Loo T W, Clarke D M
Medical Research Council Group in Membrane Biology, Department of Medicine, University of Toronto, Ontario M5S 1A8, Canada.
J Biol Chem. 1999 Aug 27;274(35):24759-65. doi: 10.1074/jbc.274.35.24759.
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.
人类多药耐药性P-糖蛋白（P-gp）由两个串联重复序列组成，每个重复序列包含一个N端疏水区，该疏水区含有六个潜在的跨膜片段，随后是一个含有核苷酸结合结构域的亲水区。一系列缺失突变体与体内药物结合试验一起用于测试缺失突变体是否与底物相互作用或是否转运到细胞表面。我们发现，仅由跨膜结构域组成的缺失突变体（残基1-379加681-1025）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、长春碱、辣椒素或环孢素A存在的情况下表达，会产生一种对胰蛋白酶和内切糖苷酶H有抗性的突变蛋白。然后在细胞表面检测到该突变体，并且它对内切糖苷酶F的消化敏感。相比之下，单独的N端跨膜结构域（残基1-379）不与药物底物相互作用，因为它仅对内切糖苷酶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转运到细胞表面不需要核苷酸结合结构域。
