<br>OATP1B3 表达和功能通过与 OCT1、OATP1B1 和 NTCP 的共表达进行调节。,Drug Metabolism and Disposition

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OATP1B3 表达和功能通过与 OCT1、OATP1B1 和 NTCP 的共表达进行调节。
Drug Metabolism and Disposition ( IF 4.4 ) Pub Date : 2020-06-01 , DOI: 10.1124/dmd.119.089847
Yuchen Zhang ₁ , Melissa Ruggiero ₁ , Bruno Hagenbuch ₂

Affiliation

有机阴离子转运多肽（OATP） 1B3 是一种在人肝细胞基底外侧膜上表达的药物转运蛋白。与其他转运蛋白一起，包括 OATP1B1、Na +/牛磺胆酸盐共转运多肽（NTCP）和有机阳离子转运蛋白（OCT） 1，它负责将内生素和外源性物质摄取到肝细胞中。我们以前的研究表明，OATP1B3可以在人胚胎肾 293T （HEK293）细胞中形成带有 OATP1B1 的异源寡聚体，在 HEK293 细胞和冷冻人肝切片中与 NTCP 形成异源寡聚体。为了进一步表征 OATP1B3 的异源寡聚化，我们研究了 OCT1 作为潜在的相互作用伙伴，并确定了 OATP1B3异源寡聚化的功能后果。我们通过与来自人肝细胞的抗 OATP1B3 抗体进行免疫共沉淀来证明 OATP1B3 和 OCT1 之间的相互作用。此外，我们使用邻位连接测定法在 HEK293 细胞和冷冻人肝切片中可视化了相互作用。我们通过在 OATP1B1、 NTCP 和 OCT1 存在的情况下测量OATP1B3质膜表达和OATP1B3选择性底物胆囊收缩素-8 （CCK-8）的摄取，研究了OATP1B3异源寡聚化的功能后果。与 OCT1 共表达后观察到 OATP1B3 质膜表达显著降低，而与 OATP1B1 或 NTCP 共表达导致质膜表达增加。在转运方面，OCT1 的共表达增加了 OATP1B3 的表观周转率，而 OATP1B1 或 NTCP 的共表达降低了它的表观周转率。这些发现表明，OATP1B3 与 OATP1B1、NTCP 和 OCT1 在 HEK293 细胞中的共表达导致 OATP1B3 介导的 CCK-8 转运的转运蛋白依赖性修饰，并表明在过表达细胞系的单个转运蛋白中获得的功能结果高估或低估了OATP1B3人肝细胞中的功能。意义声明：有机阴离子转运多肽（OATP） 1B3 与有机阳离子转运蛋白（OCT） 1、Na + /牛磺胆酸盐共转运多肽或 OATP1B1 在人胚胎肾 293T 细胞中的共表达会影响其表达水平和功能。当 OCT1 在人肝细胞中被敲低时，OATP1B3 的功能会上升。这些结果表明，蛋白质-蛋白质相互作用会影响相关蛋白质的表达和功能，因此单个转运蛋白表达系统可能导致药物-药物相互作用的高估或低估。

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OATP1B3 Expression and Function is Modulated by Coexpression with OCT1, OATP1B1, and NTCP.

Organic anion transporting polypeptide (OATP) 1B3 is a drug transporter expressed at the basolateral membrane of human hepatocytes. Along with other transporters, including OATP1B1, Na+/taurocholate cotransporting polypeptide (NTCP), and organic cation transporter (OCT) 1, it is responsible for the uptake of endo- and xenobiotics into hepatocytes. Our previous studies demonstrated that OATP1B3 can form hetero-oligomers with OATP1B1 in human embryonic kidney 293T (HEK293) cells and with NTCP in both HEK293 cells and frozen human liver sections. To further characterize the hetero-oligomerization of OATP1B3, we investigated OCT1 as a potential interacting partner and determined the functional consequences of OATP1B3 hetero-oligomerization. We demonstrated interactions between OATP1B3 and OCT1 by coimmunoprecipitation with an anti-OATP1B3 antibody from human hepatocytes. In addition, we visualized the interaction using the proximity ligation assay in both HEK293 cells and in frozen human liver sections. We investigated the functional consequences of OATP1B3 hetero-oligomerization by measuring the OATP1B3 plasma membrane expression and the uptake of the OATP1B3 selective substrate cholecystokinin-8 (CCK-8) in the absence and presence of OATP1B1, NTCP, and OCT1. A significant decrease of OATP1B3 plasma membrane expression was observed after coexpression with OCT1, whereas coexpression with OATP1B1 or NTCP resulted in an increase of plasma membrane expression. With respect to transport, coexpression of OCT1 increased the apparent turnover rate of OATP1B3, whereas coexpression of OATP1B1 or NTCP decreased it. These findings demonstrated that coexpression of OATP1B3 with OATP1B1, NTCP, and OCT1 in HEK293 cells results in a transporter-dependent modification of OATP1B3-mediated CCK-8 transport and suggest that functional results obtained in single transporter overexpressing cell lines over- or underestimate OATP1B3 function in human hepatocytes. SIGNIFICANCE STATEMENT: Coexpression of organic anion transporting polypeptide (OATP) 1B3 with organic cation transporter (OCT) 1, Na+/taurocholate cotransporting polypeptide, or OATP1B1 in human embryonic kidney 293T cells affects its expression level and function. When OCT1 is knocked down in human hepatocytes, function of OATP1B3 goes up. These results suggest that protein-protein interactions can affect the expression and function of the involved proteins, and thus single transporter expression systems might lead to over- or underestimation of drug-drug interactions.

更新日期：2020-06-01

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