Comparison of the functional characteristics of the nucleotide binding domains of multidrug resistance protein 1
Comparison of the functional characteristics of the nucleotide binding domains of multidrug resistance protein 1
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DOI：
10.1074/jbc.275.17.13098
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发表时间：
2000-04-28
影响因子：
4.8
通讯作者：
Deeley, RG
中科院分区：
文献类型：
--
作者：
Gao, M;Cui, HR;Deeley, RG
多药耐药蛋白1（Multidrug Resistance Protein 1，MRP 1）是一种转运多种有机阴离子结合物并对细胞毒性外源性物质具有抗性的蛋白质，含有两个核苷酸结合结构域（NBD），具有ATP结合盒超家族成员的特征，并具有可被某些底物刺激的基础ATP酶活性。目前尚不清楚MRP 1的两个NBD在功能上是否等同。为了研究这个问题，我们使用了一个杆状病毒双表达载体编码的MRP 1的两个一半，以重建一个积极的转运蛋白，并比较了每个NBD的能力与8-叠氮基-[P-32]ATP的光亲和标记和陷阱8-叠氮基[P-32]ADP在原钒酸盐的存在。我们发现，NBD 1优先标记8-叠氮基[P-32]ATP，而捕获8-叠氮基-[P-32]ADP主要发生在NBD 2。虽然在NBD 2处的捕获依赖于MRP 1的两个半部分的共表达，但是当MRP 1的NH 2近端半部分单独表达时以及当NBD 1表达为可溶性多肽时，NBD 1对8-叠氮基-ATP的结合仍然是可检测的。保守的步行者A赖氨酸684的突变或在步行者A和B基序之间产生插入突变消除了NBD 1的结合和NBD 2处8-叠氮基-ADP的所有可检测的捕获。两种突变均使白三烯C-4（LTC 4）转运减少约70%。NBD 2步行者A赖氨酸1333的突变消除了NBD 2对8-叠氮基-ADP的捕获，但与NBD 1中的突变相反，基本上消除了LTC转运活性，而不影响NBD 1与8-叠氮基-[P-32]ATP的标记。
Multidrug Resistance Protein 1 (MRP1) transports diverse organic anionic conjugates and confers resistance to cytotoxic xenobiotics, The protein contains two nucleotide binding domains (NBDs) with features characteristic of members of the ATP-binding cassette superfamily and exhibits basal ATPase activity that can be stimulated by certain substrates. It is not known whether the two NBDs of MRP1 are functionally equivalent. To investigate this question, we have used a baculovirus dual expression vector encoding both halves of MRP1 to reconstitute an active transporter and have compared the ability of each NBD to be photoaffinity-labeled with 8-azido-[P-32]ATP and to trap 8-azido[P-32]ADP in the presence of orthovanadate. We found that NBD1 was preferentially labeled with 8-azido[P-32]ATP, while trapping of 8-azido-[P-32]ADP occurred predominantly at NBD2. Although trapping at NBD2 was dependent on co-expression of both halves of MRP1, binding of 8-azido-ATP by NBD1 remained detectable when the NH2-proximal half of MRP1 was expressed alone and when NBD1 was expressed as a soluble polypeptide. Mutation of the conserved Walker A lysine 684 or creation of an insertion mutation between Walker A and B motifs eliminated binding by NBD1 and all detectable trapping of 8-azido-ADP at NBD2. Both mutations decreased leukotriene C-4 (LTC4) transport by approximately 70%. Mutation of the NBD2 Walker A lysine 1333 eliminated trapping of 8-azido-ADP by NBD2 but, in contrast to the mutations in NBD1, essentially eliminated LTC, transport activity without affecting labeling of NBD1 with 8-azido-[P-32]ATP.