PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator
PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator
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DOI：
10.1074/jbc.m206964200
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发表时间：
2002-10-18
影响因子：
4.8
通讯作者：
Stanton, BA
中科院分区：
文献类型：
--
作者：
Swiatecka-Urban, A;Duhaime, M;Stanton, BA
CFTR 的 C 末端含有 PDZ 相互作用结构域，这是极化上皮细胞顶端质膜中囊性纤维化跨膜电导调节因子 (CFTR) 的极化表达所必需的。为了阐明 PDZ 相互作用结构域介导 CFTR 极化表达的机制，用野生型 (wt-CFTR) 或 C 端截短的人 CFTR (CFTR-DeltaTRL) 稳定转染 Madin-Darby 犬肾细胞。我们测试了以下假设：PDZ 相互作用结构域调节 CFTR 从高尔基体到顶端质膜的排序。脉冲追踪研究与域选择性细胞表面生物素化相结合表明，新合成的 wt-CFTR 和 CFTR-DeltaTRL 以非极化方式同等地靶向顶膜和基底外侧膜。因此，PDZ 相互作用结构域不是顶端分选基序。 PDZ相互作用结构域的缺失使顶膜中CFTR的半衰期从大约24小时减少到大约13小时，但对基底膜中CFTR的半衰期没有影响。因此，PDZ 相互作用结构域是顶膜保留基序。接下来，我们检验了以下假设：PDZ 相互作用结构域通过改变 CFTR 的内吞作用和/或内吞再循环来影响 CFTR 的顶膜半衰期。 wt-CFTR 和 CFTR-DeltaTRL 的内吞作用没有差异。然而，与 wt-CFTR 相比，CFTR-DeltaTRL 的内吞再循环减少。因此，PDZ相互作用结构域的缺失通过减少CFTR内吞再循环而减少了顶膜中CFTR的半衰期。我们的结果确定了 PDZ 蛋白在调节极化上皮细胞中 CFTR 内吞再循环中的新作用。
The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-DeltaTRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-DeltaTRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from similar to24 to similar to13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-DeltaTRL did not differ. However, endocytic recycling of CFTR-DeltaTRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.