| Project/Area Number |
10671056
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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 |
Metabolomics
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| Research Institution | Gunma University |
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Principal Investigator |
TAMEMOTO Hiroyuki Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, assistant professor, 生体調節研究所・遺伝子応用分野, 講師 (90292630)
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| Co-Investigator(Kenkyū-buntansha) |
IZUMI Tetsuro Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, associate professor, 生体調節研究所・遺伝子応用分野, 助教授 (00212952)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 1999: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | insulin / tyrosine kinase / beta cells / IRS / β細胞 |
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| Research Abstract |
We have screened receptor tyrosine kinases that are expressed in pancreatic beta cells by polymerase chain reaction using degenerate oligonucleotide primers. We found that insulin receptor related receptor (IRR) is specifically expressed in beta cells. To activate the kinase domain of IRR, we made a chimeric construct of IRR that is fused with extracellular part of insulin receptor and expressed in Chinese hamster ovary (CHO) cells. This chimeric receptor can be activated by insulin and phosphorylated insulin receptor substrate-1 (IRS-1) and IRS-2 (Hirayama et al 1999). To identify the natural ligand of IRR, we attempted to clone the cDNA for the ligand by expression cloning. For this purpose, we made a fusion construct of extracellular part of IRR fused with Fc portion of immunoglobulin G, which we call IRR-Ig. CHO clones that overexpress IRR-Ig were established. As a positive control, we also made insulin receptor and immunoglobulin fusionprotein, which we call IR-Ig. IRR-Ig and IR-Ig were secreted into the medium and purified on a protein-A column. This IR-Ig can bind IィイD1125ィエD1 labeled insulin added in the medium, however, when used to stain the insulin in the granule of the cultured beta cells, the staining was too weak. We concluded that expression cloning of the ligand for IRR is extremely difficult by this method. Alternatively, we screened proteins that interact with IRR-Ig by BIACORE method. We found several samples that interact with IRR-Ig. To know whether this sample can activate IRR, we semi-purified IRR from overexpressing CHO cells by lectin affinity column. We found that some fractions of the fetal calf serum on a gel filtration column can activate the autophosphorylation of the semi-purified IRR. However, we could not make reproducible data by fractions on the second column or samples identified by BIACORE, suggesting the in vitro kinase assay is not sensitive enough.
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