Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis.
Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis.
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DOI：
10.1091/mbc.e13-02-0111
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发表时间：
2013-09
影响因子：
3.3
通讯作者：
Falk MM
中科院分区：
文献类型：
--
作者：
Fong JT;Kells RM;Falk MM
在间隙连接蛋白connexin 43中鉴定出三个基于酪氨酸的分选信号，其中两个作为接头蛋白复合物-2的结合位点协同工作。这些分析为网格蛋白有效内化大质膜结构提供了分子模型，并提出了一种调节本构型和急性间隙连接内化的机制。当相邻细胞的两个半通道（连接子）在细胞外空间正面对接时，形成电和化学偶联相邻细胞的间隙连接（GJ）通道。值得注意的是，停靠的连接子在生理条件下是不可分割的，我们和其他人已经证明gj是整体内化的，利用内吞网格蛋白机制。内吞作用在其中一个对抗细胞的细胞质中产生双膜囊泡（环状GJs或连接体），这些细胞通过自噬体和潜在的内切/溶酶体途径降解。在这项研究中，我们研究了介导Cx43 GJ内吞作用的结构基序。我们在Cx43 c -末端发现了三个典型的基于酪氨酸的“YXXΦ”型排序信号，其中两个作为AP-2结合位点协同工作。我们生成了一组绿色荧光蛋白标记和未标记的Cx43突变体，它们分别或一起靶向这两个位点。这两个位点的突变完全消除了Cx43- ap -2/Dab2/网格蛋白的相互作用，导致GJ斑块大小增加，Cx43蛋白半衰期延长，GJ内化受损。有趣的是，Dab2是早期发现的对GJ内吞作用很重要的辅助网格蛋白接头，它通过AP-2间接与Cx43相互作用，允许每个Cx43蛋白募集多达四个网格蛋白复合物。我们的分析为网格蛋白有效内化大型质膜结构（如GJs）提供了一个机制模型。
Three tyrosine-based sorting signals in the gap junction protein connexin 43 were identified, two of which function cooperatively as adaptor protein complex-2 binding sites. The analyses provide a molecular model for clathrin to efficiently internalize large plasma membrane structures and suggest a mechanism for regulating constitutive versus acute gap junction internalization. Gap junction (GJ) channels that electrically and chemically couple neighboring cells are formed when two hemichannels (connexons) of apposed cells dock head-on in the extracellular space. Remarkably, docked connexons are inseparable under physiological conditions, and we and others have shown that GJs are internalized in whole, utilizing the endocytic clathrin machinery. Endocytosis generates double-membrane vesicles (annular GJs or connexosomes) in the cytoplasm of one of the apposed cells that are degraded by autophagosomal and, potentially, endo/lysosomal pathways. In this study, we investigated the structural motifs that mediate Cx43 GJ endocytosis. We identified three canonical tyrosine-based sorting signals of the type “YXXΦ” in the Cx43 C-terminus, two of which function cooperatively as AP-2 binding sites. We generated a set of green fluorescent protein–tagged and untagged Cx43 mutants that targeted these two sites either individually or together. Mutating both sites completely abolished Cx43-AP-2/Dab2/clathrin interaction and resulted in increased GJ plaque size, longer Cx43 protein half-lives, and impaired GJ internalization. Interestingly, Dab2, an accessory clathrin adaptor found earlier to be important for GJ endocytosis, interacts indirectly with Cx43 via AP-2, permitting the recruitment of up to four clathrin complexes per Cx43 protein. Our analyses provide a mechanistic model for clathrin's efficient internalization of large plasma membrane structures, such as GJs.