Structural Determinants of Substrate Specificity Differences between Human Multidrug Resistance Protein (MRP) 1 (ABCC1) and MRP3 (ABCC3)
Structural Determinants of Substrate Specificity Differences between Human Multidrug Resistance Protein (MRP) 1 (ABCC1) and MRP3 (ABCC3)
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DOI：
10.1124/dmd.108.022491
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发表时间：
2008-12-01
影响因子：
3.9
通讯作者：
Deeley, Roger G.
中科院分区：
文献类型：
--
作者：
Grant, Caroline E.;Gao, Mian;Deeley, Roger G.
多药耐药蛋白(MRPs)是三磷酸腺苷结合盒转运体超家族的“C”分支成员。人MRP1转运多种天然产物药物和结构多样的共轭和非共轭有机阴离子。它的近亲是MRP3。尽管它们的结构相似，但同系物在底物特异性上有很大的不同。值得注意的是，MRP1运输谷胱甘肽(GSH)和GSH共轭化合物，并显示GSH刺激的许多未结合和共轭化合物的运输。相反，MRP3不转运GSH，是一个很差的GSH结合物转运体。然而，这两种蛋白质都运输葡萄糖醛酸苷结合物，如17β-雌二醇17-(β-D-葡萄糖醛酸苷)。我们已经构建了一系列MRP1/MRP3杂交体，并用它们来鉴定MRP1的一个区域，该区域对GSH偶联物如白三烯C-4(LTC4)的结合和运输至关重要。这个包含跨膜螺旋8和9以及部分MRP1细胞质环4和5的区域被MRP3的等价区取代，消除了LTC4的转运。其他底物的转运不受影响或增强。我们在该区域发现了三个残基：Tyr(440)，Ile(441)和Met(443)，它们的突变对运输有不同的影响。值得注意的是，如MRP3中发现的那样，用Phe取代Tyr(440)，减少了LTC4和GSH刺激的雌酮-3-硫酸酯的转运，而不影响其他被测底物的转运。该突变使[H-3]LTC4和谷胱甘肽衍生物叠氮苯酰基-[S-35]谷胱甘肽对MRP1光标记的K-m增加5倍。这些结果表明，Tyr(440)对GSH的识别和LTC4等共轭化合物的GSH部分的识别做出了重要贡献。
Multidrug resistance proteins (MRPs) are members of the "C" branch of the ATP-binding cassette transporter superfamily. Human MRP1 transports a wide range of natural product drugs and structurally diverse conjugated and unconjugated organic anions. Its closest relative is MRP3. Despite their structural similarity, the homologs differ substantially in their substrate specificity. It is noteworthy that MRP1 transports glutathione (GSH) and GSH conjugates and displays GSH-stimulated transport of a number of unconjugated and conjugated compounds. In contrast, MRP3 does not transport GSH and is a poor transporter of GSH conjugates. However, both proteins transport glucuronide conjugates, such as 17 beta-estradiol 17-(beta-D-glucuronide). We have constructed a series of MRP1/MRP3 hybrids and used them to identify a region of MRP1 that is critical for binding and transport of GSH conjugates such as leukotriene C-4 (LTC4). Substitution of this region encompassing transmembrane helices 8 and 9 and portions of cytoplasmic loops 4 and 5 of MRP1 with the equivalent region of MRP3 eliminated LTC4 transport. Transport of other substrates was either unaffected or enhanced. We identified three residues in this region: Tyr(440), Ile(441), and Met(443), mutation of which differentially affected transport. It is noteworthy that substitution of Tyr(440) with Phe, as found in MRP3, reduced LTC4 and GSH-stimulated estrone-3-sulfate transport without affecting transport of other substrates tested. The mutation increased the K-m for LTC4 5-fold and substantially reduced photolabeling of MRP1 by both [H-3] LTC4 and the GSH derivative, azidophenacyl-[S-35] GSH. These results suggest that Tyr(440) makes a major contribution to recognition of GSH and the GSH moiety of conjugates such as LTC4.