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

该项目在四个不同领域取得了显著进展。[1]各种重要生理受体的G蛋白偶联能力的综合分析。我们最初的G蛋白杂交法证明，生长抑素受体3型(SSTR3)与GαI、GαQ/11、Gα16偶联，但不与Gα0、Gαz、Gα12、Gα13或Gα14偶联。同样，本研究阐明了血管紧张素II受体1(AT1)与除Gα13以外的所有G蛋白偶联，AT2与GαI3偶联。[2]一种确定受体中G蛋白偶联结构域的新方法。利用我们最初的方法，我们在降钙素受体上发现了两个Gs偶联结构域，其中一个在第三细胞内环中起主要作用，另一个在C末端尾部起辅助作用。通过结合两种方法[1]和[2]，在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氧化酶介导已建立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也有这一途径。出乎意料的是，另一个FAD基因M146L-PS1在类似的框架内产生神经毒性信号，但只是在细节上不同：M146L-PS1激活一个未知的百日咳毒素靶标，然后没有合成酶。我们还发现，被载脂蛋白E4(AD危险因子)结合的LRP可通过GαI中的任何一个触发神经毒性机制，但不能通过Gαo.[4]靶向破坏Gβ2亚单位。我们已经明确了它的基因组结构，构建了合适的靶向载体，并将其显微注射到ES细胞中，从而产生了嵌合体小鼠。我们将继续他们的分析。

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.