TOPOLOGY PROFILE FOR A GLUTAMATE-RECEPTOR - 3 TRANSMEMBRANE DOMAINS AND A CHANNEL-LINING REENTRANT MEMBRANE LOOP
TOPOLOGY PROFILE FOR A GLUTAMATE-RECEPTOR - 3 TRANSMEMBRANE DOMAINS AND A CHANNEL-LINING REENTRANT MEMBRANE LOOP
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DOI：
10.1016/0896-6273(95)90293-7
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发表时间：
1995-02-01
期刊：
影响因子：
16.2
通讯作者：
DINGLEDINE, R
中科院分区：
文献类型：
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作者：
BENNETT, JA;DINGLEDINE, R
我们研究了在补充有微粒体膜的兔网织红细胞中翻译的 GluR3 亚基的跨膜拓扑。催乳素报告表位在六个位置与 GluR3 融合，将每个提议的跨膜结构域括起来。然后通过蛋白酶 K 敏感性评估微粒体膜中表位的侧面性。通过该方法，N末端以及M3和M4之间的整个区域是细胞外的，而C末端是细胞内的。利用氨基末端的 4 个天然 N 连接糖基化位点以及 M3 和 M4 之间的 1 个引入位点，证实了这些区域的细胞外位置。插入 N12 上游和下游的表位对蛋白酶敏感，因此位于细胞内。我们的结果支持谷氨酸受体亚基的拓扑模型，该模型由三个跨膜结构域 M1、M3 和 M4 以及另一个结构域（所提出的通道衬里 M2）组成，它形成两端面向细胞质的可重入膜片段。
We investigated the transmembrane topology of the GluR3 subunit that was translated in rabbit reticulocytes supplemented with microsomal membranes. A prolactin reporter epitope was fused to GluR3 at six locations, bracketing each of the proposed transmembrane domains. The sidedness of the epitope in the microsomal membrane was then assessed by proteinase K sensitivity. The N terminus and the entire region between M3 and M4 was extracellular, and the C terminus was intracellular by this method. Four native N-linked glycosylation sites in the amino terminus and one introduced site between M3 and M4 were utilized, confirming the extracellular location of these regions. Epitopes inserted upstream and downstream of N12 were protease sensitive and thus intracellular. Our results support a topological model for glutamate receptor subunits that consists of three transmembrane domains, M1, M3, and M4, and another domain, the proposed channel-lining M2, which forms a reentrant membrane segment with both ends facing the cytoplasm.