Mapping transmembrane residues of proteinase activated receptor 2 (PAR₂) that influence ligand-modulated calcium signaling

Proteinase-activated receptor 2 (PAR₂) is a G protein-coupled receptor involved in metabolism, inflammation, and cancers. It is activated by proteolysis, which exposes a nascent N-terminal sequence that becomes a tethered agonist. Short synthetic peptides corresponding to this sequence also activate PAR₂, while small organic molecules show promising PAR₂ antagonism. Developing PAR₂ ligands into pharmaceuticals is hindered by a lack of knowledge of how synthetic ligands interact with and differentially modulate PAR₂. Guided by PAR₂ homology modeling and ligand docking based on bovine rhodopsin, followed by cross-checking with newer PAR₂ models based on ORL-1 and PAR₁, site-directed mutagenesis of PAR₂ was used to investigate the pharmacology of three agonists (two synthetic agonists and trypsin-exposed tethered ligand) and one antagonist for modulation of PAR₂ signaling. Effects of 28 PAR₂ mutations were examined for PAR₂-mediated calcium mobilization and key mutants were selected for measuring ligand binding. Nineteen of twenty-eight PAR₂ mutations reduced the potency of at least one ligand by >10-fold. Key residues mapped predominantly to a cluster in the transmembrane (TM) domains of PAR₂, differentially influence intracellular Ca²⁺ induced by synthetic agonists versus a native agonist, and highlight subtly different TM residues involved in receptor activation. This is the first evidence highlighting the importance of the PAR₂ TM regions for receptor activation by synthetic PAR₂ agonists and antagonists. The trypsin-cleaved N-terminus that activates PAR₂ was unaffected by residues that affected synthetic peptides, challenging the widespread practice of substituting peptides for proteases to characterize PAR₂ physiology.