Biochemical reconstitution of heterotrimeric G proteins in the Wnt pathway

描述（由申请人提供）：Wnt信号转导通路在动物发育和人类肿瘤发生的许多方面起着关键作用。Wnts（卷曲受体）的受体预计包含7个跨膜结构域，让人想起G蛋白偶联受体。最近，在果蝇和培养的哺乳动物细胞中进行的遗传实验表明异源三聚体G蛋白参与了Wnt信号的转导。我们建议使用生化方法来阐明G蛋白转导Wnt信号的机制。所提出的实验利用爪蟾卵提取物的能力来生化重现复杂的反应。利用这个系统，我们最近重建了Wnt通路的细胞质规范臂，以研究无细胞系统中的Wnt信号转导。这项工作建立了Wnt通路的数学模型，以及Wnt配体如何通过促进Dsh与b -连环蛋白降解复合物的直接相互作用在细胞内转导信号的分子描述。我们现在已经从G蛋白（Goto和God）水平重建了Wnt通路的信号传导，我们将系统地测试其他Ga亚家族成员在我们的生化系统中激活Wnt通路的能力。我们已经证明，Goco对Wnt通路的激活依赖于Dsh，我们将确定Dsh是否直接与Goco相互作用，并将测试Gao调节下游激酶活性的能力。最后，我们建议评估Goto是否在功能和物理上与卷曲受体Fz2相互作用。

DESCRIPTION (provided by applicant): The Wnt signal transduction pathway plays a critical role in many aspects of animal development as well as tumorigenesis in humans. The receptors for Wnts (Frizzled receptors) are predicted to contain seven transmembrane domains, reminiscent of G protein coupled receptors. Recently, genetic experiments in Drosophila and cultured mammalian cell experiments have implicated heterotrimeric G proteins in transduction of a Wnt signal. We propose to use biochemical approaches to elucidate the mechanisms by which G proteins transduce a Wnt signal. The proposed experiments take advantage of the capacity of Xenopus egg extracts to biochemically recapitulate complex reactions. Using this system, we recently reconstituted the cytoplasmic canonical arm of the Wnt pathway in order to study Wnt signal transduction in a cell-free system. This work led to a mathematical model of the Wnt pathway as well as a molecular description of how Wnt ligands transduce their signals inside the cell by promoting direct interaction of Dishevelled (Dsh) with the B-catenin degradation complex. We have now reconstituted signaling of the Wnt pathway from the level of G proteins (Goto and God) and we will systematically test other Ga subfamily members for their ability to activate the Wnt pathway in our biochemical system. We have shown that Wnt activation of the pathway by Goco is Dsh dependent, and we will determine whether Dsh interacts directly with Goco and will test Gao's ability to regulate downstream kinase activities. Finally, we propose to assess whether Goto functionally and physically interacts with the Frizzled receptor, Fz2.