Genome-edited uveal melanoma cell lines for investigating constitutively active GNAQ and GNA11

 描述(由申请人提供)： 异三聚体G蛋白(αβγ)具有连接G蛋白偶联受体(GPCRs)和细胞内反应的功能，在多种细胞外信号的传递和无数生理过程的调控中发挥着重要作用。G蛋白起着分子开关的作用，通过GPC促进α亚单位(Gα)释放gdp，随后Gα结合gtp，Gα和Gβγ解离。Gα-gtp和Gβγ可以调节多种信号蛋白，直到Gα的gtp水解酶活性通过产生失活的Gα-gdp而关闭信号，然后与Gβγ重新结合，完成激活和失活的循环。然而，在葡萄膜黑色素瘤中，这个严格控制的G蛋白周期被密切相关的Gα，αQ或α1突变破坏。这种突变发生在超过90%的葡萄膜黑色素瘤中，产生具有结构性活性的αQ或α11，其中α水解酶活性被取消，从而将Gα锁定在活跃的α结合状态，使GGTP Q或GTP 11成为该癌的致癌驱动因素。葡萄膜黑色素瘤是成人最常见的眼癌，在高达50%的葡萄膜黑色素瘤患者中会发生转移，一旦发生转移，它总是致命的，平均生存期不到 六个月。目前尚无有效的治疗转移性葡萄膜黑色素瘤的方法，因此迫切需要更好地了解促进葡萄膜黑色素瘤发生发展的分子机制。大量对G蛋白功能的基础研究表明，突变激活的Gα具有重要的细胞生物学特性，如亚细胞定位、转运、脂质修饰、降解和激活的改变。 独特的信号通路，使它们有别于野生型Gα。在葡萄膜黑色素瘤中，很难将突变激活的αQ或α11与野生型αQ或 α11是由于缺乏区分和有效研究内源突变体和野生型蛋白的工具。因此，本项目的目标是开发这样一种工具，通过使用葡萄膜黑色素瘤细胞系的基因编辑方法来生成内源性αQ或α11的细胞，在该细胞中，野生型或突变激活的形式与绿色荧光蛋白框架内融合。这些经基因组编辑的葡萄膜黑色素瘤细胞系将是一种新的和急需的工具，可以更好地了解突变激活的αQ或α11在葡萄膜黑色素瘤中的功能，并识别突变激活的αQ或α11与WLD型αQ或α11不同的调节的关键方面。

 DESCRIPTION (provided by applicant): Heterotrimeric G proteins (αβγ) are well known for their function in linking G protein- coupled receptors (GPCRs) to intracellular responses, and thereby playing essential roles in transmitting a wide variety of extracellular signals into regulation of countless physiological process. G proteins function as molecular switches whereby a GPCR promotes GDP release from the α subunit (Gα), followed by subsequent binding of GTP by Gα and dissociation of Gα and Gβγ. Gα-GTP and Gβγ can then regulate a variety of signaling proteins until the GTP hydrolysis activity of Gα turns off signaling by generating the inactive Gα-GDP, which then re- associates with Gβγ to complete the cycle of activation and inactivation. However, in uveal melanoma this tightly controlled G protein cycle is corrupted by a mutation in the closely related Gα, αq or α1. Such mutations, occurring in over 90% of uveal melanoma, generate a constitutively active αq or α11 in which GTP hydrolysis activity is abrogated, thereby locking the Gα in an active GTP bound state and turning αq or α11 into an oncogenic driver of this cancer. Uveal melanoma, the most common cancer of the eye in adults, metastasizes in up to 50% of uveal melanoma patients, and, once metastasis occurs, it is invariably fatal with an average survival of less than six months. There are currently no effective therapies for metastatic uveal melanoma, and thus there exists an urgent need to better understand the molecular mechanisms that promote the development of uveal melanoma. Based on numerous basic research studies on G protein function, it is clear that mutationally activated Gα exhibit key cell biology properties, such as changes in subcellular localization, trafficking, lipid modification, degradation and activation of unique signaling pathways, that set them apart from the wild type Gα counterpart. In uveal melanoma, it has been difficult to compare mutationally activated αq or α11 with wild type αq or α11 due to a lack of tools to differentiate between and effectively study the endogenous mutant versus wild type protein. Therefore, the objective of this project is to develop such a tool by using gene-editing approaches with uveal melanoma cell lines to generate cells in which the endogenous αq or α11, either the wild type or mutationally activated form, is fused in-frame to GFP. These genome-edited uveal melanoma cell lines will be a novel and much-needed tool to better understand how mutationally activated αq or α11 functions in uveal melanoma and to identify key aspects of regulation of mutationally activated αq or α11 that differ from that of wld type αq or α11.