TY - JOUR
T1 - Binding of a highly potent protease-activated receptor-2 (PAR2) activating peptide, [ 3H]2-furoyl-LIGRL-NH 2, to human PAR2
AU - Kanke, Toru
AU - Ishiwata, Hiroyuki
AU - Kabeya, Mototsugu
AU - Saka, Masako
AU - Doi, Takeshi
AU - Hattori, Yukio
AU - Kawabata, Atsufumi
AU - Plevin, Robin
PY - 2005/5/1
Y1 - 2005/5/1
N2 - 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.
AB - 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.
KW - [ H]2-furoyl-LIGRL- NH
KW - agonist
KW - HCT-15 cells
KW - HUVEC
KW - NCTC7244 cells
KW - protease-activated receptor-2 (PAR2)
KW - radioligand-binding
KW - trypsin
UR - http://www.scopus.com/inward/record.url?scp=19944369803&partnerID=8YFLogxK
U2 - 10.1038/sj.bjp.0706189
DO - 10.1038/sj.bjp.0706189
M3 - Article
C2 - 15765104
AN - SCOPUS:19944369803
SN - 0007-1188
VL - 145
SP - 255
EP - 263
JO - British Journal of Pharmacology
JF - British Journal of Pharmacology
IS - 2
ER -