Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation.
Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation.
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囊性纤维化跨膜电导调节剂的组成性内化通过网格蛋白依赖性内吞作用发生，并受到蛋白质磷酸化的调节。
DOI：
10.1042/bj3280353
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
F. Zhang
中科院分区：
文献类型：
--
作者：
G. Lukács;G. Segal;N. Kartner;S. Grinstein;F. Zhang
尽管囊性纤维化跨膜传导调节剂（CFTR）主要与质膜氯离子通透性的调节有关，但免疫定位和功能研究表明，CFTR存在于内体腔室中。CFTR从细胞表面传递到核内体的机制尚不清楚。为了描述CFTR的内化途径，我们在稳定转染的中国仓鼠卵巢（CHO）细胞中监测了CFTR在核内体中积累的速度和程度。在暴露于高渗介质、钾耗尽或细胞内酸负荷的细胞中，评估了网格蛋白依赖的内吞作用。这些处理抑制网格蛋白依赖的内吞作用bbb90 %，通过测量125i -转铁蛋白摄取证实。CFTR与新形成的核内体的功能关联是通过核内体pH耗散方案确定的[Lukacs， Chang, Kartner, Rotstein, Riordan and Grinstein(1992)]。化学学报，1997,26(2):568- 572。作为第二种方法，CFTR的内吞作用是在细胞表面生物素化后用可切割的磺基琥珀酰亚基-2-（生物素胺）乙基-1,3-二硫代丙酸进行测定。生化和功能分析均表明，阻止网格蛋白包被囊泡的形成可抑制CFTR从质膜向核内体的回收。膜交通的全面停止不能解释CFTR内化的抑制，因为所使用的处理不影响液相内吞作用。因此，表面CFTR的有效、组成性内在化（每分钟5%）主要通过网格蛋白依赖的内吞作用发生。camp依赖性蛋白激酶A和蛋白激酶C对蛋白磷酸化的刺激降低了细胞表面生物素化CFTR的内化率，与未刺激的细胞相比，导致内部CFTR池大幅减少。这些结果表明，CFTR内化率可能参与了CFTR通道密度的决定，从而决定了camp刺激的质膜氯离子电导。
Although the cystic fibrosis transmembrane conductance regulator (CFTR) is primarily implicated in the regulation of plasma-membrane chloride permeability, immunolocalization and functional studies indicate the presence of CFTR in the endosomal compartment. The mechanism of CFTR delivery from the cell surface to endosomes is not understood. To delineate the internalization pathway, both the rate and extent of CFTR accumulation in endosomes were monitored in stably transfected Chinese hamster ovary (CHO) cells. The role of clathrin-dependent endocytosis was assessed in cells exposed to hypertonic medium, potassium depletion or intracellular acid-load. These treatments inhibited clathrin-dependent endocytosis by >90%, as verified by measurements of 125I-transferrin uptake. Functional association of CFTR with newly formed endosomes was determined by an endosomal pH dissipation protocol [Lukacs, Chang, Kartner, Rotstein, Riordan and Grinstein (1992) J. Biol. Chem. 267, 14568-14572]. As a second approach, endocytosis of CFTR was determined after cell-surface biotinylation with the cleavable sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithio- propionate. Both the biochemical and the functional assays indicated that arresting the formation of clathrin-coated vesicles inhibited the retrieval of the CFTR from the plasma membrane to endosomes. An overall arrest of membrane traffic cannot account for the inhibition of CFTR internalization, since the fluid-phase endocytosis was not effected by the treatments used. Thus the efficient, constitutive internalization of surface CFTR (5% per min) occurs, predominantly by clathrin-dependent endocytosis. Stimulation of protein phosphorylation by cAMP-dependent protein kinase A and by protein kinase C decreased the rate of internalization of cell-surface biotinylated CFTR, and contributed to a substantial diminution of the internal CFTR pool compared with that of unstimulated cells. These results suggest that the rate of CFTR internalization may participate in the determination of the CFTR channel density, and consequently, of the cAMP-stimulated chloride conductance of the plasma membrane.