Abstract. The cell adhesion receptor integrin αVβ8 is important for vascular and cardiac morphogenesis, lung epithelial homeostasis, CNS development, immune function, and latent transforming growth factor beta (TGF-β) activation. Ligand binding and integrin activation are coupled in most integrins to two distinct, large conformational changes: extension at the bent integrin knees and opening of the ligand-binding headpiece. Thus, most integrins can equilibrate between two low affinity conformations, bent-closed and extended-closed, and a high affinity extended-open conformation. However, integrin αVβ8 is atypical because it is constitutively extended and resistant to ligand-induced headpiece opening. Moreover, the β8 cytoplasmic domain lacks binding motifs for the cytoskeletal adaptors talin and kindlin, but binds a distinct cytoskeletal protein associated with lung adenocarcinoma, DAL-1. We will structurally and functionally characterize αVβ8 to determine the molecular mechanisms that govern its atypical behavior. In Aim 1, we obtain an overview of αVβ8 ectodomain and headpiece conformational states compared to αVβ6 by EM. We investigate whether binding of pro-TGF-β1 or a crossreacting, de novo designed αVβ6-binding protein (αVβ6-BP) induces headpiece opening of αVβ8. We mutationally examine whether divergent sequences in β8 metal-binding sites or in the βI-hybrid interface stabilize the closed headpiece and whether divergent sequence at the β-knee stabilizes the extended conformation. In Aim 2, we obtain crystal structures of the αVβ8 headpiece, its complexes with peptide ligand, pro-TGF-β1, or αVβ6-BP and a structure of the αVβ8 ectodomain. Structures will demonstrate how αVβ8 resists ligand-induced headpiece opening and maintains constitutive extension. In Aim 3, we investigate affinity and functional regulation of αVβ8 and transition to developing a potential therapeutic. We characterize the structure of the β8 cytoplasmic domain bound to DAL-1 and investigate the role of this interaction in αVβ8-mediated activation of latent pro-TGF-β1. We hypothesize that in vivo, the αVβ8 headpiece may be opened by inside-out signaling, and increase affinity for ligand. To establish whether headpiece opening of αVβ8 increases affinity, we will use mutations to release restraints on αVβ8 headpiece opening, measure their effect on affinity for ligand, and correlate increases in affinity with headpiece opening measured by EM. Structures of αVβ8 bound to current, highly potent αVβ6 and αVβ8 small molecule antagonists will explain their limited selectivity for αVβ6 and αVβ8. The insights obtained will guide development of second generation, highly selective, pure antagonist therapeutics to treat diseases such as chronic obstructive pulmonary disease.