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Comprehensive analysis of hetrotrimeric G protein subunits for the purpose of elucidation and treatment of human diseases

综合分析异源三聚体 G 蛋白亚基，以阐明和治疗人类疾病

基本信息

批准号：
09307002
负责人：
NISHIMOTO Ikuo
金额：
$ 20.48万
依托单位：
KEIO university
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

This project has yielded remarkable progresses in four different fields. [1] Comprehensive analysis of the G protein-coupling ability in various physiologically important receptors. Our original G protein hybrid method proved that somatostatin receptor type 3 (SSTR3) couples to Gαi, Gαq/11, Gα16, but not to Gαo, Gαz, Gα12, Gα13, or Gα14. Likewise, the study clarified that angiotensin II receptor type 1 (AT1) couples to all G proteins except for Gα13, and that AT2 does so to Gαi3. [2] Development of a new method to specify the G protein-coupling domains in receptors. Using our original method, we have identified two Gs coupling domains in the calcitonin receptor, in which one in the third intracellular loop plays a major role and another in the C-terminal tail an assisting role. By combining both methods [1] and [2], it is theoretically possible to analyze all kinds of G protein linkage in all receptors. [3] Analysis of novel biological functions mediated by G protein subunits. We have clarified that Gao subunit and associated Gβ2 subunit mediate the neurotoxic signal of the well-established familial Alzheimer's disease (FAD) gene V642I-APP through NADPH oxidase, and that this pathway is shared by other FAD genes K595N/M596L-APP and N141I-PS2. Unexpectedly, another FAD gene M146L-PS1 generates a neurotoxic signal in a similar framework, but differing only in details : M146L-PS1 activates an unknown target of pertussis toxin and then NO synthase. We have also found that LRP bound by ApoE4 (the most established AD risk factor) triggers a neurotoxic mechanism through any of Gαi, but not Gαo. [4] targeted disruption of Gβ2 subunit. We have clarified its genomic structure, constructed an appropriate targeting vector, and microinjected it to ES cells, using which chimeric mice have been born. We will continue their analysis.

该项目在四个不同领域取得了显着进展。 [1] 综合分析各种生理重要受体的G蛋白偶联能力。我们最初的 G 蛋白杂交方法证明，生长抑素受体 3 型 (SSTR3) 与 Gαi、Gαq/11、Gα16 偶联，但不与 Gαo、Gαz、Gα12、Gα13 或 Gα14 偶联。同样，该研究阐明，血管紧张素 II 受体 1 型 (AT1) 与除 Gα13 之外的所有 G 蛋白偶联，而 AT2 则与 Gαi3 偶联。 [2] 开发一种新方法来指定受体中的 G 蛋白偶联结构域。使用我们最初的方法，我们在降钙素受体中鉴定了两个 Gs 偶联结构域，其中第三个细胞内环中的一个起主要作用，而 C 端尾部中的另一个起辅助作用。通过结合方法[1]和[2]，理论上可以分析所有受体中的各种G蛋白连接。 [3] G蛋白亚基介导的新生物学功能分析。我们已经阐明，Gao 亚基和相关的 Gβ2 亚基通过 NADPH 氧化酶介导成熟的家族性阿尔茨海默病 (FAD) 基因 V642I-APP 的神经毒性信号，并且其他 FAD 基因 K595N/M596L-APP 和 N141I-PS2 共享该通路。出乎意料的是，另一个 FAD 基因 M146L-PS1 在类似的框架中产生神经毒性信号，但仅在细节上有所不同：M146L-PS1 激活百日咳毒素的未知靶标，然后激活 NO 合酶。我们还发现，与 ApoE4（最确定的 AD 危险因素）结合的 LRP 可通过任何 Gαi 触发神经毒性机制，但不会通过 Gαo 触发神经毒性机制。 [4] Gβ2 亚基的靶向破坏。我们阐明了其基因组结构，构建了合适的靶向载体，并将其显微注射到ES细胞中，由此诞生了嵌合小鼠。我们将继续他们的分析。