Transmembrane Topology of Mammalian Planar Cell Polarity Protein Vangl1
Transmembrane Topology of Mammalian Planar Cell Polarity Protein Vangl1
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DOI：
10.1021/bi101767a
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发表时间：
2011-03-29
期刊：
影响因子：
2.9
通讯作者：
Gros, Philippe
中科院分区：
文献类型：
--
作者：
Iliescu, Alexandra;Gravel, Michel;Gros, Philippe
Vangl 1和Vangl 2是在神经发生中起重要作用的膜蛋白，并且Vangl 1/Vangl 2突变导致小鼠和人类的神经管缺陷。在细胞水平上，Vangl蛋白调节平面细胞极性（PCP）的建立，这是一个需要将极性信息传递到邻近细胞的不对称分布的多蛋白复合物的膜组装的过程。Vangl蛋白的膜拓扑结构和多蛋白膜PCP复合物的结构和功能方面所需的蛋白质片段是未知的。我们已经使用表位标记和免疫荧光来建立Vangl蛋白的二级结构，包括跨膜结构域的数量、位置和极性。将抗原性血凝素A（HA）肽（YYDVPDYS）插入预测的Vangl 1的18、64、139、178、213和314位的细胞内或细胞外环中，并在MDCK极化细胞的膜上稳定表达单个突变变体。通过在完整的（细胞外表位）和透化的（细胞内表位）细胞中的免疫荧光组合和通过表面标记来确定外表面HA标签的膜拓扑结构。结果表明，Vangl蛋白具有四跨膜结构域结构，其中蛋白的N-末端部分（HA 18和HA 64）和大的C-末端部分（HA 314）在细胞内。拓扑图和亲水性分析显示，由TMD 1 -2（HA 139）和TMD 3 -4（HA 213）描绘的环是细胞外的，而分离预测的TMD 2 -3（HA 178）的片段是细胞内的。这种二级结构揭示了一个紧凑的膜相关部分，预测非常短的细胞外和细胞内的循环，而蛋白质窝藏一个大的细胞内结构域。
Vangl1 and Vangl2 are membrane proteins that play an important role in neurogenesis, and Vangl1/Vangl2 mutations cause neural tube defects in mice and humans. At the cellular level, Vangl proteins regulate the establishment of planar cell polarity (PCP), a process requiring membrane assembly of asymmetrically distributed multiprotein complexes that transmit polarity information to neighboring cells. The membrane topology of Vangl proteins and the protein segments required for structural and functional aspects of multiprotein membrane PCP complexes is unknown. We have used epitope tagging and immunofluorescence to establish the secondary structure of Vangl proteins, including the number, position, and polarity of transmembrane domains. Antigenic hemagglutinin A (HA) peptides (YYDVPDYS) were inserted in predicted intra- or extracellular loops of Vangl1 at positions 18, 64, 139, 178, 213, and 314, and individual mutant variants were stably expressed at the membrane of MDCK polarized cells. The membrane topology of the exofacial HA tag was determined by a combination of immunofluorescence in intact (extracellular epitopes) and permeabilized (intracellular epitopes) cells and by surface labeling. Results indicate that Vangl proteins have a four-transmembrane domain structure with the N-terminal portion (HA18 and HA64) and the large C-terminal portion (HA314) of the protein being intracellular. Topology mapping and hydropathy profiling show that the loop delineated by TMD1-2 (HA139) and TMD3-4 (HA213) is extracellular while the segment separating predicted TMD2-3 (HA178) is intracellular. This secondary structure reveals a compact membrane-associated portion with very short predicted extra- and intracellular loops, while the protein harbors a large intracellular domain.