The protease activated receptor 2 (PAR2) polymorphic variant F240S constitutively activates PAR2 receptors and...

AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide] are the first two small-molecule agonists described for the G protein-coupled receptor protease-activated receptor 2 (PAR2), but whether they activate PAR2 through a similar mechanism as its tethered peptide ligand or soluble peptide mimetics of its tethered peptide ligand is unclear. Extracellular loop 2 (ECL2) has been shown to play a critical role in the activation mechanism of PAR2. Therefore, we constructed a series of PAR2 receptors mutated in ECL2, including a previously described polymorphic variant of PAR2 (F240S), and compared AC-55541 and AC-264613 to SLIGRL and a potent analog of SLIGRL called 2-furoyl LIGRLO in a series of functional assays, including cellular proliferation, phosphatidylinositol hydrolysis, and β-arrestin recruitment assays. Surprisingly, receptors with the F240S mutation were constitutively active in all functional assays tested. Furthermore, AC-55541 and AC-264613 were potentiated over 30-fold at the receptors with the F240S mutation, whereas SLIGRL and 2-furoyl LIGRLO were much less affected. In contrast, mutagenesis of charged residues in ECL2 confirmed their important role in the actions of peptide agonists of PAR2, whereas these mutations did not significantly affect activation of PAR2 by AC-55541 or AC-264613. These results suggest that F240S PAR2 receptors may be useful in screens to detect novel small-molecule PAR2 modulators and that further work on the biological importance of the F240S PAR2 variant is warranted.