| Project/Area Number |
15590221
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General pharmacology
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| Research Institution | Chiba University |
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Principal Investigator |
OGURA Takehiko Chiba University, Graduate School of Medicine, research associate, 大学院・医学研究院, 助手 (00292673)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAYA Haruaki Chiba University, Graduate School of Medicine, Professor, 大学院・医学研究院, 教授 (60113594)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2003: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | ClC-3B / tyrosine kinase / T cell / proliferation / apoptosis / chloride channel / リンパ球 / チロシンリン酸化 / ClC-3 |
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| Research Abstract |
1.Expression of mRNAs for outwardly rectifying ClC channels (ClC-3,-4,-5 ) was examined in human peripheral lymphocytes by RT-PCR. ClC-3 mRNA was most abundantly expressed in both resting and activated T cells. ClC-3 mRNA was also expressed in both resting and activated B cells. ClC-4 mRNA was detected in both resting and activated B cells, and ClC-5 mRNA was detected in resting T cells and B cells.
2.When ClC-3B was co-expressed with tyrosine kinase p56^<lck> in COS1 cells, the ClC-3B protein was tyrosine-phosphorylated by p56^<lck>. Experiments using mutated ClC-3B clones revealed that the 859^<th> tyrosine residue (Y859) in the C-terminal intracellular region was specifically phosphorylated by p56^<lck>. The splicing variant ClC-3A, which dose not carry the corresponding tyrosine residue, was not phosphorylated by p56^<lck>.
3.To verify whether tyrosine phosphorylation of the ClC-3B protein has some impact on its subcellular localization, immunocytochemical experiments were conducted. In HT-1080 cells, expression of the ClC-3B protein at surface plasma membrane was enhanced when it was co-expressed with p56^<lck>.
4.Number of thymocytes was smaller in ClC-3 knockout mice(ClC-3KO)in comparison with that in wild type mice(WT). Number of spleen T cells was also smaller in ClC-3KO. The rate of proliferation triggered by T cell receptor(TCR) stimulation using anti-CD3 antibody (clone 2C11) was decelerated in T cells of ClC-3KO. Apoptotic cell death induced by TCR stimulation using anti-CD3 antibody (clone 2C11) was normal in activated T cells of ClC-3KO. Apoptotic cell death was also normally induced by fas stimulation using anti-fas antibody (clone Jo-2) in activated T cells of ClC-3KO.
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