Identification of splicing variants of rapostlin, a novel Rnd2 effector that interacts with neural Wiskott-Aldrich syndrome protein and induces neurite branching
Identification of splicing variants of rapostlin, a novel Rnd2 effector that interacts with neural Wiskott-Aldrich syndrome protein and induces neurite branching
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DOI：
10.1074/jbc.m312763200
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发表时间：
2004-04-02
影响因子：
4.8
通讯作者：
Negishi, M
中科院分区：
文献类型：
--
作者：
Kakimoto, T;Katoh, H;Negishi, M
Rho家族gtpase调节神经元形态。Rnd亚家族是Rho家族GTPases的一个新分支。在这些gtpase中，Rnd2在大脑中特异性表达。我们最近发现Rapostlin是Rnd2的一种新的效应物。Rapostlin诱导PC12细胞的神经突分支响应Rnd2。在克隆Rapostlin的过程中，我们发现Rapostlin的两个主要表达的剪接变体（重命名为RapostlinL） Rapostlin - linm和RapostlinS分别在中心唯一插入区域缺少29个和61个残基，以及三个次要变体RapostlinLd、RapostlinMd和RapostlinSd，每个变体分别与RapostlinL、RapostlinM和RapostlinS相同的5个氨基酸缺失。RapostlinL主要在大脑中表达，而RapostlinS则普遍表达。在点印迹实验中，所有的剪接变异体都以gtp依赖的方式与Rnd2结合。然而，当RapostlinM和RapostlinS在PC12细胞中与Rnd2共表达时，诱导的神经突分支较少，这表明插入区对RapostlinL的分支活性很重要。在体外和体内，所有剪接变异体都通过羧基末端的SH3结构域与N-WASP结合，而SH3结构域对于RapostlinL的分支活性至关重要。在免疫沉淀实验中，Rnd2降低了Ra-postlinL-N-WASP的相互作用，而对RapostlinM或RapostlinS与N-WASP的相互作用影响不大。因此，我们发现功能不同的Rapostlin剪接变体对Rnd2与N-WASP相关的反应不同。
Rho family GTPases regulate neuronal morphology. Rnd subfamily is a new branch of Rho family GTPases. Of these GTPases, Rnd2 is specifically expressed in brain. We recently identified Rapostlin as a novel effector of Rnd2. Rapostlin induces neurite branching in response to Rnd2 in PC12 cells. During the cloning of Rapostlin, we have found two mainly expressed splicing variants of Rapostlin (renamed as RapostlinL), Rapost-linM and RapostlinS, lacking 29 residues and 61 residues within the unique insert region at the center, respectively, and three minor variants, RapostlinLd, RapostlinMd, and RapostlinSd, each with the identical five-amino acid deletion from RapostlinL, RapostlinM, and RapostlinS, respectively. RapostlinL is predominantly expressed in brain, whereas RapostlinS is expressed ubiquitously. In a dot-blot assay, all splicing variants bind to Rnd2 in a GTP-dependent manner. However, RapostlinM and RapostlinS induce less neurite branching when coexpressed with Rnd2 in PC12 cells, indicating that the insert region is important for the branching activity of RapostlinL. All splicing variants bind to N-WASP in vitro and in vivo through the SH3 domain at the carboxyl terminus, and the SH3 domain is essential for branching activity of RapostlinL. In immunoprecipitation experiments, Rnd2 reduces Ra-postlinL-N-WASP interaction, whereas it has little effect on the interaction of RapostlinM or RapostlinS with N-WASP. Therefore, we found that functionally different splicing variants of Rapostlin have different responses to Rnd2 in association with N-WASP.