Summary/Abstract: TGF-Beta drives the fibroinflammatory processes that leads to lung and airway fibrosis. The long-term goal of this project is to acquire a deep understanding of the regulation of TGF-Beta activity to develop new strategies and treatments for fibrosing lung disease. There are few effective therapies to treat chronic fibrosing and inflammatory diseases of the lung. The cytokine TGF-Beta is a central mediator of fibrosis and pathologic inflammation and is a potential therapeutic target in fibrosing lung disease. However, the practical utility of targeting TGF-Beta itself or its receptors is limited by risk of toxicities seen in rodents, primates and humans. More specific methods to target the fibroinflammatory effects of TGF-Beta are highly desirable. A promising method to more specifically target local effects of TGF-Beta is to target its “activation” since it is always produced in a latent form (L-TGF-Beta) that must be activated in order to function. Another feature of L-TGF-Beta that could facilitate more specific targeting is that it is covalently bound to specific cell surfaces by GARP. L-TGF-Beta binding to the integrin alphavBeta8 is essential for TGF-Beta activation in vivo. For the alphavBeta8 activation mechanism, as well as all others, it has long been assumed that TGF-Beta must be released from LAP so that free TGF-Beta can diffuse and bind its receptors on target cells. Based on recent structural data obtained using single particle electron cryomicroscopy (cryo-EM), we have recently proposed a new model whereby alphavBeta8 can bind to L-TGF-Beta on cells presenting the L-TGF-Beta/GARP complex and induce signaling without release and diffusion of TGF-Beta. Here in three aims, we address three critical questions concerning this new model of L-TGF-Beta activation. (1) Which flexible domains of L-TGF-Beta of the alphavBeta8/L-TGF-Beta/GARP ternary complex shield TGF-Beta from its receptors? (2) Is flexibility of L-TGF-Beta induced by binding to alphavBeta8 necessary to mediate TGF-Beta activation? (3) Do TGF-Beta receptors (TGF-BetaRs) bind to TGF-Beta within the alphavBeta8/L-TGF-Beta/GARP complex? To answer these questions, we will use cryo-EM to determine the structure of the alphavBeta8/L-TGF-Beta/GARP complex to determine how flexibility of L-TGF-Beta contributes to TGF-Beta activation, and finally we will capture the multimeric complex of TGF-BetaRs with alphavBeta8/LTGF-Beta/GARP. These studies will improve mechanistic understanding of TGF-Beta activation and therapeutic targeting strategies to inhibit it.