Interdisciplinary Graduate Program in Genetics, Department of Biology, University of Iowa, Iowa City, Iowa, United States of America.
PLoS One. 2012;7(9):e44096. doi: 10.1371/journal.pone.0044096. Epub 2012 Sep 5.
Axin is a critical component of the β-catenin destruction complex and is also necessary for Wnt signaling initiation at the level of co-receptor activation. Axin contains an RGS domain, which is similar to that of proteins that accelerate the GTPase activity of heterotrimeric Gα/Gna proteins and thereby limit the duration of active G-protein signaling. Although G-proteins are increasingly recognized as essential components of Wnt signaling, it has been unclear whether this domain of Axin might function in G-protein regulation. This study was performed to test the hypothesis that Axin RGS-Gna interactions would be required to attenuate Wnt signaling. We tested these ideas using an axin1 genetic mutant (masterblind) and antisense oligo knockdowns in developing zebrafish and Xenopus embryos. We generated a point mutation that is predicted to reduce Axin-Gna interaction and tested for the ability of the mutant forms to rescue Axin loss-of-function function. This Axin point mutation was deficient in binding to Gna proteins in vitro, and was unable to relocalize to the plasma membrane upon Gna overexpression. We found that the Axin point mutant construct failed to rescue normal anteroposterior neural patterning in masterblind mutant zebrafish, suggesting a requirement for G-protein interactions in this context. We also found that the same mutant was able to rescue deficiencies in maternal axin1 loss-of-function in Xenopus. These data suggest that maternal and zygotic Wnt signaling may differ in the extent of Axin regulation of G-protein signaling. We further report that expression of a membrane-localized Axin construct is sufficient to inhibit Wnt/β-catenin signaling and to promote Axin protein turnover.
轴蛋白是β-连环蛋白破坏复合物的关键组成部分，对于共受体激活水平的 Wnt 信号起始也是必需的。轴蛋白含有一个 RGS 结构域，与加速异三聚体 Gα/Gna 蛋白 GTP 酶活性从而限制活性 G 蛋白信号持续时间的蛋白质相似。尽管 G 蛋白被越来越多地认为是 Wnt 信号的必需组成部分，但尚不清楚轴蛋白的这个结构域是否在 G 蛋白调节中起作用。本研究旨在检验以下假设：轴蛋白 RGS-Gna 相互作用对于衰减 Wnt 信号是必需的。我们在发育中的斑马鱼和非洲爪蟾胚胎中使用 axin1 基因突变体（masterblind）和反义寡核苷酸敲低来测试这些想法。我们产生了一个预测会减少轴蛋白-Gna 相互作用的点突变，并测试了突变形式恢复轴蛋白功能缺失的能力。这种轴蛋白点突变在体外与 Gna 蛋白的结合能力降低，并且在 Gna 过表达时无法重新定位到质膜。我们发现，突变型轴蛋白构建体不能挽救 masterblind 突变斑马鱼中正常的前后神经模式，这表明在这种情况下 G 蛋白相互作用是必需的。我们还发现，相同的突变体能够挽救非洲爪蟾中母源 axin1 功能缺失的缺陷。这些数据表明，母源和合子 Wnt 信号在轴蛋白调节 G 蛋白信号的程度上可能存在差异。我们进一步报告，表达一种膜定位的轴蛋白构建体足以抑制 Wnt/β-连环蛋白信号并促进轴蛋白蛋白周转。
