Ni Chunxu, Yu Xuan, Fang Zihui, Huang Jiujiu, Hong Mei
College of Life Sciences, South China Agricultural University , Guangzhou 541642, China.
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms , Guangzhou 541642, China.
Mol Pharm. 2017 Feb 6;14(2):359-367. doi: 10.1021/acs.molpharmaceut.6b00649. Epub 2017 Jan 12.
Organic anion-transporting polypeptides play important roles in the uptake of various endogenous and exogenous compounds. It has been proposed that OATP family members, as membrane proteins, may form oligomers. However, oligomerization status of OATPs is still largely unclear. In the present study, HEK293 cells stably expressing OATP1B1 were generated to investigate the oligomerization status of the transporter. Chemical cross-linking and coimmunoprecipitation experiments revealed that OATP1B1 may form homo-oligomers, possibly through disulfide bonds. When wild-type OATP1B1 was coexpressed with a loss-of-function mutant W258A, cells showed reduced uptake of prototypic substrate estrone-3-sulfate (ES). Interestingly, such a coexpression did not affect OATP1B1 transport activity of high concentrations ES, implicating that oligomerization status may affect only the high affinity component of ES. OATP1B1 possesses three GXXXG motifs that have been associated with protein dimerization in other membrane proteins. When glycine residues were replaced with alanine, G219A and G393A showed drastically reduced uptake function. Further studies revealed that G219A has a similar association capability to that of the wild-type, while mutation at Gly393 may affect oligomerization status of the transporter. Kinetic analysis showed that both G219A and G393A have a dramatically reduced V for ES uptake. K of G219A was increased while that of G393A exhibited a decreased value for high affinity component of ES binding. Our studies demonstrated that OATP1B1 may function as oligomers in the high affinity site of ES while acting as monomers for the low affinity binding component of the substrate.
有机阴离子转运多肽在多种内源性和外源性化合物的摄取中发挥着重要作用。有人提出，作为膜蛋白的有机阴离子转运多肽（OATP）家族成员可能形成寡聚体。然而，OATP的寡聚化状态仍不清楚。在本研究中，构建了稳定表达OATP1B1的人胚肾293（HEK293）细胞，以研究该转运蛋白的寡聚化状态。化学交联和免疫共沉淀实验表明，OATP1B1可能形成同源寡聚体，可能通过二硫键。当野生型OATP1B1与功能缺失突变体W258A共表达时，细胞对原型底物硫酸雌酮（ES）的摄取减少。有趣的是，这种共表达并不影响OATP1B1对高浓度ES的转运活性，这表明寡聚化状态可能仅影响ES的高亲和力成分。OATP1B1具有三个GXXXG基序，这些基序与其他膜蛋白中的蛋白质二聚化有关。当甘氨酸残基被丙氨酸取代时，G219A和G393A的摄取功能大幅降低。进一步研究表明，G219A与野生型具有相似的结合能力，而Gly393处的突变可能影响转运蛋白的寡聚化状态。动力学分析表明，G219A和G393A对ES摄取的Vmax均显著降低。G219A的Km增加，而G393A对ES结合高亲和力成分的Km值降低。我们研究表明，OATP1B1在ES的高亲和力位点可能以寡聚体形式发挥作用，而在底物的低亲和力结合成分方面则以单体形式发挥作用。
