Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 295, Issue 8, Pages 2520-2540Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA119.011461
Keywords
G-protein?coupled receptor (GPCR); bioluminescence; bioluminescence resonance energy transfer (BRET); mitogen-activated protein kinase (MAPK); arrestin; docking; platelet; biased signaling; G-protein; peptide; protease-activated receptor-4 (PAR4)
Categories
Funding
- Canadian Institutes of Health Research (CIHR) [376560]
- Natural Sciences and Engineering Research Council of Canada
- Doctoral Queen Elizabeth II Graduate Scholarship in Science and Technology
- Canada Graduate Scholarship
- Ontario Graduate Scholarship
Ask authors/readers for more resources
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 G?(q/11)-coupled calcium-signaling pathway, ?-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 ?-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 Asp(230) 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 ?-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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available