Dent G, Barnes P J
Department of Thoracic Medicine, National Heart and Lung Institute, London, U.K.
Life Sci. 1993;52(20):1633-40. doi: 10.1016/0024-3205(93)90045-5.
The platelet activating factor (PAF) receptor of the guinea pig peritoneal eosinophil was characterized by radioligand ([3H]WEB 2086) binding and measurement of PAF-stimulated membrane GTPase activity. Specific binding of [3H]WEB 2086 was rapid, reversible and saturable, with an equilibrium KD of 20.4 nM at 0 degrees C, and was displaced competitively by unlabelled PAF with a KI of 3.26 nM and a pseudo-Hill coefficient significantly less than unity (0.44). The affinity of PAF for these binding sites was reduced by the nonhydrolysable GTP analogue 5'-guanylylimidodiphosphate (GppNHp), suggesting the coupling of PAF receptors to intracellular effectors through a guanine nucleotide-binding protein (G protein). PAF stimulated a membrane-associated GTPase, indicating the formation of a G protein alpha subunit-GTP complex upon agonist occupation of the PAF receptor. The EC50 for PAF stimulation was 25.5 nM and the Hill coefficient was significantly less than unity (0.56), while the response to 1 microM PAF was antagonised by WEB 2086 with an IC50 of 128 nM and a slope factor not significantly different from unity (0.91), suggesting the coupling of multiple classes of PAF receptors to the G protein. The activation of GTPase by PAF was insensitive to inhibition by cholera toxin; basal GTPase activity was increased by pertussis toxin and no further stimulation was attainable with PAF.
通过放射性配体（[3H]WEB 2086）结合以及对PAF刺激的膜GTP酶活性的测定，对豚鼠腹腔嗜酸性粒细胞的血小板活化因子（PAF）受体进行了表征。[3H]WEB 2086的特异性结合迅速、可逆且具有饱和性，在0℃时平衡解离常数KD为20.4 nM，未标记的PAF可竞争性取代，其抑制常数KI为3.26 nM，伪希尔系数显著小于1（0.44）。不可水解的GTP类似物5'-鸟苷酰亚胺二磷酸（GppNHp）降低了PAF对这些结合位点的亲和力，提示PAF受体通过鸟嘌呤核苷酸结合蛋白（G蛋白）与细胞内效应器偶联。PAF刺激一种膜相关的GTP酶，表明激动剂占据PAF受体后形成了G蛋白α亚基-GTP复合物。PAF刺激的半数有效浓度（EC50）为25.5 nM，希尔系数显著小于1（0.56），而1 μM PAF的反应被WEB 2086拮抗，半数抑制浓度（IC50）为128 nM，斜率因子与1无显著差异（0.91），提示多种类型的PAF受体与G蛋白偶联。PAF对GTP酶的激活对霍乱毒素的抑制不敏感；百日咳毒素增加了基础GTP酶活性，PAF无法进一步刺激其活性。
