SPECIFIC AIMS: Precise regulation of the insulin signaling pathway is critical for multiple facets of animal physiology 1-3. Dysregulation of the insulin signaling pathway has been linked to metabolic disorders, such as diabetes 4. As type 2 diabetes affects more than 400 million people worldwide 5, understanding the signaling pathways impacting this disease is of paramount importance. Genetic mutations of the insulin receptor (IR) cause rare and severe insulin resistance 6. Yet, the causes of insulin resistance seen in type 2 diabetes are numerous and the mechanisms are multifactorial leaving many unanswered questions. Upon insulin binding at the plasma membrane (PM), IR triggers the activation of bifurcated signaling pathways: the PI3K-AKT pathway for metabolism and the MAPK pathway for growth. Active IR is then internalized by clathrin-mediated endocytosis 7. The IR endocytosis has been extensively studied for decades 7. Yet, how cell surface levels of functional IR in the basal and insulin-stimulated states are regulated, how IR trafficking affects the activation of specific signaling pathway in vivo, and how dysregulation of IR trafficking contributes to human insulin resistance remain largely unclear. Answering these questions requires identifying specific mediators and regulators of IR endocytosis, and a mechanistic understanding of the IR endocytic pathways in an animal model. Our long-term goal is to understand how systemic IR signaling controls metabolic homeostasis and genome stability. Our recent studies show that MAD2, a key mitosis regulator, cooperates with IR substrate (IRS) to promote IR endocytosis through the recruitment of the clathrin adaptor complex AP2 to the IR 8-10. Mechanistically, MAD2 constitutively binds to the IR through a well-conserved MAD2-interacting motif (MIM). The MAD2 inhibitor p31comet blocks the MAD2-dependent AP2 recruitment to IR. A phosphorylation switch of IRS controlled by MAPK and the tyrosine phosphatase SHP2 ensures selective internalization of insulin-activated IR. Targeting this feedback regulation prolongs the metabolic branch of IR signaling and improves insulin sensitivity in mice. Our preliminary data show that IR4A/4A mice (deficient for MAD2-binding and endocytosis) are resistant to diet-induced insulin resistance. We found that MAD2 is also required to keep ATP-binding-deficient IR (kinase dead) mutants in the endoplasmic reticulum (ER). We hypothesize that mitotic regulators maintain metabolic homeostasis by exerting spatiotemporal control of IR signaling inside the cell. To test this, we will: AIM 1. Establish the physiological function of IR spatiotemporal control by MAD2. Our preliminary data show that IR4A/4A mice, in which IR cannot bind to MAD2, display delayed IR endocytosis and prolonged IR signaling. We hypothesize that IR trafficking by MAD2 controls glucose and lipid metabolism. We will analyze the metabolic phenotypes of IR4A/4A mice, using wild-type (WT), liver-specific-p31-/-...