Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells
Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells
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DOI：
10.1091/mbc.e08-01-0084
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发表时间：
2009-04-15
影响因子：
3.3
通讯作者：
Bradbury, Neil A.
中科院分区：
文献类型：
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作者：
Silvis, Mark R.;Bertrand, Carol A.;Bradbury, Neil A.
囊性纤维化跨膜电导调节器(CFTR)是cAMP/PKA激活的阴离子通道，在极化上皮细胞中经历有效的顶端循环。CFTR循环的调控机制目前知之甚少，但这一过程是在顶膜上获得合适的通道拷贝数所必需的，而且在常见的CFTR突变体Delta F508中存在缺陷。在这里，我们研究了Rab11亚型在T84细胞中调节CFTR运输的功能，T84细胞是一种内源性表达CFTR的结肠上皮系。免疫分离的Rab11a或Rab11b小泡的Western blotting显示内源性CFTR在两个隔室中都有定位。对表达Rab11a或Rab11b GDP锁定的S25N突变体的T84细胞的CFTR功能分析表明，只有Rab11b突变体抑制了cAMP激活的卤化物外流的80%，只有结构性活性的Rab11b-Q70L增加了刺激卤化物外流的速率常数。同样，RNAi敲除Rab11b，而不是Rab11a，使CFTR介导的阴离子电导反应减少了50%。在极化的T84单层中，腺病毒表达Rab11b-S25N导致Forsklin刺激的跨上皮阴离子分泌抑制70%，细胞表面生物素化检测顶膜CFTR减少50%。生物素保护实验显示，在表达Rab11b-S25N的极化T84细胞中，CFTR循环受到强烈抑制，表明对Rab11b亚型的选择性要求。这是第一个详细描述在本地表达系统中的顶端CFTR循环的报告，并证明了Rab11b调节极化上皮细胞的顶端循环。
The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, Delta F508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.