The PAR4-derived pepducin P4Pal lacks effect on neutrophil GPCRs that couple to Gαq for signaling but distinctly modulates function of the Gαi-coupled FPR2 and FFAR2.

A novel mechanism of action was described for the protease-activated receptor 4 (PAR4)-derived pepducin (P4Pal), 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 P4Pal, 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 P4Pal as a tool compound and investigated its modulatory effect on several Gαq- and Gαi-coupled GPCRs expressed by human neutrophils. P4Pal had, however, no inhibitory effects on signaling downstream of the Gαq-coupled receptors for ATP (P2YR) and PAF (PAFR). Instead, P4Pal 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.