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

在非洲爪蟾卵母细胞中注射从大鼠胃中获得的低分子量mRNA，应用5-HT诱导了大量的Ca^2+激活的Cl^-电流，这在天然卵母细胞中明显观察到或无法检测到（1），这表明又一种参与5-HT受体介导的信号转导的新蛋白质从注射的mRNA中表达出来。将体外合成的cRNA注入卵母细胞系统后，重复进行基因表达的电生理测定，从大鼠胃cDNA文库中鉴定出单个cDNA克隆。根据该cDNA序列的最长开放阅读框，推导出该克隆cDNA（RS基因）的氨基酸序列，由90个氨基酸残基组成。在网织红细胞裂解物系统中由克隆cRNA体外合成的蛋白质基于SDS聚丙烯酰胺凝胶分析具有约10 kDa的分子量，并且与根据氨基酸序列计算的分子量相同。压力机辅助分析 ...更多信息 该蛋白与其它已知的G蛋白偶联受体没有明显的序列相似性（<18%）。在使用从大鼠各种组织获得的总RNA的北方印迹分析中，发现RS基因的mRNA普遍表达，但表达水平存在一些差异。为了阐明该基因在哺乳动物细胞中的功能表达，将cDNA插入表达载体pRSV-X/pRSV-neo^r中。该构建体用于在CHO细胞系（CHO-RS）中与新霉素类似物G418稳定表达。应用荧光共聚焦显微镜，用Fluo-3测定细胞内游离钙离子浓度（[Ca^（2+）]_i）的增加，筛选高表达基因的CHO-RS细胞系。北方印迹杂交检测到克隆性CHO-RS细胞系中RS mRNA的高水平表达。10 μ M 5-HT作用于CHO-RS细胞后，其荧光强度明显增强，而未转染CHO细胞（CHO-K1）和单独转染载体DNA的CHO细胞（CHO-V）荧光强度无明显增强。在CHO-V和CHO-RS细胞系中，当施加高浓度的去甲肾上腺素（NE）时，观察到相同的荧光强度增加。应用1 mM乙酰胆碱和2 μ M P物质对这三种CHO细胞系的荧光强度没有显着增加。可以得出结论，从RS基因推导的蛋白质既不是5-HT受体本身，也不是促进5-HT受体诱导的因子，但可能是天然5-HT受体介导的信号转导的调节剂（2，3）。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