Differential Effects of Filipin and Methyl-β-cyclodextrin on B Cell Receptor Signaling

• Published in: Biochemical and biophysical research communications Vol. 287; no. 1; pp. 77 - 82
• Main Authors: Awasthi-Kalia, Manjula, Schnetkamp, Paul P.M., Deans, Julie P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 14.09.2001
• Subjects: Anti-Bacterial Agents - pharmacology; B cell receptor; B-Lymphocytes - drug effects; B-Lymphocytes - metabolism; beta-Cyclodextrins; biochemistry; Biological Transport - drug effects; calcium; Calcium - metabolism; Cbp/PAG; cholesterol; cholesterol-binding reagents; cyclodextrin; Cyclodextrins - pharmacology; filipin; Filipin - pharmacology; Heat-Shock Proteins - metabolism; Humans; Membrane Microdomains - drug effects; methyl-^b-cyclodextrin; Mitogen-Activated Protein Kinases - metabolism; Peroxidases; Peroxiredoxins; rafts; Receptors, Antigen, B-Cell - drug effects; Receptors, Antigen, B-Cell - metabolism; signal transduction; Signal Transduction - drug effects; Tumor Cells, Cultured; Tyrosine - metabolism; rafts; calcium; signal transduction; Cbp/PAG; biochemistry; cyclodextrin; cholesterol; B cell receptor; filipin
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2001.5536

Methyl-β-cyclodextrin and filipin are cholesterol-binding reagents often used interchangeably to investigate functional requirements for lipid rafts in receptor-mediated signal transduction. Recently, contradictory results were reported by two groups using these reagents in different model systems to investigate the role of lipid rafts in BCR signaling. We confirm here that BCR-mediated calcium release is inhibited by filipin and enhanced by cyclodextrin. The inhibitory effect of filipin could not be attributed to raft disruption, however, because its ability to release raft-associated proteins into the detergent-soluble phase of cell lysates was less than that of cyclodextrin. In contrast, we found that filipin profoundly inhibited phosphorylation of the raft-associated adaptor protein Cbp/PAG, whereas the effect of cyclodextrin was minor. Thus, filipin and cyclodextrin modify cholesterol-rich microdomains through different mechanisms with different consequences on receptor signaling. In addition, the enhanced calcium release observed under conditions of maximum raft disruption suggests that rafts have a role in negatively regulating BCR signals.