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首页> 外文期刊>American Journal of Physiology >Role of Na~+/H~+ exchanger regulatory factor 1 in forward trafficking of the type IIa Na~+-P_i cotransporter
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Role of Na~+/H~+ exchanger regulatory factor 1 in forward trafficking of the type IIa Na~+-P_i cotransporter

机译：Na〜+ / h〜+交换器调节因子1在归属于IIA NA〜+ -P_ICOTRANSPORTER转发的作用

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摘要

Na~+/H~+ exchanger regulatory factor (NHERF1) plays a critical role in the renal transport of phosphate by binding to Na~+-P_i cotransporter (NpT2a) in the proximal tubule. While the association between NpT2a and NHERF1 in the apical membrane is known, the role of NHERF1 to regulate the trafficking of NpT2a has not been studied. To address this question, we performed cell fractionation by sucrose gradient centrif-ugation in opossum kidney (OK) cells placed in low-Pi medium to stimulate forward trafficking of NpT2a. Immunoblot analysis demonstrated expression of NpT2a and NHERF1 in the endoplasmic retic-ulum (ER)/Golgi. Coimmunoprecipitation demonstrated a NpT2a-NHERF1 interaction in the ER/Golgi. Low-P_i medium for 4 and 8 h triggered a decrease in NHERF1 in the plasma membrane with a corresponding increase in the ER/Golgi. Time-lapse total internal reflection fluorescence imaging of OK cells placed in low-Pi medium, paired with particle tracking and mean square displacement analysis, indicated active directed movement of NHERF1 at early and late time points, whereas NpT2a showed active movement only at later times. Silence of NHERF1 in OK cells expressing green fluorescent protein (GFP)-NpT2a resulted in an intracellular accumulation of GFP-NpT2a. Transfection with GFP-labeled COOH-terminal (TRL) PDZ-binding motif deleted or wild-type NpT2a in OK cells followed by cell fractionation and immunoprecipitation confirmed that the interaction between NpT2a and NHERF1 was dependent on the TRL motif of NpT2a. We conclude that appropriate trafficking of NpT2a to the plasma membrane is dependent on the initial association between NpT2a and NHERF1 through the COOH-terminal TRL motif of NpT2a in the ER/Golgi and requires redistribution of NHERF1 to the ER/Golgi.
机译：Na〜+ / H〜+交换器调节因子（NHERF1）通过在近端小管中结合Na〜+ -P_I Cotroanger（NPT2A），在磷酸盐的肾传输中起重要作用。虽然已知NPT2A和NHERF1之间的关联是已知的，但尚未研究NHERF1调节贩运NPT2A的作用。为了解决这个问题，我们通过在低PI培养基中置于低PI培养基中的挥发肾（OK）细胞中的蔗糖梯度离心 - ugation进行细胞分馏，刺激NPT2A的前向运输。免疫斑分析证明了内质视网膜（ER）/ Golgi中的NPT2a和nherf1的表达。 Coimmunopropectipitation在ER / GOLGI中表现出NPT2A-NHERF1相互作用。 4和8 H的低P_I培养基触发了浆液中的骨骼膜中的降低，同时ER / GOLGI的相应增加。延时间隔全内反射荧光成像在低pi培养基中置于低pi培养基，与粒子跟踪和均方位移分析配对，在早期和晚期时间点表明了nherf1的主动定向运动，而NPT2A仅在后面的时间显示主动运动。表达绿色荧光蛋白（GFP）-NPT2A的OK细胞中的NHERF1沉默导致GFP-NPT2A的细胞内积累。用GFP标记的CoOH-末端（TR1）转染在OK细胞中缺失或野生型NPT2A，然后进行细胞分级和免疫沉淀，证实NPT2A和NHERF1之间的相互作用取决于NPT2A的TRL基序。我们得出结论，对血浆膜的适当贩运NPT2A依赖于NPT2A和NHERF1之间的初始关联通过ER / GOLGI中NPT2A的COOH-末端TRL基序，并且需要再分布NHERF1至ER / GOLGI。

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