抄録
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 double-knockout 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.
