Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Biochem J. 2012 Jan 15;441(2):633-43. doi: 10.1042/BJ20111566.
CFTR (cystic fibrosis transmembrane conductance regulator) is expressed in the apical membrane of epithelial cells. Cell-surface CFTR levels are regulated by endocytosis and recycling. A number of adaptor proteins including AP-2 (μ2 subunit) and Dab2 (Disabled-2) have been proposed to modulate CFTR internalization. In the present study we have used siRNA (small interfering RNA)-mediated silencing of these adaptors to test their roles in the regulation of CFTR cell-surface trafficking and stability in human airway epithelial cells. The results indicate that μ2 and Dab2 performed partially overlapping, but divergent, functions. While μ2 depletion dramatically decreased CFTR endocytosis with little effect on the half-life of the CFTR protein, Dab2 depletion increased the CFTR half-life ~3-fold, in addition to inhibiting CFTR endocytosis. Furthermore, Dab2 depletion inhibited CFTR trafficking from the sorting endosome to the recycling compartment, as well as delivery of CFTR to the late endosome, thus providing a mechanistic explanation for increased CFTR expression and half-life. To test whether two E3 ligases were required for the endocytosis and/or down-regulation of surface CFTR, we siRNA-depleted CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and c-Cbl (casitas B-lineage lymphoma). We demonstrate that CHIP and c-Cbl depletion have no effect on CFTR endocytosis, but c-Cbl depletion modestly enhanced the half-life of CFTR. The results of the present study define a significant role for Dab2 both in the endocytosis and post-endocytic fate of CFTR.
CFTR（囊性纤维化跨膜电导调节因子）表达于上皮细胞的顶膜。细胞表面 CFTR 水平受内吞和循环调节。一些衔接蛋白，包括 AP-2（μ2 亚基）和 Dab2（Disabled-2），被认为可以调节 CFTR 的内化。在本研究中，我们使用 siRNA（小干扰 RNA）介导的这些衔接蛋白沉默，以测试它们在调节人呼吸道上皮细胞 CFTR 细胞表面转运和稳定性中的作用。结果表明，μ2 和 Dab2 发挥部分重叠但不同的功能。虽然μ2 耗尽显著减少 CFTR 内吞作用，但对 CFTR 蛋白半衰期影响不大，而 Dab2 耗尽则使 CFTR 半衰期增加约 3 倍，同时抑制 CFTR 内吞作用。此外，Dab2 耗尽抑制 CFTR 从分拣内体到再循环隔室的转运，以及 CFTR 向晚期内体的输送，从而为 CFTR 表达和半衰期增加提供了机制解释。为了测试两个 E3 连接酶是否需要内化和/或下调表面 CFTR，我们用 siRNA 耗尽了 CHIP（Hsc（热休克同源蛋白）70 相互作用蛋白的 C 端）和 c-Cbl（casitas B 细胞淋巴瘤）。我们证明 CHIP 和 c-Cbl 耗尽对 CFTR 内吞作用没有影响，但 c-Cbl 耗尽适度增强了 CFTR 的半衰期。本研究的结果定义了 Dab2 在 CFTR 内化和内吞后命运中的重要作用。
