R-Ras与C3G的相互作用及生物学意义的研究

发布时间：2018-01-01 02:05

  本文关键词：R-Ras与C3G的相互作用及生物学意义的研究　出处：《哈尔滨工业大学》2009年硕士论文　论文类型：学位论文

  更多相关文章： R-Ras C3GdelC 相互作用 细胞黏附

【摘要】： R-Ras属于Ras家族小分子量G蛋白,与Ras具有55%的氨基酸序列相似性,R-Ras和Ras家族的其他成员一样,结合GTP时处于激活状态,并通过其下游效应物,将胞外信号转导到胞内,调节细胞的生物学功能。C3G为Rap1的特异性的鸟苷酸交换因子,近年的研究表明C3G能与R-Ras相互作用。本研究的前期工作也表明全长的C3G能与R-Ras相互作用,而C端催化结构域与R-Ras相互作用较弱。为了进一步研究R-Ras与C3G的相互作用极其两者相互作用的生物学意义,首先检测了R-Ras与C3G缺失C末端催化结构域的缺失突变体C3GdelC的体外相互作用,通过在HEK293中瞬时转染C3GdelC,收获其蛋白并将其与外源表达GST-R-Ras进行GST-pull down及Western blot,结果表明R-Ras与C3GdelC在体外是相互作用的。同时将R-Ras与C3GdelC共转染HEK293细胞,通过免疫共沉淀及Western blot检测出在细胞内R-Ras与C3GdelC也是相互作用的。接着通过Overlay的方法,研究了R-Ras与C3GdelC相互作用的作用方式,将转移到PVDF膜上的细胞内表达的C3GdelC与外源表达的GST-R-Ras蛋白共孵育,Western blot的结果表明,GST-R-Ras能与膜结合的C3GdelC相互作用,R-Ras与C3G的相互作用是直接的。最后通过GST-pull down及Western blot的方法,找到了R-Ras与C3G相互作用的最小结构域为第210-406氨基酸残基。为了研究R-Ras与C3GdelC相互作用的生物学意义,首先将R-Ras与C3GdelC共表达于NIH3T3细胞中,通过细胞免疫荧光实验研究两者在细胞中的定位。结果发现C3GdelC主要分布在细胞质中,少部分在细胞膜上也有分布,而R-Ras主要分布在细胞质、细胞膜,二者主要共定位于胞质和部分质膜区附近。其次通过GST-RBD-pull down实验对R-Ras进行活性化分析,结果表明C3GdelC能促进R-Ras的活化。最后通过MTT比色实验研究了R-Ras与C3GdelC的相互作用对细胞黏附能力的影响。综上,R-Ras与C3GdelC的相互作用及生物学意义的研究对深入理解C3G-R-Ras信号转导途径具有重要意义。
[Abstract]:R-Ras belongs to the Ras family of small molecular weight G protein, amino acid sequence has 55% similarity with Ras, R-Ras and other members of the Ras family, is activated when combined with GTP, and through its downstream effectors, extracellular to intracellular signal transduction, regulation of biological function of.C3G cells is a guanine nucleotide specificity Rap1 exchange factor, recent studies suggest that C3G can interact with R-Ras. The preliminary work of this study also showed that the full-length C3G can interact with R-Ras domain and R-Ras terminal catalytic structure C interaction is weak. In order to further study the biological significance of the interaction of R-Ras and C3G is the interaction of the two, first detection the interaction between C3GdelC R-Ras and C3G terminal deletion mutants lacking C catalytic domain in vitro by transiently transfected into HEK293 C3GdelC, the protein and the harvest GST-R-Ras GST-pull and exogenous D expression Own Western and blot, the results showed that R-Ras and C3GdelC interact in vitro. At the same time, R-Ras and C3GdelC were transfected into HEK293 cells by immunoprecipitation and Western blot detected in cells R-Ras and C3GdelC also interact with each other. Then by Overlay method, studied the interaction between R-Ras and C3GdelC interaction. Will transfer to C3GdelC and exogenous PVDF on the membrane of the intracellular expression of GST-R-Ras protein was incubated with Western blot, the results show that the interaction between GST-R-Ras and membrane binding C3GdelC, the interaction between R-Ras and C3G is direct. Finally through the method of GST-pull down and Western blot, to find the minimum domain R-Ras interacts with C3G for the 210-406 amino acid residues. In order to study the biological significance of the interaction between C3GdelC and R-Ras, the R-Ras and C3GdelC co expression in NIH3T3 cells, through Study on the two immunofluorescence experiments localization in cells. The results showed that C3GdelC mainly distributed in the cytoplasm, few are also distributed in the cell membrane, while R-Ras is mainly distributed in the cytoplasm, cell membrane, two were mainly located near the cytoplasmic membrane and part area. Then based on R-Ras activity analysis GST-RBD-pull down experimental results show that C3GdelC can promote the activation of R-Ras. Finally, MTT colorimetric experiment was conducted to study the effect of interaction between R-Ras and C3GdelC on cell adhesion ability. Therefore, research on interaction and biological significance of R-Ras and C3GdelC is of great significance for understanding the C3G-R-Ras signal transduction pathway.

【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

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