Drebrin is a novel connexin-43 binding partner that links gap junctions to the submembrane cytoskeleton
Drebrin is a novel connexin-43 binding partner that links gap junctions to the submembrane cytoskeleton
复制标题
DOI：
10.1016/j.cub.2004.03.063
复制
发表时间：
2004-04-20
期刊：
影响因子：
9.2
通讯作者：
Majoul, I
中科院分区：
文献类型：
--
作者：
Butkevich, E;Hülsmann, S;Majoul, I
背景：连接蛋白形成间隙连接，介导细胞间离子、代谢物和第二信使的传递。连接蛋白功能的许多方面，例如细胞转运、斑块组装和稳定性以及通道传导性，都是精细调节的，并且可能涉及与连接蛋白的胞质结构域结合的蛋白质。然而，人们对这些调节蛋白知之甚少。为了确定新的蛋白质相互作用的COOH-末端结构域的连接蛋白-43（Cx43），最广泛表达的连接蛋白家族成员，我们采用了蛋白质组学方法筛选馏分小鼠组织匀浆的binding partners.Results：drepletin回收作为一个结合伴侣的Cx43 COOH-末端结构域从小鼠脑匀浆。Dreplastin以前被描述为一种肌动蛋白结合蛋白，在阿尔茨海默病期间在大脑中减少。通过蛋白质组学鉴定的新型Drebrin-Cx43相互作用通过星形胶质细胞和Vero细胞中内源性蛋白质的共定位、共免疫沉淀、电子显微镜、电生理学、两种蛋白质与荧光标签的共表达和活细胞FRET分析来证实。耗尽drepletin在细胞与siRNA的结果受损的细胞-细胞耦合，间隙连接的内化，并针对Cx43的degradative pathway.Conclusions：我们得出结论，drepletin是必要的维持Cx43的间隙连接在其功能状态在质膜。因此，可能的是，dreplastin可能与间隙连接区的细胞-细胞接触的调节方式响应细胞外信号。连接蛋白和含有Drebrin的亚膜细胞骨架之间相互作用的重排或破坏将连接蛋白引导至降解细胞途径。
Background: Connexins form gap junctions that mediate the transfer of ions, metabolites, and second messengers between contacting cells. Many aspects of connexin function, for example cellular transport, plaque assembly and stability, and channel conductivity, are finely tuned and likely involve proteins that bind to connexins' cytoplasmic domains. However, little is known about such regulatory proteins. To identify novel proteins that interact with the COOH-terminal domain of Connexin-43 (Cx43), the most widely expressed connexin family member, we applied a proteomics approach to screen fractions of mouse tissue homogenates for binding partners.Results: Drebrin was recovered as a binding partner of the Cx43 COOH-terminal domain from mouse brain homogenate. Drebrin had previously been described as an actin binding protein that diminishes in brains during Alzheimer's disease. The novel Drebrin-Cx43 interaction identified by proteomics was confirmed by colocalization of endogenous proteins in astrocytes and Vero cells, coimmunoprecipitation, electron microscopy, electrophysiology, coexpression of both proteins with fluorescent tags, and live-cell FRET analysis. Depletion of Drebrin in cells with siRNA results in impaired cell-cell coupling, internalization of gap junctions, and targeting of Cx43 to a degradative pathway.Conclusions: We conclude that Drebrin is required for maintaining Cx43-containing gap junctions in their functional state at the plasma membrane. It is thus possible that Drebrin may interact with gap junctions in zones of cell-cell contacts in a regulated fashion in response to extracellular signals. The rearrangement or disruption of interactions between connexins and the Drebrin-containing submembrane cytoskeleton directs connexins to degradative cellular pathways.