Georges Elias
Institute of Parasitology, McGill University, Quebec H9X 1C0, Canada.
Biochemistry. 2007 Jun 26;46(25):7337-42. doi: 10.1021/bi7006228. Epub 2007 May 27.
P-Glycoprotein (or ABCB1) has been shown to cause multidrug resistance in tumor cell lines selected with lipophilic anticancer drugs. ABCB1 encodes a duplicated molecule with two hydrophobic and hydrophilic domains linked by a highly charged region of approximately 90 amino acids, the "linker domain" with as yet unknown function(s). In this report, we demonstrate a role for this domain in binding to other cellular proteins. Using overlapping hexapeptides that encode the entire amino acid sequence of the linker domain of human ABCB1, we show a direct and specific binding between sequences in the linker domain and several intracellular proteins. Three different polypeptide sequences [617EKGIYFKLVTM627 (LDS617-627), 657SRSSLIRKRSTRRSVRGSQA676 (LDS657-676), and 693PVSFWRIMKLNLT705 (LDS693-705)] in the linker domain interacted tightly with several proteins with apparent molecular masses of approximately 80, 57, and 30 kDa. Interestingly, only the 57 kDa protein (or P57) interacted with all three different sequences of the linker domain. Purification and partial N-terminal amino acid sequencing of P57 showed that it encodes the N-terminal amino acids of alpha- and beta-tubulins. The identity of the P57 interacting protein as tubulins was further confirmed by Western blotting using monoclonal antibodies to alpha- and beta-tubulin. Taken together, the results of this study provide the first evidence for ABCB1 protein interaction mediated by sequences in the linker domain. These findings are likely to provide further insight into the functions of ABCB1 in normal and drug resistant tumor cells.
P-糖蛋白（或ABCB1）已被证明在选用亲脂性抗癌药物筛选的肿瘤细胞系中会导致多药耐药。ABCB1编码一种重复分子，该分子具有两个疏水和亲水结构域，由大约90个氨基酸的高电荷区域连接，即功能尚不清楚的“连接子结构域”。在本报告中，我们证明了该结构域在与其他细胞蛋白结合中的作用。使用编码人ABCB1连接子结构域完整氨基酸序列的重叠六肽，我们展示了连接子结构域中的序列与几种细胞内蛋白之间的直接和特异性结合。连接子结构域中的三种不同多肽序列[617EKGIYFKLVTM627（LDS617 - 627）、657SRSSLIRKRSTRRSVRGSQA676（LDS657 - 676）和693PVSFWRIMKLNLT705（LDS693 - 705）]与几种表观分子量约为80、57和30 kDa的蛋白紧密相互作用。有趣的是，只有57 kDa的蛋白（或P57）与连接子结构域的所有三种不同序列相互作用。P57的纯化和部分N端氨基酸测序表明它编码α-和β-微管蛋白的N端氨基酸。使用针对α-和β-微管蛋白的单克隆抗体进行蛋白质印迹进一步证实了与P57相互作用的蛋白为微管蛋白。综上所述，本研究结果为连接子结构域中的序列介导的ABCB1蛋白相互作用提供了首个证据。这些发现可能会进一步深入了解ABCB1在正常和耐药肿瘤细胞中的功能。
