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

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