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- Title
Binding of a highly potent protease-activated receptor-2 (PAR2) activating peptide,[<sup>3</sup>H]2-furoyl-LIGRL-NH<sub>2</sub>, to human PAR2.
- Authors
Kanke, Toru; Ishiwata, Hiroyuki; Kabeya, Mototsugu; Saka, Masako; Doi, Takeshi; Hattori, Yukio; Kawabata, Atsufumi; Plevin, Robin
- Abstract
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-NH2), whole-cell binding assays were performed utilising a radioactive ligand,[3H]2-furoyl-LIGRL-NH2.Specific binding of[3H]2-furoyl-LIGRL-NH2 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 Kd of 122±26.1?nM and a corresponding Bmax of 180±6?f?mol in 3.0×105?cells.The relative binding affinities of a series of modified PAR2 agonist peptides obtained from competition studies paralleled their relative EC50 values for Ca2+ 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-NH2, 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-NH2 was displaced by addition of unlabeled ligands, Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) or 2-furoyl-LIGRL-NH2. The relative binding affinity of 2-furoyl-LIGRL-NH2 to SLIGKV-OH was comparable to its relative EC50 value for Ca2+ 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-NH2 binds to human PAR2 at its ligand-binding site. The use of this radioligand will be valuable for characterising chemicals that interact to PAR2.British Journal of Pharmacology (2005) 145, 255-263. doi:10.1038/sj.bjp.0706189
- Subjects
PROTEOLYTIC enzymes; CHEMICAL agonists; PROTEINS; PEPTIDES; LIGANDS (Biochemistry); PHARMACOLOGY
- Publication
British Journal of Pharmacology, 2005, Vol 145, Issue 2, p255
- ISSN
0007-1188
- Publication type
Article
- DOI
10.1038/sj.bjp.0706189