Abstract
A novel mechanism of action was described for the protease-activated receptor 4 (PAR4)-derived pepducin (P4Pal10), when it was shown to exhibit inhibitory efficacy towards G protein coupling to multiple Gαq-coupled receptors (Carr, R., 3rd et al., Mol. Pharmacol. 2016(89) 94). We could confirm that P4Pal10, similar to an earlier-characterized Gαq inhibitor (YM-254890), inhibited platelet aggregation induced by agonists for the Gαq-coupled receptors PAR1 and PAR4. Next, we applied P4Pal10 as a tool compound and investigated its modulatory effect on several Gαq- and Gαi-coupled GPCRs expressed by human neutrophils. P4Pal10 had, however, no inhibitory effects on signaling downstream of the Gαq-coupled receptors for ATP (P2Y2R) and PAF (PAFR). Instead, P4Pal10 inhibited signaling downstream the Gαi-coupled FPR2. The inhibition was not due to a direct effect on Gαi as the closely related FPR1 was unaffected. In addition, we found that the pepducin activated allosterically modulated short chain fatty acid receptor (FFAR2), a Gαi/Gαq coupled GPCR that is functionally expressed in neutrophils. Taken together, we show that pepducins are unique tool-compounds for mechanistic studies of GPCR signaling and modulation in neutrophils. The data presented add also lipopeptides into the known ligand recognition lists for the two pattern recognition receptors FPR2 and FFAR2, receptors that primarily sense formylated peptides and short free fatty acids, respectively, inflammatory mediators of microbial origin.
Highlights
We show that no inhibition was induced by P4Pal10 on the signals generated by Gαq-coupled receptors, whereas the responses triggered by the Gαicoupled formyl peptide receptor 2 (FPR2) and FFA2R was selectively inhibited and amplified, respectively
We examined the effect of P4Pal10 on activation of the O2-generating NADPH-oxidase, an electron transporting enzyme system in neutrophils that is assembled/activated by many agonist-occupied G-protein coupled receptors (GPCRs) [24, 25]
We have investigated the effects of the presumed Gαq-inhibitory pepducin P4Pal10 using human neutrophils and well-characterized agonists specific for Gαq- and Gαi-coupled receptors
Summary
In this study, conducted at the Sahlgrenska Academy in Sweden, buffy coats obtained from the blood bank at Sahlgrenska University Hospital, Gothenburg, Sweden were used to isolate neutrophils. According to the Swedish legislation section code 4§ 3p SFS 2003:460, no ethical approval was needed since the buffy coats were provided anonymously. Dextran and Ficoll-Paque were obtained from GE-Healthcare Bio-Science (Uppsala, Sweden). The tripeptide fMLF, and bovine serum albumin, were purchased from Millipore Sigma (Burlington, MA, USA). The hexapeptide WKYMVM, isoluminol, TNFα, propionate, dimethyl sulfoxide (DMSO) and ATP were obtained from Sigma (St. Louis, MO, USA). The FPR2 peptidomimetic agonist Compound 14 [13] and the FPR2 peptidomimetic antagonist CN6 [14] were kind gifts from Henrik Franzyk (Copenhagen, Denmark). MCT-ND4 was a kind gift from Hidehito Mukai
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