Demeule M, Wenger R M, Béliveau R
Laboratoire d'Oncologie Moléculaire, Département de Chimie-biochimie, Université du Québec-Hopital Ste-Justine, Montréal, Québec H3C 3P8, Canada.
J Biol Chem. 1997 Mar 7;272(10):6647-52. doi: 10.1074/jbc.272.10.6647.
The interaction between P-glycoprotein (140-180 kDa) from the multidrug-resistant Chinese hamster ovary cell line CHRC5 and cyclosporin A was characterized using three different photoactivable cyclosporin A analogs. Two monoclonal antibodies, which are able to discriminate between two major domains of cyclosporin A (the cyclophilin and calcineurin binding domains), were used to detect the photolabeled proteins. A protein of 155 kDa corresponding to P-glycoprotein was much more strongly photolabeled in membranes of CHRC5 cells than in membranes of their drug-sensitive parent cell line AuxB1. The antitumor drug vinblastine and the reversal agents verapamil and cyclosporin A inhibited the photolabeling, and the nonimmunosuppressive derivative PSC-833 caused a stronger inhibition than cyclosporin A. P-glycoprotein photolabeled with cyclosporin A analogs was only detected with the monoclonal antibody that recognizes cyclosporin A and its metabolites, indicating that the calcineurin binding domain recognized specifically by the other antibody is not exposed. These results suggest that the portion of cyclosporin A that binds to calcineurin plays a role in the interaction of cyclosporin A with P-glycoprotein.
使用三种不同的可光活化环孢菌素A类似物，对多药耐药的中国仓鼠卵巢细胞系CHRC5中的P-糖蛋白（140 - 180 kDa）与环孢菌素A之间的相互作用进行了表征。使用两种单克隆抗体来检测光标记的蛋白质，这两种抗体能够区分环孢菌素A的两个主要结构域（亲环蛋白结合结构域和钙调神经磷酸酶结合结构域）。与P-糖蛋白相对应的155 kDa蛋白质在CHRC5细胞的膜中比在其药物敏感的亲本细胞系AuxB1的膜中被更强地光标记。抗肿瘤药物长春碱以及逆转剂维拉帕米和环孢菌素A抑制了光标记，并且非免疫抑制衍生物PSC - 833比环孢菌素A产生更强的抑制作用。用环孢菌素A类似物光标记的P-糖蛋白仅用识别环孢菌素A及其代谢物的单克隆抗体检测到，这表明另一种抗体特异性识别的钙调神经磷酸酶结合结构域未暴露。这些结果表明，环孢菌素A与钙调神经磷酸酶结合的部分在环孢菌素A与P-糖蛋白的相互作用中起作用。
