over-activation through MMP-1 cleavage facilitates breast cancer Boire et al. 2005, Cell Ossovskaya and Bunnett. 2004, Physiology Rev

2. GPCR and PAR 101 Bockaert and Pin. 1999, EMBO PAR1: Potential cleavage site for thrombin PAR2: 30% amino acid identity to PAR1. Putative trypsin cleavage site PAR3: 28% sequence homology to human PAR1 and PAR2. Thrombin cleavage site. PAR4:. 33% homologous to the other human PARs, Cleavage site for thrombin and trypsin PARs -N-terminus cleaved by soluble proteases -New N-terminus exposed -Tethered ligand domain, binds intramolecularly to induce intracellular signal transduction - activates the cleaved receptor OR - generate a disabled receptor GPRCRs of the largest families of proteins in the mammalian genome Mediate extracellular messages by interacting with G proteins Large variety of ligands Wide range of proteases cleave PARs Adams et al. 2011, Pharmacology & Therapeutics

3. Receptor structure and downstream signaling N-terminus Signal- and pro-peptide- regions Tethered ligand Hirudin-like domains (PAR1/3) N-glycosylation sites C-terminus Putative palmitylation sites Tyrosin based motif for regulation of receptor trafficking PTM sites Transmembrane region Three extracellular loops (ECL) Three intracellular loops (ICL) Cystein for disulfide-link (ECL2- TM3) PTM sites N-glycosylation sites Adams et al. 2011, Pharmacology & Therapeutics Coughlin. 2000, Nature Adams et al. 2011, Pharmacology & Therapeutics β-arrestin pathway PAR2 most completely characterized. Phosphorylated c- terminus of PAR binds to the scaffold protein β-arrestin. Mediate downstream signaling by also binding other peptide, permitting complex formations. G-protein pathway PAR1/2: Gαq, Gαi, Gα12/13, Gβγ, NFκB (inflammatory response, mostly PAR2) PAR3: unclear, serves as co-factor for PAR4 but also seems to have own signaling pathways activating ERK among others. PAR4: Gαq, Gαi/o

4. Implications for cancer PAR receptors have been implicateted in a number of diseases Function of PAR is important for normal development Mis-regulation has been linked to several forms of cancer Activation of PAR1 and PAR2 most studied Study on the different activation by Thrombin and MMP1 Epitope cleavage is specific for both proteases Other proteases don't cleave specific Clevage causes downstream activation through Ca² ⁺ signaling Boire et al. 2005, Cell Ossovskaya and Bunnett. 2004, Physiology Rev

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