本文關(guān)鍵詞：R-Ras與C3G的相互作用及生物學(xué)意義的研究　出處：《哈爾濱工業(yè)大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： R-Ras C3GdelC 相互作用 細(xì)胞黏附

【摘要】： R-Ras屬于Ras家族小分子量G蛋白,與Ras具有55%的氨基酸序列相似性,R-Ras和Ras家族的其他成員一樣,結(jié)合GTP時(shí)處于激活狀態(tài),并通過其下游效應(yīng)物,將胞外信號(hào)轉(zhuǎn)導(dǎo)到胞內(nèi),調(diào)節(jié)細(xì)胞的生物學(xué)功能。C3G為Rap1的特異性的鳥苷酸交換因子,近年的研究表明C3G能與R-Ras相互作用。本研究的前期工作也表明全長的C3G能與R-Ras相互作用,而C端催化結(jié)構(gòu)域與R-Ras相互作用較弱。為了進(jìn)一步研究R-Ras與C3G的相互作用極其兩者相互作用的生物學(xué)意義,首先檢測了R-Ras與C3G缺失C末端催化結(jié)構(gòu)域的缺失突變體C3GdelC的體外相互作用,通過在HEK293中瞬時(shí)轉(zhuǎn)染C3GdelC,收獲其蛋白并將其與外源表達(dá)GST-R-Ras進(jìn)行GST-pull down及Western blot,結(jié)果表明R-Ras與C3GdelC在體外是相互作用的。同時(shí)將R-Ras與C3GdelC共轉(zhuǎn)染HEK293細(xì)胞,通過免疫共沉淀及Western blot檢測出在細(xì)胞內(nèi)R-Ras與C3GdelC也是相互作用的。接著通過Overlay的方法,研究了R-Ras與C3GdelC相互作用的作用方式,將轉(zhuǎn)移到PVDF膜上的細(xì)胞內(nèi)表達(dá)的C3GdelC與外源表達(dá)的GST-R-Ras蛋白共孵育,Western blot的結(jié)果表明,GST-R-Ras能與膜結(jié)合的C3GdelC相互作用,R-Ras與C3G的相互作用是直接的。最后通過GST-pull down及Western blot的方法,找到了R-Ras與C3G相互作用的最小結(jié)構(gòu)域?yàn)榈?10-406氨基酸殘基。為了研究R-Ras與C3GdelC相互作用的生物學(xué)意義,首先將R-Ras與C3GdelC共表達(dá)于NIH3T3細(xì)胞中,通過細(xì)胞免疫熒光實(shí)驗(yàn)研究兩者在細(xì)胞中的定位。結(jié)果發(fā)現(xiàn)C3GdelC主要分布在細(xì)胞質(zhì)中,少部分在細(xì)胞膜上也有分布,而R-Ras主要分布在細(xì)胞質(zhì)、細(xì)胞膜,二者主要共定位于胞質(zhì)和部分質(zhì)膜區(qū)附近。其次通過GST-RBD-pull down實(shí)驗(yàn)對(duì)R-Ras進(jìn)行活性化分析,結(jié)果表明C3GdelC能促進(jìn)R-Ras的活化。最后通過MTT比色實(shí)驗(yàn)研究了R-Ras與C3GdelC的相互作用對(duì)細(xì)胞黏附能力的影響。綜上,R-Ras與C3GdelC的相互作用及生物學(xué)意義的研究對(duì)深入理解C3G-R-Ras信號(hào)轉(zhuǎn)導(dǎo)途徑具有重要意義。
[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.

【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R341

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