Lectin Site Ligation of CR3 Induces Conformational Changes and Signaling*

Background: CR3 is a β2 integrin that contains a lectin-like domain that binds the fungal pathogen-associated molecular pattern β-glucan. Results: Soluble β-glucan stabilizes an intermediate CR3 conformation that induces differential intracellular phosphorylation. Conclusion: CR3 is a signaling pattern recognition receptor for β-glucan. Significance: The CR3 receptor is a target for the design of novel immune modulators. Neutrophils provide an innate immune response to tissues infected with fungal pathogens such as Candida albicans. This response is tightly regulated in part through the interaction of integrins with extracellular matrix ligands that are distributed within infected tissues. The β2 integrin, CR3 (CD11b/CD18), is unique among integrins in containing a lectin-like domain that binds the fungal pathogen-associated molecular pattern β-glucan and serves as the dominant receptor for recognition of fungal pathogens by human granulocytes. β-Glucan, when isolated in soluble form, has been shown to be a safe and effective immune potentiator when administered therapeutically. Currently a pharmaceutical grade preparation of β-glucan is in several clinical trials with an anti-cancer indication. CR3 binding of extracellular matrix, carbohydrate, or both ligands simultaneously differentially regulates neutrophil function through a mechanism not clearly understood. Using FRET reporters, we interrogated the effects of soluble β-glucan on intracellular and extracellular CR3 structure. Although the canonical CR3 ligand fibrinogen induced full activation, β-glucan alone or in conjunction with fibrinogen stabilized an intermediate conformation with moderate headpiece extension and full cytoplasmic tail separation. A set of phosphopeptides differentially regulated by β-glucan in a CR3-dependent manner were identified using functional proteomics and found to be enriched for signaling molecules and proteins involved in transcriptional regulation, mRNA processing, and alternative splicing. These data confirm that CR3 is a signaling pattern recognition receptor for β-glucan and represent the first direct evidence of soluble β-glucan binding and affecting a signaling-competent intermediate CR3 conformation on living cells.