Altered polarization of PAR-2 signaling during airway epithelial remodeling

Ryan M. Carey; Jenna R. Freund; Benjamin M. Hariri; Nithin D. Adappa; James N. Palmer; Robert J. Lee

Aspergillus fumigatus also activates PAR-2 in the airway 7 and cornea. 34 A. fumigatus is a common cause of allergic bronchopulmonary aspergillosis or allergic fungal rhinosinusitis, 21 and can cause fatal invasive aspergillosis. 35 To confirm if A. fumigatus produces PAR-2-activating proteases, we utilized a fluorogenic assay (Trio assay) to directly visualize PAR-2 activation by its interaction with b-arrestin, 36 which associates with activated and phosphorylated GPCRs and mediates further signal transduction and/or internalization to endosomes 37 (Fig 1A) . This assay is based on a tripartite GFP, with the 11 th b strand fused to the C-terminus of PAR-2, the 10 th b-strand fused to barrestin, and b strands 1-9 expressed as a soluble protein. 36 When PAR-2 is activated, association with b-arrestin allows a complete GFP to form and increases fluorescence (Fig 1B) . This approach eliminates other effects that may impinge on downstream signaling (e.g., toll-like receptor activation during exposure to fungal conditioned media).