β-Arrestin Interacts with the Beta/Gamma Subunits of Trimeric G-Proteins and Dishevelled in the Wnt/Ca2+ Pathway in Xenopus Gastrulation

β-Catenin independent, non-canonical Wnt signaling pathways play a major role in the regulation of morphogenetic movements in vertebrates. The term non-canonical Wnt signaling comprises multiple, intracellularly divergent, Wnt-activated and β-Catenin independent signaling cascades including the Wnt/Planar Cell Polarity and the Wnt/Ca2+ cascades. Wnt/Planar Cell Polarity and Wnt/Ca2+ pathways share common effector proteins, including the Wnt ligand, Frizzled receptors and Dishevelled, with each other and with additional branches of Wnt signaling. Along with the aforementioned proteins, β-Arrestin has been identified as an essential effector protein in the Wnt/β-Catenin and the Wnt/Planar Cell Polarity pathway. Our results demonstrate that β-Arrestin is required in the Wnt/Ca2+ signaling cascade upstream of Protein Kinase C (PKC) and Ca2+/Calmodulin-dependent Protein Kinase II (CamKII). We have further characterized the role of β-Arrestin in this branch of non-canonical Wnt signaling by knock-down and rescue experiments in Xenopus embryo explants and analyzed protein-protein interactions in 293T cells. Functional interaction of β-Arrestin, the β subunit of trimeric G-proteins and Dishevelled is required to induce PKC activation and membrane translocation. In Xenopus gastrulation, β-Arrestin function in Wnt/Ca2+ signaling is essential for convergent extension movements. We further show that β-Arrestin physically interacts with the β subunit of trimeric G-proteins and Dishevelled, and that the interaction between β-Arrestin and Dishevelled is promoted by the beta/gamma subunits of trimeric G-proteins, indicating the formation of a multiprotein signaling complex.

不依赖β-连环蛋白（β-Catenin）的非经典Wnt信号通路（non-canonical Wnt signaling）在脊椎动物的形态发生运动调控中发挥核心作用。非经典Wnt信号通路涵盖多种胞内通路各异、由Wnt激活且不依赖β-Catenin的信号级联反应，包括Wnt/平面细胞极性（Wnt/Planar Cell Polarity）通路与Wnt/钙离子（Wnt/Ca²⁺）级联反应。Wnt/平面细胞极性与Wnt/钙离子通路共享核心效应蛋白，包括Wnt配体、卷曲蛋白（Frizzled）受体与散乱蛋白（Dishevelled），且这些蛋白亦可与Wnt信号通路的其他分支产生互作。除上述蛋白外，β-阻遏蛋白（β-Arrestin）已被证实是Wnt/β-连环蛋白与Wnt/平面细胞极性通路中的必需效应蛋白。本研究结果表明，β-阻遏蛋白是Wnt/钙离子信号级联中蛋白激酶C（Protein Kinase C, PKC）以及钙离子/钙调蛋白依赖性蛋白激酶II（Ca²⁺/Calmodulin-dependent Protein Kinase II, CamKII）上游的必需组分。我们通过非洲爪蟾胚胎外植体的基因敲低与功能挽救实验，进一步解析了β-阻遏蛋白在该非经典Wnt信号通路分支中的功能，并在293T细胞中分析了蛋白间的相互作用。β-阻遏蛋白、三聚体G蛋白β亚基与散乱蛋白的功能互作，是诱导PKC激活与膜转位的必要条件。在非洲爪蟾原肠胚形成过程中，β-阻遏蛋白在Wnt/钙离子信号通路中的功能对于会聚延伸运动至关重要。我们进一步证实，β-阻遏蛋白可与三聚体G蛋白β亚基及散乱蛋白发生物理互作，且三聚体G蛋白β/γ亚基可促进β-阻遏蛋白与散乱蛋白的结合，这提示存在多蛋白信号复合物的形成。