Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein–coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, β-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting β-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for β-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4.

28Proteinase Activated Receptor-4 (PAR4) is a member of the proteolytically-activated PAR 29 family of G-Protein-coupled Receptors (GPCRs). PARs are activated following proteolytic 30 cleavage of the receptor N-terminus by enzymes such as thrombin, trypsin, and cathepsin-G to 31 reveal the receptor-activating motif termed the tethered ligand. The tethered ligand binds 32 intramolecularly to the receptor and triggers receptor signalling and cellular responses. In spite 33 of this unusual mechanism of activation, PARs are fundamentally peptide receptors and can 34 also be activated by exogenous application of short synthetic peptides derived from the tethered 35 ligand sequence. In order to gain a better understanding of the molecular basis for PAR4-36 dependent signalling, we examined signalling responses to a library of peptides derived from 37 the canonical PAR4 activating peptide (PAR4-AP), AYPGKF-NH2. We examined peptide 38 residues involved in activation of the Gaq/11-coupled calcium signalling pathway, b-arrestin 39 recruitment, and mitogen-activated protein kinase pathway activation. The peptide N-methyl-40 alanine-YPGKF-NH2 was identified as a compound that is a poor activator of PAR4-dependent 41 calcium signalling but was fully competent in recruiting b-arrestin-1 and -2. In order to gain a 42 better understanding of the ligand-binding pocket, we used in silico docking to identify key 43 residues involved in PAR4 interaction with AYPGKF-NH2. The predicted interactions were 44 verified by site-directed mutagenesis and analysis of calcium signalling and b-arrestin-1/-2 45 recruitment following proteolytic activation (with thrombin) or activation with the synthetic 46 agonist peptide (AYPGKF-NH2). We determined that a key extracellular loop-2 aspartic acid 47 Introduction: 58 G-Protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and 59 regulate a host of important physiological responses (Fredriksson and Schioth, 2005; Pierce et 60 al., 2002). GPCRs respond to a variety of extracellular signals and regulate cellular behaviour 61 through engaging intracellular effector molecules to activate various cell-signalling pathways 62 (Erlandson et al., 2018; Hilger et al., 2018). In drug discovery, GPCRs are a valuable and highly 63 tractable class of drug targets with over 30% of all currently approved drugs acting on GPCRs 64 (Sriram and Insel, 2018). GPCR activation occurs typically through binding of a soluble ligand 65 and conformational changes that enable engagement of G-protein-dependent or b-arrestin-66 mediated signal transduction. The Proteinase Activated Receptors (PARs) are a four-member 67family of GPCRs with a distinct mechanism of activation that involves limited proteolysis and 68 unmasking of a receptor activating motif called the tethered ligand. PARs were first discovered 69 in an effort to identify the cellular receptors responsible for actions of thrombin that were 70 independent from its role in the coagulation cascade . Various proteinases have 71 now been de...

Proteinase-activated receptor 4 (PAR4) is a member of the proteolytically-activated PAR family of G-Protein Coupled Receptors. PARs are activated following proteolytic cleavage of the receptor N-terminus by enzymes such as thrombin, trypsin, and Cathepsin-G to reveal the receptor activating motif termed the tethered ligand. The tethered ligand binds intramolecularly to the receptor to trigger receptor signaling and trafficking cascades. PARs can also be activated by short synthetic peptides derived from the tethered ligand sequence. Here we examined PAR4 signaling and trafficking following proteolytic activation (with thrombin) and activation with a synthetic agonist peptide (AYPGKF-NH2). We used calcium sensitive fluorescent dyes to monitor elevations in intracellular calcium levels, Bioluminescence Resonance Energy Transfer (BRET) to monitor beta-arrestin-1 and 2 recruitment as well as G-protein activation, and western blotting to determine activation of p44/42 MAPK and Akt. PAR4 activation with thrombin or AYPGKF-NH2 triggers Gq coupled calcium signal and recruitment of beta-arrestin 1 and 2. Activated receptors trafficked to Rab5 positive vesicles and to lysosomes in a clathrin-and dynamin-dependent manner. To examine if beta-arrestins were essential for PAR4 internalization we generated a beta-arrestin doubleknockout cell line using CRISPR/Cas9 targeting. We were unable to detect any PAR4 internalization in these cells with any of the agonists tested while reconstitution of beta-arrestins rescued receptor internalization. To further dissect PAR4-dependent signaling pathways we sought to develop novel biased agonists for PAR4 and generated a library of peptides derived from AYPGKF-NH2. Peptides were synthesized using solid phase Fmoc-peptide chemistry, purified by preparative high-performance liquid chromatography (HPLC), and characterized by liquid chromatography mass spectrometry (LCMS). Twenty-five peptides were synthesized at greater than 95% purity. Of the 25 peptides screened, 18 showed decreased beta-arrestin recruitment compared to AYPGKF-NH2. A subset of peptides also showed decreased calcium signaling compared to AYPGKF-NH2. Interestingly, we find 4 peptides that activate PAR4 recruitment of beta-arrestins yet are unable to trigger PAR4-dependent calcium signaling. Platelet aggregation assays were performed to link the signaling bias in PAR4 to platelet function. Ongoing work is aimed at characterizing the bias of this library of PAR4 targeted compounds on multiple signaling pathway and cellular behavior.