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首页> 外文期刊>Cell communication & adhesion >Connexin43 PDZ2 Binding Domain Mutants Create Functional Gap Junctions and Exhibit Altered Phosphorylation
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Connexin43 PDZ2 Binding Domain Mutants Create Functional Gap Junctions and Exhibit Altered Phosphorylation

机译：Connexin43 PDZ2结合域突变体创建功能性缺口连接并展示改变的磷酸化

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Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions -3 and -2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43?I382) or the last five amino acids (Cx43?378–382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43?I382 and Cx43?378–382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation.
机译：连接蛋白43（Cx43）是表达最丰富的间隙连接蛋白。 Cx43的C末端尾部对于通过特定酪氨酸和丝氨酸残基的磷酸化以及与细胞蛋白的相互作用调节间隙连接很重要。已显示Cx43的C末端与紧密和粘附连接相关的Zona咬合蛋白1（ZO-1）蛋白的PDZ2域相互作用。 Cx43的PDZ2结合结构域的分析表明，位置-3和-2以及C末端的最终疏水氨基酸对ZO-1结合至关重要。此外，连接蛋白40和45的C末端与ZO-1相互作用，但Cx32没有。要评估Cx43-ZO-1相互作用的功能意义，需要Cx43野生型（Cx43wt）和缺少C端疏水异亮氨酸（Cx43？I382）或最后五个氨基酸（Cx43？378-382）的突变体为了体外结合ZO-1，将其引入Cx43缺陷的MDCK细胞系。体外结合研究和免疫共沉淀试验表明，这些Cx43突变体无法与ZO-1相互作用。共焦和反卷积显微镜显示，一部分Cx43wt与ZO-1共定位在质膜上。对于Cx43？I382和Cx43？378-382突变体，观察到了类似的共定位模式，这些突变体易位至质膜并形成功能性间隙连接通道。 wt和突变体Cx43似乎具有相似的周转率。但是，与Cx43wt相比，Cx43突变体的P2和P3磷酸同工型显着降低。这些研究表明，Cx43与ZO-1的相互作用可能有助于Cx43磷酸化的调节。

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