Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator ΔF508
Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator ΔF508
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DOI：
10.1074/jbc.m205644200
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发表时间：
2002-09-27
影响因子：
4.8
通讯作者：
Teem, JL
中科院分区：
文献类型：
--
作者：
deCarvalho, ACV;Gansheroff, LJ;Teem, JL
编码囊性纤维化跨膜传导调节因子（CFTR）的基因在囊性纤维化（CF）患者中发生突变，CFTR是一种ATP结合盒（ABC）转运蛋白，其功能是磷酸化和核苷酸调节的氯离子通道。在第一核苷酸结合结构域（NBD 1）中氨基酸位置508（AF 508）处的苯丙氨酸缺失是最普遍的CF引起突变，并且导致蛋白质加工缺陷和CFTR功能降低，导致CF上皮和异源系统中的氯离子不渗透性。使用STE 6/CFTRAF 508嵌合体系统在酵母中，我们分离出两个新的AF 508回复突变体，I539 T和G550 E，分别接近和内的保守的ABC签名基序的NBD 1，。在哺乳动物细胞中的蛋白质印迹和功能分析表明，突变1539 T和G550 E各自部分挽救CFTRAF 508缺陷。此外，两种回复突变的组合导致CFTRAF 508介导的氯离子电流增加38倍，代表野生型通道活性的29%。G550 E突变增加了CFTRAF 508和野生型CFTR对cAMP激动剂激活的敏感性，并通过2 mm 3-异丁基-1-甲基黄嘌呤阻断了CFTRAF 508通道活性的增强。数据显示，AF 508缺陷可以通过核苷酸结合结构域1区域中的第二位点突变来显著挽救，该区域包括LSGGQ共有基序。
The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylationand nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (AF508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTRAF508 chimera system in yeast, we isolated two novel AF508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations 1539T and G550E each partially rescue the CFTRAF508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTRAF508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTRAF508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTRAF508 channel activityby.2 mm 3-isobutyl-1-methylxanthine. The data show that the AF508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.