Lee Young-Min A, Cui Yunhai, König Jörg, Risch Angela, Jäger Birgit, Drings Peter, Bartsch Helmut, Keppler Dietrich, Nies Anne T
Division of Tumour Biochemistry, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany.
Pharmacogenetics. 2004 Apr;14(4):213-23. doi: 10.1097/00008571-200404000-00001.
The human multidrug resistance protein 3 (MRP3, symbol ABCC3) is an ATP-binding cassette transporter that mediates the efflux of organic anions, including lipophilic substances conjugated with glucuronate, sulphate or glutathione, across the basolateral membrane of polarized cells (e.g. hepatocytes) into blood. Genetic variants of MRP3 may affect the transport of these substances out of cells. The aims of this study were: (i) to identify MRP3 polymorphisms; (ii) to functionally characterize one relatively frequent MRP3 polymorphism; and (iii) to establish whether MRP3 transports bilirubin glucuronosides. Exonic nucleotide variants in the ABCC3 gene were identified by single-strand conformation polymorphism analysis. The 3890G>A mutation, resulting in MRP3-ArgHis, was introduced into the ABCC3 cDNA which was stably transfected into MDCKII cells. For the functional characterization of MRP3-ArgHis in comparison with MRP3, ATP-dependent transport was analysed in isolated membrane vesicles. Two non-synonymous MRP3 variants were identified with an allele frequency of 0.003 for 1643T>A (MRP3-LeuGln) and 0.08 for 3890G>A (MRP3-ArgHis). Because of the high frequency of the 3890G>A mutation, and because of the close proximity of Arg to the second nucleotide-binding domain, we pursued the functional characterization of the MRP3-ArgHis polymorphic variant. MRP3-ArgHis was correctly localized to the basolateral membrane of polarized MDCKII cells. We identified monoglucuronosyl bilirubin, bisglucuronosyl bilirubin and leukotriene C4 as substrates for both MRP3 and MRP3-ArgHis. Dehydroepiandrosterone-3-sulphate and 17beta-glucuronosyl oestradiol were transported with similar kinetics by MRP3 and MRP3-ArgHis. This experimental setup provides a useful tool to analyse the functional consequences of polymorphic variants of MRP3.
人类多药耐药蛋白3（MRP3，符号为ABCC3）是一种ATP结合盒转运蛋白，可介导有机阴离子（包括与葡萄糖醛酸、硫酸盐或谷胱甘肽结合的亲脂性物质）通过极化细胞（如肝细胞）的基底外侧膜进入血液。MRP3的基因变异可能会影响这些物质从细胞内的转运。本研究的目的是：（i）鉴定MRP3多态性；（ii）对一种相对常见的MRP3多态性进行功能表征；（iii）确定MRP3是否转运胆红素葡萄糖醛酸苷。通过单链构象多态性分析鉴定ABCC3基因中的外显子核苷酸变异。将导致MRP3-ArgHis的3890G>A突变引入稳定转染到MDCKII细胞中的ABCC3 cDNA中。为了与MRP3相比对MRP3-ArgHis进行功能表征，在分离的膜囊泡中分析了ATP依赖性转运。鉴定出两个非同义MRP3变异体，1643T>A（MRP3-LeuGln）的等位基因频率为0.003，3890G>A（MRP3-ArgHis）的等位基因频率为0.08。由于3890G>A突变的频率较高，且精氨酸与第二个核苷酸结合结构域距离较近，我们对MRP3-ArgHis多态性变异体进行了功能表征。MRP3-ArgHis正确定位于极化MDCKII细胞的基底外侧膜。我们鉴定出单葡萄糖醛酸胆红素、双葡萄糖醛酸胆红素和白三烯C4是MRP3和MRP3-ArgHis的底物。硫酸脱氢表雄酮和17β-葡萄糖醛酸雌二醇由MRP3和MRP3-ArgHis以相似的动力学进行转运。该实验设置为分析MRP3多态性变异体的功能后果提供了一个有用的工具。
