| Project/Area Number |
18370079
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | Osaka University |
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Principal Investigator |
MEKADA Eisuke Osaka University, Research Institute for Microbial Disease, Professor (20135742)
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| Co-Investigator(Kenkyū-buntansha) |
MIZUSHIMA Hiroto Osaka University, Research Institute for Microbial Disease, Assistant Professor (30379094)
ASANO Shiro Osaka University, Research Institute for Microbial Disease, Designated Researcher (00397613)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥17,270,000 (Direct Cost: ¥15,500,000、Indirect Cost: ¥1,770,000)
Fiscal Year 2007: ¥7,670,000 (Direct Cost: ¥5,900,000、Indirect Cost: ¥1,770,000)
Fiscal Year 2006: ¥9,600,000 (Direct Cost: ¥9,600,000)
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| Keywords | ectodomain shedding / SiRNA / HB-EGF / DNA aray / シェディング / レンチウィルス |
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| Research Abstract |
The extracellular domain of a number of membrane proteins can be proteolytically cleaved causing release into the medium. This proteolytic processing, also referred to as "ectodomain shedding", is observed in growth factors and other membrane proteins. Ectodomain shedding of membrane proteins affects the biological activities of membrane proteins. Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family. The soluble mature HB-EGF is generated from proHB-EGF by ectodomain shedding. TPA is a strong stimulater of HB-EGF ectodomain shedding, and ADAM family metalloprotease is also involved in this cleavage. However, the mechanism of the ectodomain shedding of membrane proteins remains totally obscure. To understand the mechanism of the TPA-induced shedding of the proHB-EGF ectodomain, we attempted to identify a new candidate of s proteins which is involved in the HB-EGF ectodomain shedding by loss-of-function genetic screenings using Lentiviral siRNA Library. After treatment with TPA, cells expressing strong proHB-EGF signal were collected, and RNA is recovered from the cells and amplified by PCR with oligonucleotides specific to the lentiviral vector. siRNA templetes which were specifically concentrated during selection were identified. Three independent screenings suggested that more than 10 genes are candidate genes involved in HB-EGF shedding. ADAM17 was also included in these 10 genes. These results indicate that the loss-of-function genetic screening by siRNA library is useful to identify genes for the purpose.
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