Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
Mol Biol Cell. 2011 Dec;22(23):4503-12. doi: 10.1091/mbc.E11-05-0438. Epub 2011 Sep 30.
The three connexins expressed in the ocular lens each contain PDZ domain-binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell-cell communication in HeLa cells, whereas the addition of a seven-amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain-binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.
在眼部晶状体中表达的三种连接蛋白都含有 PDZ 结构域结合基序，这些基序指导与支架蛋白 ZO-1 的物理相互作用，但这种相互作用的意义尚不清楚。我们发现，具有 PDZ 结合基序突变的 Cx50 不能在 HeLa 细胞中形成间隙连接斑或诱导细胞间通讯，而添加一个七氨基酸 PDZ 结合基序可以恢复缺乏整个 C 末端胞质结构域的 Cx50 的正常功能。C 末端缺失对 Cx46 也有类似的影响，尽管较弱，但对 Cx43 的靶向和功能影响很小。此外，用小干扰 RNA 敲低 ZO-1 可完全抑制野生型 Cx50 在 HeLa 细胞中形成间隙连接。因此，PDZ 结合基序和 ZO-1 对于 Cx50 在 HeLa 细胞中的细胞间通道形成都是必需的。表达 PDZ 结合基序突变的 Cx50 基因敲入小鼠表现出与 Cx50 基因敲除小鼠相似的表型。此外，在基因敲入的分化晶状体纤维中，广泛存在细胞内 Cx50，而成熟纤维中的斑痕仅含有 Cx46。因此，Cx50 在体内的正常功能也需要完整的 PDZ 结构域结合基序。这是首次证明连接蛋白相互作用蛋白在间隙连接组装中对连接蛋白具有特异性要求。
