Molecular cloning of a gene involved in serotohin receptor-mediated signal transduction

在注射大鼠胃小分子mRNAs的非洲爪哇卵母细胞中，5-HT2+和gt；-激活的氯离子电流在天然卵母细胞中明显地被忽略或检测不到(1)，这表明从注入的mRNAs中又表达了一种参与5-HTR介导的信号转导的新蛋白。将体外合成的CRNAs注射到卵母细胞系统后，重复进行基因表达的电生理学分析，从大鼠胃cDNA库中鉴定出一个单一的cDNA克隆。克隆基因(RS基因)的氨基酸序列由90个氨基酸残基组成，由最长的开放阅读框确定。克隆的cRNA在网织红细胞裂解液体系中体外合成的蛋白质经SDS-PAGE分析，其相对分子质量为-10 kDa，与根据氨基酸序列计算的结果一致。公关…的计算机辅助分析更多的药物蛋白没有显示出与任何其他已知的G蛋白偶联受体有任何明显的序列相似性(&lt；18%)。用从大鼠不同组织中获得的总RNA进行Northern杂交分析，发现RS基因的mRNA普遍表达，但表达水平存在一定的差异。为了阐明该基因在哺乳动物细胞中的功能表达，将其克隆到表达载体pRSV-X/pRSV-neo^r中，并用新霉素类似物G418在CHO-RS细胞中稳定表达。用共聚焦荧光显微镜检测5-羟色胺处理后细胞内游离钙离子浓度([Ca^&lt；2+&gt；]i)的变化，筛选高表达基因的CHO-RS细胞系。Northern印迹杂交检测到克隆株CHO-RS中RS基因的高水平表达。10-微米5-羟色胺作用后，CHO-RS细胞的共聚焦荧光强度增强，而未转染CHO-K1细胞和单纯载体DNA转染组CHO-RS细胞的共聚焦荧光强度未见明显增强。在CHO-V和CHO-RS细胞系中，高浓度去甲肾上腺素(NE)可引起相同的荧光强度增强。1 mM乙酰胆碱和2µM P物质对上述三种CHO细胞的荧光强度无明显增强作用。结论：RS基因编码的蛋白质既不是5-羟色胺受体本身，也不是促进5-羟色胺受体诱导的因子，而可能是天然5-羟色胺受体介导的信号转导的调节物(2，3)。较少

In Xenopus oocytes injected with low molecular weight mRNAs obtained from rat stomach, application of 5-HT induced substantial Ca^<2+>-activated Cl^- current which was clearly obsrved or undetectably small in native oocytes(1), suggesting that one more novel proteins involved in 5-HT receptor-mediated signal transduction were expressed from the injected mRNAs. Repeating electrophysiological assay of gene expression following injection of the cRNAs synthesized in vitro into the oocyte system, a sigle cDNA clone was identified from a rat stomach cDNA library. The deduced amino-acid sequence of the clonal cDNA (RS gene) which was assigned from the longest openreading frame of the cDNA sequence consisted of 90 amino-acid residues. The protein synthesized in vitro in the reticulocyte lysate system from the clonal cRNA had a molecular weight of -10kDa based upon SDS polyacrylamide gel analysis, and is identical to that calculate from the amino-acid sequence. Computer-aided analysis of the pr … More edicted protein dose not show any obvious sequence similarities (<18%) to any other known G protein-coupled receptors. In a Northern blot analysis using total RNAs obtained from various tissues of the rat, it was found that mRNA of RS gene is ubiquitously expressed with some variance of expression levels. To clarify the functional expression of this gene in mammalian cells, the cDNA insert was in the expression vector pRSV-X/pRSV-neo^r. This construct was used for stable expression in CHO cell lines (CHO-RS) with neomycin analog, G418. CHO-RS cell lines expressing high levels of gene were screened by measuring the increase in the intracellular free calcium concentration ([Ca^<2+>]_i) with fluo-3 in responese to the application of 5-HT,by means of a confocal fluorescence microscope. High level expression of RS mRNA in the clonal CHO-RS cell lines was detected by Northern blot hybridization analysis. The confocal flurescense intensity of CHO-RS cell lines was increased after application of 10muM 5-HT.In contrast, significant increase in fluorescence intensity was not observed in nontransfected CHO cell lines (CHO-K1) or CHO cell lines transfected with vector DNA alone (CHO-V). In CHO-V and CHO-RS cell lines, same fluorescence intensity increase was observed when high concentrations of norepinephrine (NE) was applied. Application of 1mM acetylcholine and 2muM substance P did not significantly increase the fluorescence intensity in these three CHO cell lines. It can be concluded that the protein deduced from RS gene is neither a 5-HT receptor itself nor a factor facilitating the induction of 5-HT receptors but may be a modulator of native 5-HT receptor-mediated signal transduction (2,3). Less