Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein
Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein
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DOI：
10.1073/pnas.94.20.10594
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发表时间：
1997-09-30
影响因子：
11.1
通讯作者：
Ambudkar, SV
中科院分区：
文献类型：
--
作者：
Dey, S;Ramachandra, M;Ambudkar, SV
人类p -糖蛋白（Pgp）通过atp依赖性挤出许多结构不同的疏水化合物，从而赋予对癌细胞的多药耐药性，Pgp识别这些不同底物的方式尚不清楚。该蛋白在其N端和c端半部分之间显示出内部同源性，每个半部分由六个假设的跨膜螺旋和一个一致的ATP结合/利用位点组成，某些Pgp底物的光活性衍生物特异性标记两个区域，每个区域在蛋白质的一半上。在本研究中，使用[I-125]iodoarylazidoprazosin ([I-125]IAAP)，一种吡唑嗪的光活性类似物，我们已经证明了Pgp中存在两个不相同的药物相互作用位点。利用Pgp连接区的胰蛋白酶高度敏感的切割位点，我们表征了[I-125]IAAP与Nand c -末端半部分的结合，顺式(Z)-氟苯替索，Pgp功能调节剂，通过将K-d从20降低到6 nM而优先增加[I-125]IAAP对蛋白质c -末端半部分（c -位点）的亲和力（K-d = 42-46 nM），而不改变n -末端半部分（n -位点）的标记或亲和力（K-d = 42-46 nM）。顺式(Z)-氟哌噻醇使[I-125]IAAP与c位点结合50%的抑制作用所需的vinblastine （Pgp底物）和cyclosporin A （Pgp调节剂）的浓度增加了5- 6倍，而对n位点没有任何影响。此外，[I-125]IAAP与n位点的结合比c位点对钒酸盐的抑制作用更不敏感，钒酸盐阻断ATP水解和药物运输，这些数据表明Pgp中至少存在两个不同的底物相互作用位点。
Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds, The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site, Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein, In this study, using [I-125]iodoarylazidoprazosin ([I-125]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [I-125]IAAP binding to the Nand C-terminal halves, cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [I-125]IAAP for the C-terminal half of the protein (C-site) by reducing the K-d from 20 to 6 nM without changing the labeling or affinity (K-d = 42-46 nM) of the N-terminal half (N-site), Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [I-125]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site, In addition, [I-125]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport, These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.