Signaling molecules differentiate single versus dual ligation of complement receptor 3

Complement Receptor 3 (CR3), an integrin found on neutrophils, regulates firm cell adhesion to the extracellular matrix and serves as a pattern recognition receptor for the fungal Pathogen Associated Molecular Pattern (PAMP) β‐glucan. Coincident ligation of CR3 with fibronectin and β‐glucan is possible due to spatially distinct ligand binding domains. Current work demonstrates that dual ligation of CR3 with fibronectin at its I‐domain and β‐glucan at its lectin‐like site causes homotypic aggregation of primed human neutrophils. The downstream signaling pathways resulting from differential CR3 ligation are unknown. Using phosphoproteomics, we have identified several proteins that are significantly phosphorylated when ligated with fibronectin and β‐Glucan but not either ligand alone. Phosphoinositide 3 Kinase (PI3K), Protein Kinase Cδ (PKCδ), Glycogen Synthase Kinase 3β (GSK3β), and ERK show increased phosphorylation and each may play a role in the homotypic aggregation of neutrophils via CR3. Western Blotting has validated our initial phosphoproteomic findings for each protein. Use of inhibitors obviated homotypic aggregation of neutrophils for all aforementioned proteins except for GSK3β. Thus PI3K, PKCδ, and ERK are all components of the signaling pathway of dually ligated CR3 that are also relevant to homotypic aggregation.