1089-OR: The Antiviral Response Proteins IFITM2/IFITM3 Bind to the Insulin Receptor and Drive Both Ligand and Tyrosine Kinase-Dependent and -Independent Functions

Introduction and Objective: Insulin classically signals through the tyrosine kinase activity of the insulin receptor (IR) to regulate metabolic and mitogenic processes. We recently identified a second IR signaling pathway that is mediated by IR’s intracellular domain but functions independently of ligand binding and tyrosine kinase activity (LYK-I) to control apoptotic sensitivity, modulate extracellular matrix signaling, and suppress immune and senescence-associated pathways. Here, we sought to define the molecular mechanisms underlying novel IR functions. Methods: Using affinity purification mass spectrometry, we identified classic LYK-D interactors (e.g., IRS-1, Grb10), as well as novel proteins that associate with IR in a LYK-I manner. Among these were the immunoregulatory proteins IFITM2 and IFITM3. Interactions between IR and IFITM2/3 were validated by co-immunoprecipitation and proximity ligation assays. To define the interaction interface, we generated a panel of IFITM3 mutants and assessed binding to wild-type human IR (hIR) and IR mutants (K1030R, dCT, JMO) in preadipocytes lacking endogenous IR/IGF1R. Results: Deletion of amino acids 72-92 in the cytosolic intracellular loop of IFITM3 abolished binding to IR constructs containing an intact intracellular domain, indicating this region is essential for interaction. Functionally, silencing IFITM3 enhanced serum starvation-induced apoptosis, whereas overexpression suppressed apoptosis in cells expressing either WT or kinase-dead IR, but not in cells lacking IR/IGF1R. Additionally, IFITM3 knockdown augmented insulin-stimulated IR and AKT phosphorylation, while the IFITM3-Y20A mutant increased basal and stimulated canonical signaling. Conclusion: Collectively, these findings demonstrate that both classical and LYK-I actions of the IR are mediated through a novel IR-IFITM2/3 signaling complex, providing a new mechanism for IR function beyond traditional kinase signaling in control of metabolism and apoptosis. Disclosure A. Ghosh: None. V.R. Muñoz: None. Y. Watanabe: None. M. Lino: None. C. Kahn: Consultant; Current; TIXiMED, Alnylam Pharmaceuticals, Inc. Board Member; Current; 1825 Therapeutics. Consultant; Ended; Cellarity. Funding National Institutes of Health