Mutations in congenital myasthenic syndromes reveal an ε subunit C-terminal cysteine, C470, crucial for maturation and surface expression of adult AChR
Mutations in congenital myasthenic syndromes reveal an ε subunit C-terminal cysteine, C470, crucial for maturation and surface expression of adult AChR
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DOI：
10.1093/hmg/11.24.3087
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发表时间：
2002-11-15
影响因子：
3.5
通讯作者：
Beeson, D
中科院分区：
文献类型：
--
作者：
Ealing, J;Webster, R;Beeson, D
许多先天性肌无力综合征（CMS）与编码乙酰胆碱受体（AChR）的基因突变有关，AChR是一种结构为α（2）β-D-内酰胺酶的寡聚蛋白。AChR缺陷通常是由于AChR β亚基中的纯合或杂等位基因突变，其中大多数导致多肽链截短和AChR表面表达丧失。在此，我们鉴定了突变ε 1369 delG和ε Y 458 X，它们位于位于M4跨膜结构域胞外的18个氨基酸的β亚基C-末端。然后，我们将绿色荧光蛋白（GFP）掺入突变型或野生型AChR亚基的M3和M4之间的细胞内环中，并在RD或HEK 293细胞中表达AChR。含有野生型GFP标记的AChR亚基被纳入表面膜，而GFP标记的AChR突变体AChR亚基与内质网（ER）标记物共定位，并且不在细胞表面上表达。此外，突变乙酰胆碱受体没有达到细胞表面，作为测量的标记与I-125-α-银环蛇毒素和沉淀与ε-亚基特异性抗血清的完整细胞。突变研究表明，半胱氨酸470，位于4个氨基酸的C-末端，是必不可少的α/β组装和成人AChR的表面表达。用丝氨酸取代半胱氨酸470不会恢复α/β组装或表面表达。我们的研究结果提供了第一次使用GFP标记的AChR作为CMS的调查工具，并证明了一个以前未确定的作用，为二硫键结合的胱氨酸在AChR亚基C-末端，这起着至关重要的作用，成人AChR的表达。
Many congenital myasthenic syndromes (CMS) are associated with mutations in the genes encoding the acetylcholine receptor (AChR), an oligomeric protein with the structure alpha(2)betadeltaepsilon. AChR deficiency is frequently due to homozygous or heteroallelic mutations in the AChR epsilon subunit, most of which cause truncation of the polypeptide chain and loss of surface expression of AChR. Here we identified mutations epsilon1369delG and epsilonY458X, located in the 18 amino acid epsilon subunit C-terminus that lies extracellular to the M4 transmembrane domain. We then incorporated green fluorescent protein (GFP) into the intracellular loop between M3 and M4 of mutant or wild-type epsilon subunits and expressed the AChRs in RD or HEK 293 cells. AChR containing wild-type GFP-tagged epsilon subunits were incorporated into the surface membrane, whereas the GFP-tagged AChR mutant epsilon subunits co-localized with an endoplasmic reticulum (ER) marker and were not expressed on the cell surface. In addition, mutant AChRs did not reach the cell surface, as measured by labelling of intact cells with I-125-alpha-bungarotoxin and precipitation with an epsilon-subunit-specific antiserum. Mutagenesis studies showed that cysteine 470, located four amino acids from the C-terminus, is essential for alpha/epsilon assembly and surface expression of adult AChR. Replacement of cysteine 470 by serine does not restore alpha/epsilon assembly or surface expression. Our results provide the first use of GFP-tagged AChR as a tool for investigation of CMS and demonstrate a previously undetermined role for a disulphide-bonded cystine in the epsilon subunit C-terminus, which plays a crucial role in expression of the adult AChR.