Abstract 1509 Antagonist-Induced Post-Translational Modifications Result in ANTXR2/CMG2 Degradation and Angiogenesis Inhibition

Pathological angiogenesis contributes to a myriad of diseases, including cancer, endometriosis, and corneal neovascularization. Current anti-angiogenic treatments (such as drugs targeting VEGF and VEGFR) have limited efficacy. Previously published work from our lab has shown that the anthrax protective antigen (PA) inhibits growth factor-induced angiogenesis by blocking ANTXR2/CMG2-mediated chemotaxis. This study aims to identify potential therapeutic targets to inhibit angiogenesis by elucidating how PA halts chemotaxis and resulting angiogenesis. We focused on the role of the ANTXR2/CMG2 cytosolic tail to help identify the signaling pathway responsible for the blockade of chemotaxis toward vascular growth factors. Several ANTXR2/CMG2 mutants, including a cytosolic tail domain deletion, tyrosine "phospho-null," and lysine "ubiquitin-null" constructs, were tested. Plasmids coding for these mutants were created using PCR-based mutagenesis or gene synthesis. A stable cell line was created for each ANTXR2/CMG2 construct under a doxycycline promoter via lentiviral transduction into EAhy.926 cells, followed by puromycin selection and cell sorting. Using Ibidi μ-slide chemotaxis devices, we characterized the effects of each mutation by measuring growth factor-induced chemotaxis. We observed no chemotaxis toward multiple growth factors in the cytosolic tail deletion cell line. This result strongly supports the requirement of the ANTXR2/CMG2 cytosolic tail for chemotaxis. However, mutating all cytosolic tail tyrosine residues to phenylalanine resulted in no inhibition of chemotaxis, even when PA was added to the assay. PA inhibition of chemotaxis was restored in specific tyrosine add-back mutants. Similarly, the ubiquitin-null mutant showed normal chemotaxis independent of PA treatment. These results align with the mechanism of ANTXR2/CMG2 internalization via endocytosis. We also showed that treatment of endothelial cells with PA results in a significant decrease in CMG2 protein levels, presumably due to the degradation of ANTXR2/CMG2 in lysosomes. Hence, antagonists that bind the extracellular domain of ANTXR2/CMG2 trigger a signaling cascade mediated by the cytosolic tail that results in endocytosis and eventual degradation. This prevents normal cell signaling and results in loss of growth factor-induced chemotaxis and resulting pathological angiogenesis. We acknowledge funding from the National Institutes of Health (R01 EY033354).