Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels.
Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels.
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DOI：
10.1091/mbc.e11-05-0438
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发表时间：
2011-12
影响因子：
3.3
通讯作者：
Paul DL
中科院分区：
文献类型：
--
作者：
Chai Z;Goodenough DA;Paul DL
调节间隙连接组装的机制尚未明确。结果表明，含有Cx 50的间隙连接的组装需要连接蛋白C-末端的PDZ结构域结合基序和支架蛋白ZO-1。在眼透镜中表达的三种连接蛋白各自含有PDZ结构域结合基序，其指导与支架蛋白ZO-1的物理关联，但相互作用的意义尚不清楚。我们发现，Cx 50与PDZ结合基序突变没有形成间隙连接斑块或诱导HeLa细胞中的细胞间通讯，而添加一个7个氨基酸的PDZ结合基序恢复正常功能的Cx 50缺乏其整个C-末端胞质结构域。C-末端缺失对Cx46具有类似的作用，尽管作用较弱，但对Cx43的靶向和功能几乎没有影响。此外，ZO-1的小干扰RNA敲低完全抑制了HeLa细胞中野生型Cx 50的间隙连接的形成。因此，PDZ结合基序和ZO-1都是HeLa细胞中Cx 50细胞间通道形成所必需的。敲入表达具有PDZ结合基序突变的Cx 50的小鼠表型模仿Cx 50敲除。此外，分化透镜纤维在敲入显示广泛的细胞内Cx 50，而斑块中的成熟纤维只包含Cx46。因此，体内正常的Cx 50功能也需要完整的PDZ结构域结合基序。这是第一次证明间隙连接组装中连接蛋白相互作用蛋白的连接蛋白特异性要求。
The mechanisms regulating assembly of gap junctions are not well defined. It is shown that assembly of gap junctions containing Cx50 requires both a PDZ domain–binding motif at the connexin C-terminus and the scaffolding protein ZO-1. 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.