Molecular Basis for G-protein-Coupled Receptor (GPCR) Activation and Biased Signalling at the Platelet Thrombin Receptor Proteinase Activated Receptor-4 (PAR4)

Abstract

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...

Role of the Helix-8 and C-Terminal Tail in Regulating Proteinase Activated Receptor 2 Signaling

“…Human embryonic kidney (HEK) cells (HEK-293; ATCC), PAR2-knockout HEK-293, and β-arrestin-1/-2-knockout HEK-293 (β-arrestin-knockout HEK-293) cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% sodium pyruvate, and penicillin streptomycin solution (50 000 units penicillin, 50 000 μg streptomycin). 26 Since trypsin activates the PARs, cells were routinely subcultured using enzyme-free isotonic phosphate-buffered saline (PBS) containing EDTA (1 mM). Cells were transfected with PAR2-YFP or mutated PAR2-YFP receptor vectors using calcium phosphate [nuclease-free water, 2.5 M CaCl 2 , and 2× HEPES-buffered saline (HBS)] or Fugene6 transfection methods (Promega, Madison, WI).…”

“…Agonist-stimulated calcium signaling was recorded in PAR2-knockout HEK-293 cells as previously described. 26,36,68 Cells were detached in enzyme-free cell dissociation buffer, centrifuged to form a pellet (1000 rpm, 5 min), and resuspended in Fluo-4 NW (no wash) calcium indicator dye (Thermo Fisher Scientific). Following a 30 min incubation at ambient temperature, intracellular fluorescence (excitation 480 nm; emission recorded at 530 nm) was monitored before and after addition of agonists (trypsin or SLIGRL-NH 2 ) on a PTI spectrophotometer (Photon Technology International, Birmingham, NJ).…”

“…Role of Gα q/11 and β-Arrestin in PAR2-Stimulated ERK Activation. Since GPCR-mediated ERK activation can occur downstream of Gα q/11 -dependent and/or β-arrestin-dependent/G-protein-independent pathways, we employed pharmacological inhibition of Gα q/11 with the compound YM254890 and CRISPR/Cas9 β-arrestin-knockout HEK-293 cells 26 to probe these pathways downstream of PAR2. Cells were stimulated with SLIGRL-NH 2 (30 μM) for various time points to record ERK phosphorylation (p-p44/42).…”

Mice Lacking PECAM-1 and Ceacam1 Have Enhanced Platelet Secretion and Thrombus Growth: Novel Link with PAR4

“…23 Alternatively, G 12/13 recruitment signals through RhoA that further controls shape change and dense granule release. 23 36 In this study, we showed that the hyper-responsiveness of thrombin or PAR4-mediated α-granule release in DKO Pecam-1/Ceacam1 platelets was linked with PAR4/G αq /PLC-β but not with the PAR4/G α12/13 /RhoA signaling pathway. These results suggest that these classical ITIM-bearing receptors, PECAM-1 and CEACAM1, work in concert to negatively regulate the PAR4/G αq /PLCβ signaling pathway in platelets.…”

“…33 GPCR signaling in mouse platelets works through proto-enzymatic cleavage of the N-terminal extracellular domain via thrombin, which then acts as a ligand to the PAR4 receptor leading to the recruitment of the G proteins, G αq , and G 12/13 . 36 Upon ligation, G αq recruitment leads to PLCβ activation and downstream activation that results in calcium mobilization, shape change, and granule secretion. 23 Alternatively, G 12/13 recruitment signals through RhoA that further controls shape change and dense granule release.…”