MATURATION AND FUNCTION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR VARIANTS BEARING MUTATIONS IN PUTATIVE NUCLEOTIDE-BINDING DOMAIN-1 AND DOMAIN-2
MATURATION AND FUNCTION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR VARIANTS BEARING MUTATIONS IN PUTATIVE NUCLEOTIDE-BINDING DOMAIN-1 AND DOMAIN-2
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DOI：
10.1128/mcb.11.8.3886
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发表时间：
1991-08-01
影响因子：
5.3
通讯作者：
SMITH, AE
中科院分区：
文献类型：
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作者：
GREGORY, RJ;RICH, DP;SMITH, AE
迄今为止发现的与囊性纤维化（CF）疾病相关的突变的一个特征是它们中的许多聚集在CF跨膜传导调节因子（CFTR）的第一个核苷酸结合结构域（NBD）内。 我们试图通过在CFTR突变的两个NBD中引入模拟与CF相关的氨基酸变化或改变高度保守基序内的残基来发现这种聚类的分子基础。 通过在COS细胞中瞬时表达研究突变CFTR的合成和成熟。 通过使用6-甲氧基-N-（磺丙基）喹啉（SPQ）在HeLa细胞表达突变质粒的荧光测量的能力的改变的蛋白质产生环AMP刺激的阴离子流出进行了评估。 结果显示，（i）所有CF相关突变体，除了一个例外，缺乏如SPQ测定中测量的功能活性，（ii）NBD 1中的突变比NBD 2中的相应突变对相同氨基酸变化的影响更敏感，（iii）用携带CF相关突变的质粒转染的细胞通常但不完全缺乏成熟CFTR，（iv）缺乏成熟CFTR的NBD突变体不能激活Cl-通道，和（v）CFTR的糖基化本身不是CFTR功能所需的。 我们的理由是NBD 1本身或周围结构域的结构使其对突变变化特别敏感。 因此，大多数NBD 1突变体，但只有少数NBD 2突变体，不能成熟或缺乏功能活性。 这些发现与观察到的CF患者的NBD 1和NBD 2之间CFTR错义突变的不均匀分布一致。
One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP-stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients.