F-domain valency determines outcome of signaling through the angiopoietin pathway.

Angiopoietins 1 and 2 (Ang1 and Ang2) regulate angiogenesis through their similar F-domains by activating Tie2 receptors on endothelial cells. Despite the similarity in the underlying receptor-binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of AKT, strengthens cell-cell junctions, and enhances endothelial cell survival while Ang2 can antagonize these effects, depending on cellular context. To investigate the molecular basis for the opposing effects, we examined the phenotypes of a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: Scaffolds presenting 3 or 4 F-domains have Ang2-like activity, upregulating pFAK and pERK but not pAKT, while scaffolds presenting 6, 8, 12, 30, or 60 F-domains have Ang1-like activity, upregulating pAKT and inducing migration and vascular stability. The scaffolds with 6 or more F-domains display super-agonist activity, producing stronger phenotypes at lower concentrations than Ang1. Tie2 super-agonist nanoparticles reduced blood extravasation and improved blood-brain barrier integrity four days after a controlled cortical impact injury.

F-domain valency determines outcome of signaling through the angiopoietin pathway.

Angiopoietin 1 and 2 (Ang1 and Ang2) modulate angiogenesis and vascular homeostasis through engagement of their very similar F-domain modules with the Tie2 receptor tyrosine kinase on endothelial cells. Despite this similarity in the underlying receptor binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of protein kinase B (AKT), strengthens cell-cell junctions and enhances endothelial cell survival while Ang2 antagonizes these effects 1-4 . To investigate the molecular basis for the opposing effects, we examined the protein kinase activation and morphological phenotypes produced by a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: scaffolds presenting 4 F-domains have Ang2 like activity, upregulating pFAK and pERK but not pAKT, and failing to induce cell migration and tube formation, while scaffolds presenting 6 or more F-domains have Ang1 like activity, upregulating pAKT and inducing migration and tube formation. The scaffolds with 8 or more F-domains display superagonist activity, producing stronger phenotypes at lower concentrations than Ang1. When examined in vivo , superagonist icosahedral self-assembling nanoparticles caused significant revascularization in hemorrhagic brains after a controlled cortical impact injury.