Oligomerization Study of Human Organic Anion Transporting Polypeptide 1B1
Oligomerization Study of Human Organic Anion Transporting Polypeptide 1B1
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人有机阴离子转运多肽1B1的寡聚化研究
DOI：
10.1021/acs.molpharmaceut.6b00649
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发表时间：
2017-02-01
影响因子：
4.9
通讯作者：
Hong, Mei
中科院分区：
文献类型：
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作者：
Ni, Chunxu;Yu, Xuan;Hong, Mei
有机阴离子转运多肽在各种内源性和外源性化合物的摄取中起重要作用。已经提出OATP家族成员作为膜蛋白，可以形成寡聚体。然而，OATP的寡聚化状态在很大程度上仍不清楚。在本研究中，产生了稳定表达OATP 1B 1的HEK 293细胞，以研究转运蛋白的寡聚化状态。化学交联和免疫共沉淀实验表明，OATP 1B 1可能通过二硫键形成同源寡聚体。当野生型OATP 1B 1与功能丧失突变体W258 A共表达时，细胞显示原型底物雌酮-3-硫酸酯（ES）的摄取减少。有趣的是，这种共表达并不影响高浓度ES的OATP 1B 1转运活性，这意味着寡聚化状态可能只影响ES的高亲和力组分。OATP 1B 1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，G219 A和G393 A显示出显著降低的摄取功能。进一步的研究表明，G219 A具有与野生型相似的缔合能力，而G1 y393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，G219 A和G393 A都具有显著降低的Vma。对于ES摄取。G219 A对ES结合的高亲和力组分的Km值增加，而G393 A的Km值降低。我们的研究表明，OATP 1B 1可能在ES的高亲和力位点作为寡聚体发挥作用，同时作为底物的低亲和力结合组分的单体发挥作用。
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 G1y393 may affect oligomerization status of the transporter. Kinetic analysis showed that both G219A and G393A have a dramatically reduced Vma. for ES uptake. Km 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.