| Project/Area Number |
63480269
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
内分泌・代謝学
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| Research Institution | Shiga University of Medical Science |
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Principal Investigator |
KOBAYASHI Masashi Shiga University of Medical Science, Assistant Professor., 医学部, 講師 (80115758)
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| Co-Investigator(Kenkyū-buntansha) |
TERAOKA Hiroshi Shionogi Research Institute Senior Researcher., 主任研究員
KOBAYASHI Masashi Shiga University of Medical Science, Assistant Professor. (80115758)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1990: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1989: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1988: ¥3,300,000 (Direct Cost: ¥3,300,000)
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| Keywords | Insulin receptor disease / PCR / point mutation / Insulin receptor / Insulin resistance / インスリン抵抗性 / 点突然変異 / 糖尿病 / インスリン誘導体 / インスリン持抗性糖尿病 / インスリンレセプター異常症 / プロレセプター |
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| Research Abstract |
We reported the siblings with severe insulin resistance because of unprocessed insulin proreceptors. To determine the structural abnormalities, we analyzed DNA sequence of the cleavage site by direct sequence method using PCR technique. The structural change of the cleavage site from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser due to G-T point mutation appeared to be the cause for failure to process the proreceptors. To study whether the mutation of insulin proreceptors at the cleavage site was responsible for unprocessed insulin proreceptors and to elucidate the structural and binding characteristics of the proreceptors, we performed transfection of cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. At 72 hours after transfection, insulin binding increased to the maximum in both cells transfected with normal cDNA or mutated cDNA, and they were 40 times and 14 times of control, non-transfected cells, respectively. Affinity cross-linking and surface labeling showed 135 kDa normal alpha subunit in cells transfected with normal cDNA and 210 kDa proreceptor in mutant cells. The proreceptors were cleaved by trypsin to alpha and beta subunit at the concentration of 0.025%. Auto-phosphorylation study showed decreased ^<32>P incorporation to proreceptors of cells transfected with mutated cDNA at both basal and insulin stimulated states without change in the sensitivity to insulin compared with cells transfected with normal cDNA. Competitive binding study with insulin, proinsulin and miniproinsulin showed that the proreceptors had lower relative affinity to proinsulin, but this characteristics disappeared by trypsin treatment. These results suggest that the mutation was the cause for unprocessed insulin proreceptors in the patients with insulin resistance, and that the expressed proreceptors of the COS 7 cells transfected with the mutated cDNA had similar binding specificity and sensitivity to trypsin.
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