Abstract
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.
Highlights
Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated proteinase-activated receptors (PARs) family of G-protein– coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics
G␣q/11-coupled calcium signaling, -arrestin-1/2 recruitment (-arr-1–rluc and -arr-2– rluc, respectively), and p44/42 mitogen-activated protein kinase (MAPK) signaling by each of these peptides was monitored and compared with responses triggered by PAR4-AP (Table 1)
Thrombin has been described as a PAR1 and PAR4 activator, and trypsin is described as a PAR2 and PAR4 activator
Summary
To understand the rules governing agonist peptide activation of PAR4, we synthesized 37 hexapeptides through modification to the synthetic PAR4 agonist peptide AYPGKF-NH2. This peptide was able to initiate -arrestin-1/2 recruitment comparable with levels obtained with PAR4-AP (-arr-1, 119.2 Ϯ 11.9%; EC50 ϭ 6.8 Ϯ 2.6 M; -arr-2, 80.6 Ϯ 10.8%; EC50 ϭ 2.0 Ϯ 0.7 M) These results suggest that increasing steric bulk and hydrophobicity of the backbone at amide positions through the addition of an N-terminal methyl group drives PAR4 signaling toward -arrestin pathways and causes only modest activation of calcium signaling. 1, through substitution with norleucine, completely abolished calcium signaling (Nle-YPGKF-NH2, EC50 ϭ ND) and resulted in partial agonism of -arrestin recruitment (-arr-1, 40.2 Ϯ 9.7%; -arr-2, 36.5 Ϯ 8.8%) Together, these data indicate that the precise steric bulk of the side chain in position 1 is important for PAR4 agonism. It is apparent that the precise position of the side chain in position 1 is important for agonism
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