The transmembrane domains of the human multidrug resistance P-glycoprotein are sufficient to mediate drug binding and trafficking to the cell surface
The transmembrane domains of the human multidrug resistance P-glycoprotein are sufficient to mediate drug binding and trafficking to the cell surface
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DOI：
10.1074/jbc.274.35.24759
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发表时间：
1999-08-27
影响因子：
4.8
通讯作者：
Clarke, DM
中科院分区：
文献类型：
--
作者：
Loo, TW;Clarke, DM
人类多药耐药性 P-糖蛋白 (P-gp) 由两个串联重复序列组成，每个重复序列由一个包含六个潜在跨膜片段的 N 端疏水结构域和一个包含核苷酸结合折叠的亲水结构域组成。使用一系列缺失突变体和体内药物结合测定来测试缺失突变体是否与底物相互作用或被转运到细胞表面。我们发现仅由跨膜结构域（残基 1-379 加 681-1025）组成的缺失突变体保留了与药物底物相互作用的能力。在没有药物底物的情况下，缺失突变体对胰蛋白酶和内切糖苷酶 H 敏感。然而，在维拉帕米、长春花碱、辣椒素或环孢菌素 A 存在的情况下表达，会产生对胰蛋白酶和内切糖苷酶 H 具有抗性的突变蛋白。然后在细胞表面检测到该突变体，并且对内切糖苷酶 F 的消化敏感。相比之下，N 端跨膜结构域（残基1-379）单独不与药物底物相互作用，因为它仅对糖苷内切酶 H 敏感并且在细胞表面未检测到。这些结果表明，P-gp 与底物相互作用或 P-gp 运输至细胞表面不需要核苷酸结合结构域。
The human multidrug resistance P-glycoprotein (P-gp) is organized in two tandem repeats with each repeat consisting of an N-terminal hydrophobic domain containing six potential transmembrane segments followed by a hydrophilic domain containing a nucleotide-binding fold. A series of deletion mutants together with an in vivo drug-binding assay were used to test whether the deletion mutants interacted with substrates or were transported to the cell surface. We found that a deletion mutant consisting of only the transmembrane domains (residues 1-379 plus 681-1025) retained the ability to interact with drug substrates. In the absence of drug substrates, the deletion mutant was sensitive to trypsin and endoglycosidase H. Expression in the presence of verapamil, vinblastine, capsaicin, or cyclosporin A, however, resulted in a mutant protein that was resistant to trypsin and endoglycosidase H. The mutant was then detected at the cell surface and was sensitive to digestion by endoglycosidase F, By contrast, the N-terminal transmembrane domain (residues 1-379) alone did not interact with drug substrates, since it was sensitive to only endoglycosidase H and was not detected at the cell surface. These results show that the nucleotide-binding domains are not required for interaction of P-gp with substrate or for trafficking of P-gp to the cell surface.