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首页> 外文学位 >Ankyrin-G in renal epithelia: Identification of novel isoforms and the interaction between ankyrin-G and H+/K+-ATPase alpha 2 subunit.
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Ankyrin-G in renal epithelia: Identification of novel isoforms and the interaction between ankyrin-G and H+/K+-ATPase alpha 2 subunit.

机译：肾上皮中的锚蛋白-G：新型同工型的鉴定以及锚蛋白-G与H + / K + -ATPaseα2亚基之间的相互作用。

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摘要

Ankyrins are multifunctional membrane adaptors which link diverse membrane proteins to the spectrin-based membrane cytoskeleton. Ankyrin-G (Ank-G, encoded by the Ank-3 gene) is broadly expressed and exhibits alternative splicing in different tissues, giving rise to isoforms ranging in Mr from 100-480 kDa. However, little is known about the alternative promoter usage and alternative splicing patterns of the Ank-3 gene in kidney, particularly at the N-terminus. In the first study, a novel full-length Ank-G cDNA and two novel Ank-G amino termini were isolated from mouse kidney by cDNA library screening and RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). Their distinct 5' splicing patterns and their transcription start sites were also defined. Putative promoter regions were cloned and their activities were examined by using sequential deletion analysis in different kidney cell lines. Our data demonstrate that alternative promoter usage and alternative splicing create the transcriptome complexity of Ank-G in mouse kidney.;The apical localization of H+/K+-ATPase alpha2 (HKalpha2) in renal epithelial cells is required for its role in acid-base and K+ homeostasis. There is limited information on the mechanisms contributing to the segregation of HKalpha2 to the apical membrane domain of the collecting duct epithelial cells. Ank-G is localized to both the apical and lateral domains in renal epithelia and has been reported to contribute to the specific domain localization of integral membrane proteins. In the second study, we performed overlay assays and pull-down experiments to demonstrate a direct interaction between the HKalpha2 and Ank-G. It was found that the ankyrin repeat domain, spectrin-binding domain of Ank-G, and amino-terminus, the first cytoplasmic loop (Loop1) of HK alpha2 are involved in the interaction. An interaction between Ank-G and HKalpha2 was corroborated by co-immunoprecipitation and co-localization studies. These data demonstrate a direct interaction between Ank-G and HKalpha2 which contributes to the apical localization of HKalpha2.;Our studies not only revealed that multifaceted usage of Ank-3 gene contributes to the production of Ank-G transcriptome complexity, but also demonstrated a direct interaction between Ank-G and apical membrane associated integral proteins, significantly extending our current understanding of Ank-G structural and functional diversities.
机译：锚蛋白是多功能的膜衔接子，其将多种膜蛋白连接至基于血影蛋白的膜细胞骨架。锚蛋白-G（Ank-G，由Ank-3基因编码）广泛表达，并在不同的组织中表现出可变的剪接，产生的异构体范围从100-480 kDa不等。但是，关于肾脏，特别是在N末端的Ank-3基因的替代启动子用法和替代剪接模式知之甚少。在第一项研究中，通过cDNA文库筛选和RNA连接酶介导的cDNA末端快速扩增（RLM-RACE），从小鼠肾脏中分离出一个新的全长Ank-G cDNA和两个新的Ank-G氨基末端。还定义了它们独特的5'剪接模式和转录起始位点。克隆假定的启动子区域，并通过在不同肾细胞系中使用顺序缺失分析来检查其活性。我们的数据表明，替代启动子的使用和替代剪接在小鼠肾脏中产生了Ank-G的转录组复杂性。; H + / K + -ATPaseα2（HKalpha2）在肾上皮细胞中的顶端定位是其在酸碱和磷酸中的作用所必需的K +动态平衡。关于有助于HKalpha2分离到收集导管上皮细胞的顶膜域的机制的信息有限。 Ank-G定位于肾上皮的顶端和外侧结构域，据报道有助于整体膜蛋白的特定结构域定位。在第二项研究中，我们进行了重叠测定和下拉实验，以证明HKalpha2和Ank-G之间的直接相互作用。发现锚蛋白重复结构域，Ank-G的血影蛋白结合结构域和氨基末端，HK alpha2的第一个细胞质环（Loop1）参与了相互作用。共同免疫沉淀和共同定位研究证实了Ank-G和HKalpha2之间的相互作用。这些数据证明了Ank-G和HKalpha2之间的直接相互作用，这有助于HKalpha2的顶端定位。;我们的研究不仅揭示了Ank-3基因的多面性用法有助于Ank-G转录组复杂性的产生，而且还证明了Ank-G与顶膜相关的整合蛋白之间的直接相互作用，大大扩展了我们目前对Ank-G结构和功能多样性的了解。

著录项

  • 作者

    Li, Jun.;

  • 作者单位

    The University of Alabama at Birmingham.;

  • 授予单位 The University of Alabama at Birmingham.;
  • 学科 Biology Cell.;Biology Animal Physiology.
  • 学位 Ph.D.
  • 年度 2008
  • 页码 165 p.
  • 总页数 165
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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