Roles of activin/follistatin in neural induction

激活素/卵泡抑素在神经诱导中的作用

• 批准号: 10044298
• 负责人: SUGINO Hiromu
• 金额: $ 3.07万
• 依托单位: University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10044298/
• 关键词: activin; activin receptor; PDZ domain; WW domain; Smad; synapse; follistatin; 神経細胞; TSC-36; 中胚葉

The aim of this project is to elucidate the roles of activin and follistatin in neural induction in embryogenesis. In this period, we have obtained the following findings.(1) Identification and characterization of a PDZ protein.We have identified a mouse PDZ protein that interacts with the activin type IIA receptor (ActRIIA), which we named ARIP1 (activin receptor interacting protein 1 ). By using yeast two-hybrid screening, we isolated a cDNA clone of ARIP1 from a mouse brain cDNA library. ARIPI had one guanylate kinase domain (Guk) in the NH2-terminal region, followed by two WW domains and five PDZ domains (PDZ1-5). ARIP1 interacted with ActRIIA through PDZ5. The COOH-terminal residues of ActRIIA (E-S-S-L) agree with a PDZ-binding consensus motif, and ARIP1 recognized the consensus sequence. ARIP1 interacts specifically with ActRIIA among the receptors for TGF-β family. Interestingly, ARIP1 also interacted with Smad3, which is an activin/TGF-β intracellular signaling molecule. The mR … More NA of ARIP1 was more abundant in the brain that in other tissues. Overexpressin of ARIP1 controls activin-induced and Smad3-induced transcription in activin-responsive cell lines. These findings suggest that ARIP1 has a significant role in assembling activin signaling molecules at specific subcellular sites and in regulating signal transduction in neuronal cells.(2) Characterization of a novel follistatin-like protein as an activin-binding protein.We have identified a novel follistatin-like protein in humans and mice, which was found to differ from known follistatin gene products. Follistatin has three follistatin domains that are presumed to be growth factor binding motifs. Both the human and mouse follistatin-like proteins possessed only two follistatin domains. Northen blotting revealed that mRNAs for the novel follistatin-like proteins were abundantly expressed in placenta, adrenal gland, lung, heart, pituitary, testis and ovary, tissues where activins are also abundantly expressed. The recombinant proteins were found to have an activin-bindig activity as revealed by coimmunoprecipitation analysis. Furthermore, the recombinant protein inhibited activin-induced transcriptional responses in activin-responsive cells I n a dose-dependent manner. Ligand blotting using ィイD1125ィエD1I-activin and GST-pull down analysis revealed that the second follistatin domain in the C-terminal region on the molecule was responsible for the activin-binding activity. In summary, we have identified a novel follistatin possessing activin-binding activity. Our finding implies that cellular signaling of activin is tightly regulated by multiple members of the follistatin family. Less

本课题旨在阐明激活素和卵泡抑素在胚胎发育神经诱导中的作用。在此期间，我们获得了以下发现。(1)PDZ蛋白的鉴定和表征我们已经鉴定了与激活素IIA型受体（ActRIIA）相互作用的小鼠PDZ蛋白，我们将其命名为ARIP 1（激活素受体相互作用蛋白1）。利用酵母双杂交技术，从小鼠脑cDNA文库中克隆了ARIP 1基因。ARIPI在NH 2-末端区域具有一个鸟苷酸激酶结构域（Guk），随后是两个WW结构域和五个PDZ结构域（PDZ 1 -5）。ARIP 1通过PDZ 5与ActRIIA相互作用。ActRIIA（E-S-S-L）的COOH末端残基与PDZ结合共有基序一致，并且ARIP 1识别共有序列。在TGF-β家族的受体中，ARIP 1与ActRIIA特异性相互作用。有趣的是，ARIP 1还与Smad 3相互作用，Smad 3是一种激活素/TGF-β细胞内信号分子。的mR ...更多信息 ARIP 1的NA在脑组织中比在其他组织中更丰富。ARIP 1过表达控制激活素应答细胞系中激活素诱导和Smad 3诱导的转录这些发现表明，ARIP 1在特定的亚细胞位点组装激活素信号分子和调节神经元细胞中的信号转导中具有重要作用。(2)一种新的卵泡抑素样蛋白作为激活素结合蛋白的特征我们已经在人类和小鼠中鉴定出一种新的卵泡抑素样蛋白，它被发现不同于已知的卵泡抑素基因产物。卵泡抑素具有三个卵泡抑素结构域，推测是生长因子结合基序。人和小鼠卵泡抑素样蛋白都只有两个卵泡抑素结构域。Northen印迹显示，新卵泡抑素样蛋白的mRNA在胎盘、肾上腺、肺、心脏、垂体、睾丸和卵巢中大量表达，这些组织中激活素也大量表达。免疫共沉淀分析表明重组蛋白具有激活素结合活性。此外，重组蛋白以剂量依赖性方式抑制激活素应答细胞中激活素诱导的转录应答。用荧光素D1125荧光素D1 I-激活素和GST-下拉分析的配体印迹显示，在C-末端区域的第二个卵泡抑素结构域的分子是负责激活素结合活性。总之，我们已经确定了一种新的卵泡抑素具有激活素结合活性。我们的发现表明激活素的细胞信号传导受到卵泡抑素家族多个成员的严格调控。少

项目成果

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Sumitomo, K. 等人：“TGF-β 诱导基因 TSC-36 的表达会抑制人肺癌细胞系的生长。”Cancer Letters（出版中）。

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Yamaoka,T.,et al.：“表达激活素受体突变体的转基因小鼠胰岛发育不全。”

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Miyamoto,S.et al.：“激活素对信号雌性大鼠垂体细胞激素分泌的影响：细胞免疫印迹分析。”J.Endocrinol.. 161. 375-382 (1999)

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Hashimoto,O.,et al.：“激活素 I 型受体在激活素 A 诱导的小鼠细胞生长停滞和凋亡中的作用。”

Sumitomo, K. et al.: "Expression of TGF-b inducible gene, TSC-36, causes growth inhibition in human lung cancer cell lines."Cancer letters. (in press). (2000)

Sumitomo, K. 等人：“TGF-b 诱导基因 TSC-36 的表达会抑制人肺癌细胞系的生长。”癌症信件。