Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes
Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes
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DOI：
10.1074/jbc.m414337200
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发表时间：
2005-04-22
影响因子：
4.8
通讯作者：
Young, JD
中科院分区：
文献类型：
--
作者：
Baldwin, SA;Yao, SYM;Young, JD
第一个哺乳动物的平衡型核苷转运蛋白家族的特点，hENT 1和hENT 2，被动转运主要位于人细胞的质膜。我们现在报告的功能表征的第三个小组的家庭成员，从人类和小鼠，这在其性质上有很大的不同，从以前的哺乳动物核苷转运蛋白。分别命名为hENT 3和mENT 3的475个残基的人和小鼠蛋白质在氨基酸序列上具有73%的同一性，并且具有长的N-末端亲水结构域，其具有典型的（DE）XXXL（LI）内体/溶酶体靶向基序。ENT 3转录物和蛋白质广泛分布于人类和啮齿动物组织中，在胎盘中特别丰富。然而，与ENT 1和ENT 2相反，发现全长hENT 3蛋白的内源性和绿色荧光蛋白标记形式主要是细胞内蛋白，其部分地与培养的人细胞中的溶酶体标志物共定位。截断的亲水性N-末端区域或突变的双亮氨酸基序丙氨酸引起的蛋白质被重新定位到细胞表面在人类细胞和非洲爪蟾卵母细胞，允许其运输活性的表征在后者。该蛋白被证明是一个广泛选择性的，低亲和力的核苷转运蛋白，也可以转运腺嘌呤。转运活性是相对不敏感的经典核苷转运抑制剂nitrobenzylthioinosine，双嘧达莫，地拉卓和钠离子的依赖性。然而，它强烈依赖于pH值，最佳pH值为5.5可能反映了转运蛋白在酸性细胞内隔室中的位置。
The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical ( DE) XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent protein-tagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell surface both in human cells and in Xenopus oocytes, allowing characterization of its transport activity in the latter. The protein proved to be a broad selectivity, low affinity nucleoside transporter that could also transport adenine. Transport activity was relatively insensitive to the classical nucleoside transport inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep and was sodium ion-independent. However, it was strongly dependent upon pH, and the optimum pH value of 5.5 probably reflected the location of the transporter in acidic, intracellular compartments.