Investigation of the role of insulin receptor in chromosome stability.

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 In our unpublished results, we found that disruption of IR-MAD2 interaction in mice increases aneuploidy in the immune cells and promotes T-cell lymphoma.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.