Molecular cloning of a gene involved in serotohin receptor-mediated signal transduction
| Project/Area Number |
06807170
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Nagoya City University |
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Principal Investigator |
IMAIZUMI Yuji Nagoya City University, Chemical Pharmacology, Associate Professor, 薬学部, 助教授 (60117794)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | xenopus oocyte / rat stomach / 5-HT / CDNA library / CHO cell / confocal fluorescence microscope / [Ca^<2+>]_i / signal transduction / ラット / 胃 / アフリカツメガエル卵母細胞 / 細胞内カルシウム濃度 |
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| Research Abstract |
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
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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
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