Binding of a highly potent protease-activated receptor-2 (PAR2) activating peptide, [ ₃H]2-furoyl-LIGRL-NH ₂, to human PAR2

To determine the binding characteristics of a highly potent agonist for protease-activated receptor-2 (PAR2), 2-furoyl-Leu-Ile-Gly-Arg-Leu-amide (2-furoyl-LIGRL-NH ₂), whole-cell binding assays were performed utilising a radioactive ligand, [ ₃H]2-furoyl-LIGRL-NH ₂. Specific binding of [ ₃H]2-furoyl-LIGRL-NH ₂ was observed in NCTC2544 cells, dependent upon PAR2 expression, and competitively displaced by the addition of unlabeled PAR2 agonists. Scatchard analysis of specific saturation binding suggested a single binding site, with K _d of 122 ± 26.1 nM and a corresponding B _max of 180 ± 6 f mol in 3.0 × 10 ⁵ cells. The relative binding affinities of a series of modified PAR2 agonist peptides obtained from competition studies paralleled their relative EC ₅₀ values for Ca ²⁺ mobilisation assays, indicating improved binding affinities by substitution with 2-furoyl at the N-terminus serine. Pretreatment of cells with trypsin reduced specific binding of [ ³H]2-furoyl-LIGRL-NH ₂, demonstrating direct competition between the synthetic agonist peptide and the proteolytically revealed tethered ligand for the binding site of the receptor. In HCT-15 cells endogenously expressing PAR2, the binding of [ ³H]2-furoyl-LIGRL- NH ₂ was displaced by addition of unlabeled ligands, Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) or 2-furoyl-LIGRL-NH ₂. The relative binding affinity of 2-furoyl-LIGRL-NH ₂ to SLIGKV-OH was comparable to its relative EC ₅₀ value for Ca ²⁺ mobilisation assays. The binding assay was successfully performed in monolayers of PAR2 expressing NCTC2544 and human umbilical vein endothelial cells (HUVEC), in 96- and 24-well plate formats, respectively. These studies indicate that [ ³H]2-furoyl-LIGRL-NH ₂ binds to human PAR2 at its ligand-binding site. The use of this radioligand will be valuable for characterising chemicals that interact to PAR2.