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

Proteinase-activated receptor 2 (PAR) 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 PAR, while small organic molecules show promising PAR antagonism. Developing PAR ligands into pharmaceuticals is hindered by a lack of knowledge of how synthetic ligands interact with and differentially modulate PAR. Guided by PAR homology modeling and ligand docking based on bovine rhodopsin, followed by cross-checking with newer PAR models based on ORL-1 and PAR, site-directed mutagenesis of PAR was used to investigate the pharmacology of three agonists (two synthetic agonists and trypsin-exposed tethered ligand) and one antagonist for modulation of PAR signaling. Effects of 28 PAR mutations were examined for PAR-mediated calcium mobilization and key mutants were selected for measuring ligand binding. Nineteen of twenty-eight PAR 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 PAR, differentially influence intracellular Ca 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 PAR TM regions for receptor activation by synthetic PAR agonists and antagonists. The trypsin-cleaved N-terminus that activates PAR was unaffected by residues that affected synthetic peptides, challenging the widespread practice of substituting peptides for proteases to characterize PAR physiology.