Conserved amino acids in the region connecting membrane spanning domain 1 to nucleotide binding domain 1 are essential for expression of the MRP1 (ABCC1) transporter.
Conserved amino acids in the region connecting membrane spanning domain 1 to nucleotide binding domain 1 are essential for expression of the MRP1 (ABCC1) transporter.
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DOI：
10.1371/journal.pone.0246727
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Cole SPC
中科院分区：
文献类型：
--
作者：
Smith EE;Conseil G;Cole SPC
多药耐药蛋白1（MRP 1）（基因符号ABCC 1）是一种ATP结合盒（ABC）转运蛋白，可排出外源性和内源性有机阴离子，包括雌二醇葡糖苷酸和促炎性白三烯C4。MRP 1还通过主动外排减少细胞内药物蓄积而产生多药耐药性。MRP 1具有三个跨膜结构域（MSD）和两个核苷酸结合结构域（NBD）。MSD 1和MSD 2分别通过连接区域（CR）1和CR 2与NBD 1和NBD 2连接。在此，我们靶向CR 1中的四个残基（Ser 612、Arg 615、His 622、Glu 624）进行丙氨酸取代，并且出乎意料地发现，转染的HEK细胞中三种突变体（S612 A、R615 A、E624 A）的细胞水平显著低于野生型MRP 1。尽管CR 1-H622 A适当地运输到质膜并表现出与野生型MRP 1相当的有机阴离子转运活性，但表达差的R615 A和E624 A（以及在较小程度上的S612 A）突变蛋白保留在细胞内。MRP 1的低温电子显微镜和原子同源性模型的分析表明，Arg 615和Glu 624可能参与与附近残基的键合相互作用，以稳定转运蛋白的表达。然而，这并不支持双交换突变E624 K/K406 E，R615 D/D430 R和R615 F/F619 R，这些突变未能改善MRP 1水平。然而，这些实验表明，MSD 1的第一个胞质环中高度保守的CR 1-Phe 619和远端Lys 406也是MRP 1蛋白表达所必需的。这项研究首次证明CR 1含有几个对MRP 1质膜表达至关重要的高度保守的残基，但到目前为止，目前可用的结构和模型没有提供对潜在机制的任何见解。需要具有严格的生化验证数据的额外结构，以充分理解对这种临床重要的ABC转运蛋白的稳定表达至关重要的键合相互作用。
Multidrug resistance protein 1 (MRP1) (gene symbol ABCC1) is an ATP-binding cassette (ABC) transporter which effluxes xeno- and endobiotic organic anions including estradiol glucuronide and the pro-inflammatory leukotriene C4. MRP1 also confers multidrug resistance by reducing intracellular drug accumulation through active efflux. MRP1 has three membrane spanning domains (MSD), and two nucleotide binding domains (NBD). MSD1 and MSD2 are linked to NBD1 and NBD2 by connecting regions (CR) 1 and CR2, respectively. Here we targeted four residues in CR1 (Ser612, Arg615, His622, Glu624) for alanine substitution and unexpectedly, found that cellular levels of three mutants (S612A, R615A, E624A) in transfected HEK cells were substantially lower than wild-type MRP1. Whereas CR1-H622A properly trafficked to the plasma membrane and exhibited organic anion transport activity comparable to wild-type MRP1, the poorly expressing R615A and E624A (and to a lesser extent S612A) mutant proteins were retained intracellularly. Analyses of cryogenic electron microscopic and atomic homology models of MRP1 indicated that Arg615 and Glu624 might participate in bonding interactions with nearby residues to stabilize expression of the transporter. However, this was not supported by double exchange mutations E624K/K406E, R615D/D430R and R615F/F619R which failed to improve MRP1 levels. Nevertheless, these experiments revealed that the highly conserved CR1-Phe619 and distal Lys406 in the first cytoplasmic loop of MSD1 are also essential for expression of MRP1 protein. This study is the first to demonstrate that CR1 contains several highly conserved residues critical for plasma membrane expression of MRP1 but thus far, currently available structures and models do not provide any insights into the underlying mechanism(s). Additional structures with rigorous biochemical validation data are needed to fully understand the bonding interactions critical to stable expression of this clinically important ABC transporter.
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