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首页> 外文期刊>Cellular Signalling >Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function
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Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function

机译：功能丧失点突变和两个弗林蛋白酶结构域衍生物提供有关R-spondin2结构和功能的见解

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摘要

R-spondins (Rspos) potentiate Wnt/beta-catenin signaling, an important pathway in embryonic development that is constitutively active in many cancers. To analyze Rspo structure and function, we expressed full-length wild-type Rspo2 and Rspo2 point mutants corresponding to Rspo4 variants that have been linked to developmental defects. The Rspo2 mutants had markedly reduced potency relative to the wild-type protein, demonstrating for the first time specific amino acid residues in Rspos that are critical for beta-catenin signaling. The diminished activity of Rspo2/C78 gamma and Rspo2/C113R was attributable to a defect in their secretion, while Rspo2/Q70R exhibited a decrease in its intrinsic activity. Cysteine assignments in a Rspo2 derivative containing only the two furin-like domains (Rspo2-2F) provided the first information about the disulfide-bonding pattern of this motif, which was characterized by multiple short loops and unpaired cysteine residues, and established that the loss-of-function cysteine mutants disrupted disulfide bond formation. Moreover, Rspo2-2F demonstrated potent activity and synergized strongly with Wnt-3a in a beta-catenin reporter assay. In contrast, an Rspo2-2F derivative containing the Q70R substitution showed significantly reduced activity, although it still synergized with Wnt-3a in the reporter assay. Rspo2-2F derivatives elicited an unusually sustained phosphorylation (20 h) of the Wnt co-receptor. low density lipoprotein receptor-related protein 6 (LRP6), as well as an increase in cell surface LRP6. Co-immunoprecipitation experiments involving LRP6 and Kremens suggested that these associations contribute to Rspo2 activity, although the lack of major differences between wild-type and Q70R derivatives implied that additional interactions may be important. Published by Elsevier Inc.
机译：R-spondins（Rspos）增强Wnt /β-catenin信号传导，这是胚胎发育中的重要途径，在许多癌症中具有组成性活性。为了分析Rspo的结构和功能，我们表达了与野生型Rspo4变体相对应的全长野生型Rspo2和Rspo2点突变体，这些变体与发育缺陷有关。相对于野生型蛋白，Rspo2突变体的效力显着降低，这首次证明了Rspos中对于β-catenin信号传导至关重要的特定氨基酸残基。 Rspo2 / C78γ和Rspo2 / C113R的活性降低归因于它们的分泌缺陷，而Rspo2 / Q70R表现出其固有活性降低。仅包含两个弗林蛋白酶样结构域（Rspo2-2F）的Rspo2衍生物中的半胱氨酸分配提供了有关该基序的二硫键结合模式的第一个信息，其特征是多个短环和未配对的半胱氨酸残基，并确定了这种损失功能的半胱氨酸突变体破坏了二硫键的形成。此外，Rspo2-2F在β-catenin报告基因检测中显示出强大的活性并与Wnt-3a强烈协同。相反，含有Q70R取代基的Rspo2-2F衍生物活性显着降低，尽管在报告基因分析中仍与Wnt-3a协同作用。 Rspo2-2F衍生物引起Wnt共同受体异常持续的磷酸化（20小时）。低密度脂蛋白受体相关蛋白6（LRP6），以及细胞表面LRP6的增加。涉及LRP6和Kremens的免疫共沉淀实验表明，这些关联有助于Rspo2的活性，尽管野生型和Q70R衍生物之间没有主要区别，这意味着其他相互作用可能很重要。由Elsevier Inc.发布

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