The intracellular NPxY motif is critical in maintaining the function and expression of human organic anion transporting polypeptide 1B1
The intracellular NPxY motif is critical in maintaining the function and expression of human organic anion transporting polypeptide 1B1
复制标题
细胞内 NPxY 基序对于维持人有机阴离子转运多肽 1B1 的功能和表达至关重要
DOI：
10.1016/j.bbamem.2019.04.001
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发表时间：
2019-06-01
影响因子：
3.4
通讯作者：
Hong, Mei
中科院分区：
文献类型：
--
作者：
Wang, Xuyang;Liang, Yushuang;Hong, Mei
有机阴离子转运多肽（OATP，基因符号SLCO）介导内源性化合物如胆汁盐、激素及其缀合物以及毒素和药物的钠非依赖性转运。OATP 1B 1是在人肝细胞基底外侧膜特异性表达的主要OATP，许多临床重要药物已被证明是转运蛋白的底物。通过计算机亲水性分析发现，OATP的跨膜结构域6和7之间存在一个较大的胞内环3（IL 3），其中存在一个保守的NPxY基序。本研究利用表达HA标记的OATP 1B 1的HEK 293细胞，研究NPxY基序对OATP 1B 1转运蛋白功能和表达的影响。丙氨酸取代N335或P336保留了大量的摄取功能，而这些残基的同时突变导致几乎失去所有的运输活性的双突变体。另一方面，Y338 A显示雌酮-3-硫酸盐摄取减少> 80%。质膜蛋白分析显示N335/P336 A完全丧失其细胞表面蛋白的表达，而Y338 A的表达显著降低。进一步的药理学抑制剂和免疫细胞化学研究表明，N335/336 A被滞留在高尔基体和Y338 A表现出加速蛋白质降解速率相比，野生型。用苯丙氨酸保守替换Y338完全恢复了转运蛋白的摄取和表达。总之，观察到位于OATP 1B 1的IL 3中的NPxY基序的新作用，其可能影响转运蛋白的加工和稳定性。
Organic anion transporting polypeptides (OATPs, gene symbol SLCO) mediate sodium-independent transport of endogenous compounds such as bile salts, hormones and their conjugates as well as toxins and drugs. OATP1B1 is the major OATP specifically expressed at the basolateral membrane of human hepatocytes and many clinically important drugs have been shown to be substrates of the transporter. According to the computer-based hydropathy analysis, a large intracellular loop 3 (IL3) is situated between transmembrane domain 6 and 7 of OATPs, in which a conserved NPxY motif is found. In the current study, HEK293 cells expressing the HA-tagged OATP1B1 was utilized to investigate the role of the NPxY motif for the function and expression of the transporter. Alanine replacement of N335 or P336 retained substantial uptake function; while simultaneous mutation of these residues resulted in a double mutant that lost almost all the transport activity. On the other hand, Y338A showed > 80% reduction for estrone-3-sulfate uptake. Plasma membrane protein analysis revealed that N335/P336A completely lost its cell surface protein expression; while that of Y338A is dramatically reduced. Further investigation with pharmacological inhibitors and immunocytochemistry demonstrated that N335/336A is detained in the Golgi apparatus and Y338A exhibited accelerated protein degradation rate compared to that of the wild-type. Conservative replacement of Y338 with phenylalanine fully recovered uptake and expression of the transporter. In summary, a new role was observed for the NPxY motif located in the IL3 of OATP1B1, which may affect processing and stability of the transporter.