Endoplasmic reticulum retention and associated degradation of a GABAA receptor epilepsy mutation that inserts an aspartate in the M3 transmembrane segment of the α1 subunit
Endoplasmic reticulum retention and associated degradation of a GABAA receptor epilepsy mutation that inserts an aspartate in the M3 transmembrane segment of the α1 subunit
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DOI：
10.1074/jbc.m508305200
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发表时间：
2005-11-11
影响因子：
4.8
通讯作者：
Macdonald, RL
中科院分区：
文献类型：
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作者：
Gallagher, MJ;Shen, WZ;Macdonald, RL
GABA(A)受体α1亚单位癫痫突变(α1(A322D))将带负电荷的天冬氨酸残基引入α1亚单位的疏水M3跨膜区。我们以前曾报道，在哺乳动物细胞中异源表达α1(A322D)β2γ2受体会导致总和表面α1亚单位蛋白的减少。在这里，我们演示了这种减少的机制。总α1(A322D)亚基蛋白在单独表达时(86+/-6%)或与β2和伽马2S亚基共表达时(78+/-6%)与野生型蛋白相比减少了相似的量，这表明亚基齐聚之前表达减少。在α1β2伽马2S受体中，内切糖苷酶H仅使26+/-5%的α1亚基脱糖，这与蛋白质的大量成熟一致，但在α1(A322D)β2伽马2S受体中，内切糖苷酶H脱糖91+/-4%的α1(A322D)亚基，与蛋白质成熟失败一致。为了确定野生型和突变型亚基的细胞定位，用黄色(α1-YFP)或青色(α1-CFP)荧光蛋白标记α1亚基。共聚焦显微镜成像显示36+/-4%的α1-YFPβ2-Gamma 2与质膜共定位，而只有5+/-1%的α1(A322D)-YFPβ2-Gamma 2与质膜共定位，而其余大部分受体与内质网共定位(55+/-4%α1-YFPβ2 Gamma2S，86+/-3%α1(A322D)-YFP)。α1-CFPβ2γ2S和α1(A322D)-YFPβ2γ2S或α1-YFPβ2γ2S和α1(A322D)CFPβ2γ2S受体的杂合表达表明，膜GABA(A)受体主要含有野生型α1亚单位。这些数据表明，A322D突变在翻译后但在组装前降低了α1亚基的表达，导致内质网相关的降解和膜上几乎完全是野生型亚基的α1亚基。
A GABA(A) receptor alpha 1 subunit epilepsy mutation (alpha 1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the alpha 1 subunit. We reported previously that heterologous expression of alpha 1(A322D) beta 2 gamma 2 receptors in mammalian cells resulted in reduced total and surface alpha 1 subunit protein. Here we demonstrate the mechanism of this reduction. Total alpha 1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone ( 86 +/- 6%) or when coexpressed with beta 2 and gamma 2S subunits (78 +/- 6%), indicating an expression reduction prior to subunit oligomerization. In alpha 1 beta 2 gamma 2S receptors, endoglycosidase H deglycosylated only 26 +/- 5% of alpha 1 subunits, consistent with substantial protein maturation, but in alpha 1(A322D) beta 2 gamma 2S receptors, endoglycosidase H deglycosylated 91 +/- 4% of alpha 1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the alpha 1 subunit was tagged with yellow (alpha 1-YFP) or cyan (alpha 1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 +/- 4% of alpha 1-YFP beta 2 gamma 2 but only 5 +/- 1% alpha 1(A322D)- YFP beta 2 gamma 2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 +/- 4% alpha 1-YFP beta 2 gamma 2S, 86 +/- 3% alpha 1(A322D)-YFP). Heterozygous expression of alpha 1-CFP beta 2 gamma 2S and alpha 1(A322D)-YFP beta 2 gamma 2S or alpha 1-YFP beta 2 gamma 2S and alpha 1(A322D)CFP beta 2 gamma 2S receptors showed that membrane GABA(A) receptors contained primarily wild type alpha 1 subunits. These data demonstrate that the A322D mutation reduces alpha 1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane alpha 1 subunits that are almost exclusively wild type subunits.