Molecular basis for activation and biased signaling at the thrombin-activated GPCR proteinase activated receptor-4 (PAR4).

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, ß-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 ß-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 ß-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.

Biased signaling in platelet G-protein coupled receptors.

Platelets are small megakaryocyte-derived, anucleate, disk-like structures that play an outsized role in human health and disease. Both a decrease in the number of platelets and a variety of platelet function disorders result in petechiae or bleeding that can be life threatening. Conversely, the inappropriate activation of platelets, within diseased blood vessels, remains the leading cause of death and morbidity by affecting heart attacks and stroke. The fine balance of the platelet state in healthy individuals is controlled by a number of receptor-mediated signaling pathways that allow the platelet to rapidly respond and maintain haemostasis. G-protein coupled receptors (GPCRs) are particularly important regulators of platelet function. Here we focus on the major platelet-expressed GPCRs and discuss the roles of downstream signaling pathways (e.g., different G-protein subtypes or ß-arrestin) in regulating the different phases of the platelet activation. Further, we consider the potential for selectively targeting signaling pathways that may contribute to platelet responses in disease through development of biased agonists. Such selective targeting of GPCR-mediated signaling pathways by drugs, often referred to as biased signaling, holds promise in delivering therapeutic interventions that do not present significant side effects, especially in finely balanced physiological systems such as platelet activation in haemostasis.

Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA and ß-Arrestin.

Proteinase-activated receptors (PARs) are a four-member family of G-protein-coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin, and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types, including platelets, vascular smooth muscle cells, and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane, with spherical membrane protrusions that resemble plasma membrane blebbing. Since nonapoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release, we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gα q/11- and Gα i-signaling pathways and is dependent on signaling via the ß-arrestin-1/2 and Ras homolog family member A (RhoA) signaling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. SIGNIFICANCE STATEMENT: We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA-and ß-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4.

Targeting a Proteinase-Activated Receptor 4 (PAR4) Carboxyl Terminal Motif to Regulate Platelet Function.

Thrombin initiates human platelet aggregation by coordinately activating proteinase-activated receptors (PARs) 1 and 4. However, targeting PAR1 with an orthosteric-tethered ligand binding-site antagonist results in bleeding, possibly owing to the important role of PAR1 activation on cells other than platelets. Because of its more restricted tissue expression profile, we have therefore turned to PAR4 as an antiplatelet target. We have identified an intracellular PAR4 C-terminal motif that regulates calcium signaling and ß-arrestin interactions. By disrupting this PAR4 calcium/ß-arrestin signaling process with a novel cell-penetrating peptide, we were able to inhibit both thrombin-triggered platelet aggregation in vitro and clot consolidation in vivo. We suggest that targeting PAR4 represents an attractive alternative to blocking PAR1 for antiplatelet therapy in humans.

Protease-activated receptor 2 (PAR2)-targeting peptide derivatives for positron emission tomography (PET) imaging.

The proteolytically-activated G protein-coupled receptor (GPCR) protease-activated receptor 2 (PAR2), is implicated in various cancers and inflammatory diseases. Synthetic ligands and in vitro imaging probes targeting this receptor have been developed with low nanomolar affinity, however, no in vivo imaging probes exist for PAR2. Here, we report the strategic design, synthesis, and biological evaluation of a series of novel 4-fluorobenzoylated PAR2-targeting peptides derived from 2f-LIGRLO-NH2 (2f-LI-) and Isox-Cha-Chg-Xaa-NH2 (Isox-) peptide families, where the 4-fluorobenzoyl moiety acts as the 19F-standard of an 18F-labeled probe for potential use in in vivo imaging. We found that several of the 4-fluorobenzoylated peptides from the 2f-LI-family exhibited PAR2 selectivity with moderate potency (EC50 = 151-252 nM), whereas several from the Isox-family exhibited PAR2 selectivity with high potency (EC50 = 13-42 nM). Our lead candidate, Isox-Cha-Chg-Ala-Arg-Dpr(4FB)-NH2 (EC50 = 13 nM), was successfully synthesized with fluorine-18 with a radiochemical yield of 37%, radiochemical purity of >98%, molar activity of 20 GBq/µmol, and an end of synthesis time of 125 min. Biodistribution studies and preliminary PET imaging of the tracer in mice showed predominantly renal clearance. This 18F-labeled tracer is the first reported PAR2 imaging agent with potential for use in vivo. Future work will explore the use of this tracer in cancer xenografts and inflammation models involving upregulation of PAR2 expression.

A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy.

The FZD4:LRP5:TSPAN12 receptor complex is activated by the secreted protein Norrin in retinal endothelial cells and leads to ßcatenin-dependent formation of the blood-retina-barrier during development and its homeostasis in adults. Mutations disrupting Norrin signaling have been identified in several congenital diseases leading to hypovascularization of the retina and blindness. Here, we developed F4L5.13, a tetravalent antibody designed to induce FZD4 and LRP5 proximity in such a way as to trigger ßcatenin signaling. Treatment of cultured endothelial cells with F4L5.13 rescued permeability induced by VEGF in part by promoting surface expression of junction proteins. Treatment of Tspan12-/- mice with F4L5.13 restored retinal angiogenesis and barrier function. F4L5.13 treatment also significantly normalized neovascularization in an oxygen-induced retinopathy model revealing a novel therapeutic strategy for diseases characterized by abnormal angiogenesis and/or barrier dysfunction.

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

The C-terminal tail of G-protein-coupled receptors (GPCR) contain important regulatory sites that enable interaction with intracellular signaling effectors. Here we examine the relative contribution of the C-tail serine/threonine phosphorylation sites (Ser383-385, Ser387-Thr392) and the helix-8 palmitoylation site (Cys361) in signaling regulation downstream of the proteolytically activated GPCR, PAR2. We examined Gαq/11-coupled calcium signaling, ß-arrestin-1/-2 recruitment, and MAPK activation (p44/42 phosphorylation) by wild-type and mutant receptors expressed in a CRISPR/Cas9 PAR2-knockout HEK-293 cell background with both peptide stimulation of the receptor (SLIGRL-NH2) as well as activation with its endogenous trypsin revealed a tethered ligand. We find that alanine substitution of the membrane proximal serine residues (Ser383-385Ala) had no effect on SLIGRL-NH2- or trypsin-stimulated ß-arrestin recruitment. In contrast, alanine substitutions in the Ser387-Thr392 cluster resulted in a large (∼50%) decrease in ß-arrestin-1/-2 recruitment triggered by the activating peptide, SLIGRL-NH2, but was without an effect on trypsin-activated ß-arrestin-1/-2 recruitment. Additionally, we find that alanine substitution of the helix-8 cysteine residue (Cys361Ala) led to a large decrease in both Gαq/11 coupling and ß-arrestin-1/-2 recruitment to PAR2. Furthermore, we show that Gαq/11 inhibition with YM254890, inhibited ERK phosphorylation by PAR2 agonists, while genetic deletion of ß-arrestin-1/-2 by CRISPR/Cas9 enhanced MAPK activation. Knockout of ß-arrestins also enhanced Gαq/11-mediated calcium signaling. In line with these findings, a C-tail serine/threonine mutant that has decreased ß-arrestin recruitment also showed enhanced ERK activation. Thus, our studies point to multiple mechanisms that regulate ß-arrestin interaction with PAR2 and highlight differences in regulation of tethered-ligand- and peptide-mediated activation of this receptor.

High Affinity Fluorescent Probe for Proteinase-Activated Receptor 2 (PAR2).

PAR2 is a proteolytically activated G protein-coupled receptor (GPCR) that is implicated in various cancers and inflammatory diseases. Ligands with low nanomolar affinity for PAR2 have been developed, but there is a paucity of research on the development of PAR2-targeting imaging probes. Here, we report the development of seven novel PAR2-targeting compounds. Four of these compounds are highly potent and selective PAR2-targeting peptides (EC50 = 10 to 23 nM) that have a primary amine handle available for facile conjugation to various imaging components. We describe a peptide of the sequence Isox-Cha-Chg-ARK(Sulfo-Cy5)-NH2 as the most potent and highest affinity PAR2-selective fluorescent probe reported to date (EC50 = 16 nM, K D = 38 nM). This compound has a greater than 10-fold increase in potency and binding affinity for PAR2 compared to the leading previously reported probe and is conjugated to a red-shifted fluorophore, enabling in vitro and in vivo studies.

Single Amino Acid Replacement in G-7039 Leads to a 70-fold Increase in Binding toward GHS-R1a.

The growth hormone secretagogue receptor type 1a (GHS-R1a) is a class A rhodopsin-like G protein coupled receptor (GPCR) that is expressed in a variety of human tissues and is differentially expressed in benign and malignant prostate cancer. Previously, the peptidomimetic [1-Nal4 ,Lys5 (4-fluorobenzoyl)]G-7039 was designed as a molecular imaging tool for positron emission tomography (PET). However, this candidate was a poor binder (IC50 =69 nm), required a lengthy four-step radiosynthesis, and had a cLogP above 8. To address these challenges, we now report on changes targeted at the 4th position of G-7039. A 2-fluoropropionic acid (2-FPA) group was added on to Lys5 to determine the potential binding affinity of the [18 F]-2-FP radiolabeled analogue, which could be prepared by simplified radiochemistry. Lead candidate [Tyr4 ,Lys5 (2-fluoropropionyl)]G-7039 exhibited an IC50 of 0.28 nm and low picomolar activity toward GHS-R1a. Molecular docking revealed a molecular basis for this picomolar affinity.