Allosteric activation of mPR3 by GPR97 triggers PAR2 activation in neutrophils by unmasking novel tethered agonist peptides

A unique multi-receptor complex comprising GPR97, CD177, CD16b, and PAR2 was recently identified as a positive modulator of human neutrophil activation. GPR97, an adhesion G protein-coupled receptor (adhesion GPCR), binds to membrane-bound CD177-associated proteinase 3 (mPR3) and enhances its enzymatic activity. The GPR97-stimulated mPR3 then cleaves and activates PAR2, another GPCR, leading to robust inflammatory responses. However, the underlying GPR97-mediated PAR2 transactivation mechanism and its downstream signaling pathways remain poorly understood. Herein, we demonstrate that PAR2 is a unique lipid raft-dependent substrate of GPR97-activated mPR3. We further identify specific mPR3 cleavage sites on PAR2 and uncover novel intramolecular agonist peptides responsible for PAR2 activation. Lastly, we delineate the downstream signaling networks elicited by these peptide agonists, highlighting the roles of distinct G proteins and signaling effectors in shaping inflammatory responses. Collectively, our findings unveil a novel GPR97-mediated PAR2 transactivation mechanism that contributes to neutrophil activation.