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

 描述（由申请人提供）： 异源三聚体G蛋白（αβγ）是一类重要的G蛋白偶联受体（GPCRs），在细胞内信号转导中发挥重要作用，参与细胞外信号的调节。G蛋白作为分子开关发挥作用，由此GPCR促进GDP从α亚基（Gα）释放，随后Gα与GTP结合，Gα和Gβγ解离。然后Gα-GTP和Gβγ可以调节多种信号蛋白，直到Gα的GTP水解活性通过产生失活的Gα-GDP来关闭信号传导，然后G α-GDP与Gβγ重新缔合以完成激活和失活的循环。然而，在葡萄膜黑色素瘤中，这种严格控制的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，产生组成型活性αq或α11，其中GTP水解活性被废除，从而将Gα锁定在活性GTP结合状态，并将αq或α11转变为这种癌症的致癌驱动因子。葡萄膜黑色素瘤是成人中最常见的眼睛癌症，在高达50%的葡萄膜黑色素瘤患者中转移，并且一旦转移发生，其总是致命的，平均存活率小于50%。 六个月目前对于转移性葡萄膜黑色素瘤没有有效的治疗方法，因此迫切需要更好地了解促进葡萄膜黑色素瘤发展的分子机制。基于对G蛋白功能的大量基础研究，很明显，突变激活的Gα表现出关键的细胞生物学特性，如亚细胞定位、运输、脂质修饰、降解和激活的变化。 独特的信号通路，使它们有别于野生型Gα对应物。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进行比较。 α11，因为缺乏区分和有效研究内源性突变体与野生型蛋白的工具。因此，该项目的目的是通过使用葡萄膜黑色素瘤细胞系的基因编辑方法来开发这样一种工具，以生成其中内源性αq或α11（野生型或突变激活形式）与GFP框内融合的细胞。这些基因组编辑的葡萄膜黑色素瘤细胞系将成为一种新的和急需的工具，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，并确定与wld型αq或α 11不同的突变激活的αq或α 11调控的关键方面。

 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.