Investigation of the role of insulin receptor in chromosome stability.

项目总结 多细胞生物发展由细胞外生长因子启动的受体介导的信号转导 扩散。长期以来，胰岛素一直被认为是一种生长因子，高胰岛素血症可以促进和维持肿瘤 成长。胰岛素受体(IR)定位于细胞表面质膜，在代谢组织细胞和 高度增殖的细胞，如免疫细胞和癌细胞。IR激活两条下游信号通路， PI3K-AKT通路和MAPK通路，调节细胞代谢、增殖和生长。尽管 IR信号在系统内稳态中的有趣发现，营养信号如何维持染色体 稳定性仍不确定。我们知识上的这一鸿沟是我们理解 胰岛素在细胞增殖和分化中的作用及其对人类健康的影响 与包括2型糖尿病和癌症在内的各种疾病有关。关键的纺锤体检查点蛋白MAD2 形成有丝分裂检查点复合体(MCC)，确保染色体分离的保真度。我们最近 研究表明，MAD2与IR结合，通过组装MCC样络合物招募笼状蛋白适配器络合物， 并促进IR内吞作用。在我们未发表的结果中，我们发现IR-MAD2相互作用的中断在 小鼠会增加免疫细胞中的非整倍体，并促进T细胞淋巴瘤。这些结果表明， 纺锤体检查点调节器和IR在有丝分裂和IR信号中相互调节。IR和胰岛素- 与生长因子1受体(IGF1R)类似，IGF1R是高度同源的受体酪氨酸激酶(RTK)。IGF1R不支持 与MAD2结合，其内吞机制和信号转导结果与IR不同。我们最近 明确了IR和IGF1R不同的激活机制，提示配体特异性诱导结构 差异可能影响RTK的内吞作用和下游信号转导。与我们对角色的研究同步进行 在IR信号中的纺锤体检查点蛋白，我们也进行了无偏见的，系统性的全基因组丢失 使用CRISPR-CAS9进行功能研究。我们发现了增加或降低表面水平的新基因。 IR和IR信令。在这里，我们建议将小鼠遗传学、细胞生物学、生物化学、 基因组学和冷冻-EM研究IR在有丝分裂中的作用以及IR信号的激活机制 对细胞增殖和新陈代谢的影响。我们今后五年的目标是探索以下问题：(1) IR如何确保有丝分裂中染色体的准确分离？IR的生理功能是什么？ 在细胞分裂中；(2)IR如何选择性地激活PI3K-AKT与MAPK信号分支；以及(3)还有什么 IR在细胞增殖和分化中的作用需要哪些因子？总的来说，拟议的研究 将促进我们对胰岛素在生理上的作用、调节和机制(S)的理解 细胞增殖和分化。此外，我们的研究可能会作为进一步翻译的基础 研究和未来的治疗方法，如高胰岛素血症和2型糖尿病，与增加风险有关 可用于某些癌症，也可用于癌症免疫治疗。

PROJECT SUMMARY Multicellular organisms develop receptor-mediated signal transduction initiated by extracellular growth factors to proliferate. Insulin has long been known as a growth factor, and hyperinsulinemia can promote and sustain tumor growth. Insulin receptor (IR) localizes to the cell surface plasma membrane both in metabolic tissue cells and in highly proliferative cells such as immune cells and cancer cells. IR activates two downstream signaling pathways, the PI3K-AKT pathway and the MAPK pathway, to regulate cell metabolism, proliferation, and growth. Despite intriguing findings for IR signaling in systemic homeostasis, how the nutrient signaling maintains chromosome stability still remains uncertain. This gap in our knowledge presents a key barrier to our understanding of the function of insulin in cell proliferation and differentiation and its impact on human health, as hyperinsulinemia is associated with various diseases including type 2 diabetes and cancer. The key spindle checkpoint protein MAD2 forms a mitotic checkpoint complex (MCC) and ensures the fidelity of chromosome segregation. Our recent studies showed that MAD2 binds to IR, recruits the clathrin adaptor complex by assembling an MCC-like complex, and promotes IR endocytosis. In our unpublished results, we found that disruption of IR-MAD2 interaction in mice increases aneuploidy in the immune cells and promotes T-cell lymphoma. These results suggest that the spindle checkpoint regulators and IR mutually regulate each other in both mitosis and IR signaling. IR and insulin- like growth factor 1 receptor (IGF1R) are highly homologous receptor tyrosine kinases (RTKs). IGF1R does not bind to MAD2, and its endocytosis mechanism and signaling outcomes are different from that of IR. We recently defined the distinct activation mechanisms of IR and IGF1R, suggesting that ligand-specific induced structural differences might affect the endocytosis and downstream signaling of RTKs. In parallel to our studies on the role of spindle checkpoint proteins in IR signaling, we have also performed unbiased, systematic genome-wide loss of function studies using CRISPR-Cas9. We identified novel genes that increase or decrease the levels of surface IR and IR signaling. Here, we propose to combine approaches in mouse genetics, cell biology, biochemistry, genomics and cryo-EM to determine the function of IR in mitosis, as well as activation mechanisms of IR signaling for cell proliferation vs. metabolism. Our goals over the next five years are to explore the following questions: (1) how does IR ensure accurate chromosome segregation in mitosis, and what is the physiological function for IR in cell division; (2) how does IR selectively activate the PI3K-AKT vs. MAPK signaling branch; and (3) what other factors are required for IR function in cell proliferation and differentiation? Collectively, the proposed research will advance our understanding of the function, regulation, and mechanism(s) of insulin action in physiological cell proliferation and differentiation. Furthermore, our studies will likely serve as a basis for further translational research and future therapeutics as hyperinsulinemia and type 2 diabetes are associated with increased risks for certain cancers and may also be harnessed for cancer immunotherapies.