Abstract
Protease-activated receptor 2 (PAR2) is a transmembrane receptor that is irreversibly activated by proteolytic cleavage of its N-terminus via extracellular proteases, resulting in the release of the tethered ligand (TL), which binds to and activates the receptor. PAR2 plays a pivotal role in the inflammatory response and pain sensation and is a promising drug target for treating arthritis, asthma, and neuronal pain. Here, we present the cryo-electron microscopy structures of active PAR2 complexed with miniG(s/q) and miniG(13). Combining functional assays with structural analysis, our study revealed that TL forms a parallel β-sheet with the extracellular loop 2 of PAR2 to engage the receptor. The binding of TL triggers a conformational rearrangement in the transmembrane core, releasing the inhibitory ion lock and allowing receptor activation. Furthermore, we provide structural insights into the engagement of G(q) and G(13) with PAR2, highlighting that a hydrophobic interaction mediated by the last methionine residue of Gα(13) is crucial for G(13) coupling selectivity. In combination with molecular dynamics simulations and mutagenesis, we identified the I39(TL3)/D62(N-term) interaction at the pocket side of the receptor as a key determinant of G(13) signaling. Disrupting this interaction significantly inhibits G(13) signaling while preserving G(q) activity, enabling us to design a biased peptide ligand that selectively activates G(q) signaling. The information revealed in this study provides a framework for understanding PAR2 signaling and offers a rational basis for the design of biased PAR2 ligands.