Activation of a Frizzled-2/β-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Gαo and Gαt
Activation of a Frizzled-2/β-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Gαo and Gαt
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DOI：
10.1073/pnas.96.25.14383
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发表时间：
1999-12-07
影响因子：
11.1
通讯作者：
Wang, HY
中科院分区：
文献类型：
--
作者：
Liu, XX;Liu, T;Wang, HY
推测的Wnt受体的卷曲基因家族编码具有G蛋白连接受体特征的七跨膜基序的蛋白质。尽管没有功能丧失的研究证明卷曲信号传导需要G蛋白。我们通过使用β 2肾上腺素能受体的配体结合结构域设计了对β肾上腺素能激动剂有反应的卷曲蛋白2嵌合体。期望的是，嵌合体将对药物介导的激活和阻断敏感，从而避免纯化可溶性和活性Wnt配体以激活Frizzled的问题。在斑马鱼胚胎中的嵌合体的表达表现出异丙肾上腺素（ISO）刺激，普萘洛尔敏感的钙瞬变，从而证实了β-肾上腺素能性质的Wnt信号转导的嵌合受体。因为F9胚胎畸胎癌细胞在稳定转染Frizzled-2嵌合体并用ISO刺激后形成原始内胚层。它们经历G蛋白亚基的消耗。表达嵌合受体的F9干细胞的内胚层形成的ISO刺激被百日咳毒素和G α 0、G α t2和G β 2的反义寡脱氧核苷酸阻断。我们的研究结果表明，需要两个百日咳毒素敏感的G蛋白，G α o和G α t，通过卷曲蛋白-2受体的信号。
The frizzled gene family of putative Wnt receptors encodes proteins that have a seven-transmembrane-spanning motif characteristic of G protein-linked receptors. though no loss-of-function studies have demonstrated a requirement for G proteins for Frizzled signaling. We engineered a Frizzled-2 chimera responsive to beta-adrenergic agonist by using the ligand-binding domains of the beta(2)-adrenergic receptor. The expectation was that the chimera would be sensitive both to drug-mediated activation and blockade, thereby circumventing the problem of purifying soluble and active Wnt ligand to activate Frizzled. Expression of the chimera in zebrafish embryos demonstrated isoproterenol (ISO)-stimulated, propranolol-sensitive calcium transients, thereby confirming the beta-adrenergic nature of Wnt signaling by the chimeric receptor. Because F9 embryonic teratocarcinoma cells form primitive endoderm after stable transfection of Frizzled-2 chimera and stimulation with ISO. they were subject to depletion of G protein subunits. ISO stimulation of endoderm formation of F9 stem cells expressing the chimeric receptor was blocked by pertussis toxin and by oligodeoxynucleotide antisense to G alpha o, G alpha t2, and G beta 2. Our results demonstrate the requirement of two pertussis toxin-sensitive G proteins, G alpha o and G alpha t, for signaling by the Frizzled-2 receptor.