Kanke Toru, Ishiwata Hiroyuki, Kabeya Mototsugu, Saka Masako, Doi Takeshi, Hattori Yukio, Kawabata Atsufumi, Plevin Robin
Tokyo New Drug Research Laboratories, Kowa Company Limited, Higashimurayama, 27 Taylor Street, Tokyo 189-0022, Japan.
Br J Pharmacol. 2005 May;145(2):255-63. doi: 10.1038/sj.bjp.0706189.
1 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, [(3)H]2-furoyl-LIGRL-NH(2). 2 Specific binding of [(3)H]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 x 10(5) cells. 3 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. 4 Pretreatment of cells with trypsin reduced specific binding of [(3)H]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. 5 In HCT-15 cells endogenously expressing PAR2, the binding of [(3)H]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. 6 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. 7 These studies indicate that [(3)H]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.
1 为了确定一种高效蛋白酶激活受体 -2（PAR2）激动剂2-呋喃甲酰 - 亮氨酸 - 异亮氨酸 - 甘氨酸 - 精氨酸 - 亮氨酸 - 酰胺（2-呋喃甲酰 - LIGRL - NH₂）的结合特性，利用放射性配体[³H]2-呋喃甲酰 - LIGRL - NH₂进行了全细胞结合测定。2 在NCTC2544细胞中观察到[³H]2-呋喃甲酰 - LIGRL - NH₂的特异性结合，其依赖于PAR2的表达，并可被未标记的PAR2激动剂竞争性取代。对特异性饱和结合的Scatchard分析表明存在单一结合位点，在3.0×10⁵个细胞中，解离常数K(d)为122±26.1 nM，相应的最大结合量B(max)为180±6 fmol。3 从竞争研究中获得的一系列修饰的PAR2激动剂肽的相对结合亲和力与其在钙离子动员测定中的相对半数有效浓度（EC₅₀）值平行，表明在N端丝氨酸处用2-呋喃甲酰取代可提高结合亲和力。4 用胰蛋白酶预处理细胞可降低[³H]2-呋喃甲酰 - LIGRL - NH₂的特异性结合，这表明合成激动剂肽与经蛋白水解暴露的拴系配体在受体结合位点存在直接竞争。5 在内源性表达PAR2的HCT - 15细胞中，加入未标记的配体丝氨酸 - 亮氨酸 - 异亮氨酸 - 甘氨酸 - 赖氨酸 - 缬氨酸（SLIGKV - OH）或2-呋喃甲酰 - LIGRL - NH₂可取代[³H]2-呋喃甲酰 - LIGRL - NH₂的结合。2-呋喃甲酰 - LIGRL - NH₂对SLIGKV - OH的相对结合亲和力与其在钙离子动员测定中的相对EC₅₀值相当。6 结合测定分别在表达PAR2的NCTC2544单层细胞和人脐静脉内皮细胞（HUVEC）中成功进行，分别采用96孔板和24孔板形式。7 这些研究表明[³H]2-呋喃甲酰 - LIGRL - NH₂在其配体结合位点与人PAR2结合。这种放射性配体的使用对于表征与PAR2相互作用的化学物质将具有重要价值。
