Department of Orthopedics, University of Maryland, School of Medicine, Baltimore, Maryland, 21201.
J Cell Biochem. 2013 Nov;114(11):2542-50. doi: 10.1002/jcb.24603.
The cytoplasmic C-terminus of connexin43 (Cx43) interacts with numerous signaling complexes. We hypothesize that signal complex docking to the Cx43 C-terminus (CT) is required to propagate the molecules being shared by gap junctions. We have previously shown that Cx43 impacts the responsiveness of osteoblasts to FGF2 in a PKCδ- and ERK-dependent manner, converging on Runx2 activity. Here, we mapped the interaction domain of Cx43 and PKCδ to amino acids 243-302 of the Cx43 CT by GST pulldown assay. Using Runx2-responsive luciferase reporter assays, a Cx43 deletion construct (Cx43 S244Stop), which lacks the C-terminus (amino acids 244-382), failed to support the Cx43-dependent potentiation of transcription following FGF2 treatment in MC3T3 osteoblast-like cells. Similarly, overexpression of Cx43 S244Stop could not mimic the ability of the full length Cx43 to stimulate expression of osteoblast genes. In contrast to full length Cx43, overexpression of just the Cx43 CT (amino acids 236-382) inhibited both transcription from a Runx2 reporter and signaling via PKCδ and ERK. Inhibition of signaling by the CT did not occur in HeLa cells, which lack endogenous Cx43. In summary, the data support a model in which an intact Cx43 is required for both signal propagation/permeability (i.e., channel function) and local recruitment of signaling complexes to the CT (i.e., docking function) in order to mediate its cellular effects. Further, while the CT alone has channel independent activity, it is opposing to the effect of overexpression of the full length Cx43 channel in this cell context.
间隙连接蛋白 43（Cx43）的细胞质 C 端与许多信号复合物相互作用。我们假设，信号复合物与 Cx43 C 端（CT）的对接对于传播通过间隙连接共享的分子是必需的。我们之前已经表明，Cx43 通过 PKCδ 和 ERK 依赖性方式影响成骨细胞对 FGF2 的反应性，从而汇聚到 Runx2 活性上。在这里，我们通过 GST 下拉测定将 Cx43 和 PKCδ 的相互作用域映射到 Cx43 CT 的氨基酸 243-302。使用 Runx2 反应性荧光素酶报告基因测定，缺乏 C 端（氨基酸 244-382）的 Cx43 缺失构建体（Cx43 S244Stop）无法支持 FGF2 处理后成骨样细胞 MC3T3 中转录的 Cx43 依赖性增强。同样，全长 Cx43 能够刺激成骨基因的表达，而过表达 Cx43 S244Stop 则不能模拟这种能力。与全长 Cx43 相反，仅过表达 Cx43 CT（氨基酸 236-382）即可抑制 Runx2 报告基因的转录和 PKCδ 和 ERK 的信号传递。CT 对信号的抑制不会发生在缺乏内源性 Cx43 的 HeLa 细胞中。总之，数据支持这样一种模型，即完整的 Cx43 既需要信号的传播/通透性（即通道功能），又需要将信号复合物募集到 CT（即对接功能），以介导其细胞效应。此外，虽然 CT 本身具有独立于通道的活性，但在这种细胞环境中，它与全长 Cx43 通道的过表达的效果相反。
