Figure 1.

A. fumigatus CM directly activates PAR-2 in airway cells. a, diagram of GPCR interaction with β-arrestin after activation and phosphorylation by G-receptor kinase (GRK) b, diagram of the Trio assay (modified from Ref. 26) used to detect PAR-2 activation. A heterologously-expressed GPCR (in this case PAR-2) is tagged at the C-terminal end with the β11-strand of GFP. The receptor is co-expressed with β-arrestin tagged with the β10-strand of GFP along with soluble GFP β-strands 1–9. Association of the PAR-2 with β-arrestin allows the formation of a complete fluorescent GFP molecule; soluble mCherry is included on the plasmid as a transfection control (not shown in the model). c, representative images of A549 cells expressing PAR-2 Trio components (plus mCherry as transfection control) stimulated with 10 μm 2FLI for 0–120 min. Scale bars are 15 μm. d, normalized GFP fluorescence in cells stimulated with 2FLI or buffer alone (HBSS) at time points taken over 2 h. Data points are independent experiments imaged on three different days (n = 5–9). a and b were created with Biorender.com.