2004 Fiscal Year Final Research Report Summary
Crystallization and structural analysis of the human insulin receptor
| Project/Area Number |
15570099
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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 |
Structural biochemistry
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| Research Institution | The University of Tokushima |
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Principal Investigator |
OBATA Toshiyuki The University of Tokushima, Institute for Enzyme Research, Associate Professor, 分子酵素学研究センター, 助教授 (40325296)
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| Co-Investigator(Kenkyū-buntansha) |
EBINA Yousuke The University of Tokushima, Institute for Enzyme Research, Professor, 分子酵素学研究センター, 教授 (00112227)
KISHI Kazuhiro The University of Tokushima, Institute for Enzyme Research, Associate Professor, 分子酵素学研究センター, 助教授 (70284320)
YUASA Tomoyuki The University of Tokushima, Institute for Enzyme Research, Research Associate, 分子酵素学研究センター, 助手 (50304556)
KUSUNOKI Masami Osaka University, Research Center for Structural and Functional Proteomics, Institute for Protein Research, Associate Professor, 蛋白質研究所附属生体分子解析研究センター, 助教授 (90135749)
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| Project Period (FY) |
2003 – 2004
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| Keywords | human insulin receptor / αsubunits / crystallization / structural analysis |
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| Research Abstract |
The human insulin receptor (hIR) is a glycoprotein composed of two α subunits (735 amino acid ; 135kDa) and two β subunits (620 amino acid ; 95kDa) derived by proteolytic processing from a single polypeptide (Cell, 1985, Ebina et al.). The hIR forms a disulfide-linked heterotetramer (β-α-α-β). The extracellular region of the receptor consists of the entire α subunit and a portion of the β subunit external to the predicted transmembrane domain. Insulin binds to the α subunit and activates tyrosine kinase of the intracellular β subunit to transmit the insulin signal.
Using the expression vector of the truncated human insulin receptor (hIR), we have constructed a stable Chinese hamster ovary (CHO) cell line which secretes the His-tagged α subunit (insulin-binding domain) of hIR into medium. To examine characteristics of the His-tagged hIRα, we purified the protein secreted from the CHO cells. The His-tagged hIRα was glycosylated and processed a dimer. The molecule bound insulin with an affinity similar to that of the intact hIR. The His-tagged full length of hIR was autophosphorylated by insulin stimulation in CHO cells.
We will purify the large amount of IRα, co-crystalize it with insulin and determine the crystal structure.
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