Abstract 1360: CD40 Interacts with Filamin, But Signaling in Macrophages Does Not Require Lipid Raft Translocation

Abstract

Inhibition of CD40/CD40L interactions has been shown to reduce atherosclerosis and promote plaque stabilization in mice. However, in humans this would be accompanied by general suppression of the immune system. To enable more specific therapeutic intervention, we aim to elucidate the downstream signaling intermediates of CD40. In the present study, we focused on the initial events of CD40 signaling. Using a Yeast-two-Hybrid screening with total CD40 or the intracellular and transmembrane domain of CD40 as bait and a mouse atherosclerotic plaque cDNA or stimulated macrophages cDNA library, we identified filamin as a (intracellular) binding partner of CD40. Filamin is an actin-binding protein, involved in cell adhesion and migration, and is known to be involved in signaling by binding and clustering receptors. Interaction with CD40 has not been described before. Filamin also interacts with caveolin-1, a protein abundantly found in lipid rafts. To investigate CD40 translocation to lipid rafts in macrophages, RAW 264.7 cells were stimulated with the CD40-activating antibody 1C10 (10 μg/ml) for 0, 5, 15 and 30 min. after which the cells were lysed in 1% Triton in Mes-buffered saline pH6.5 for 30 min. Lipid rafts were extracted by ultracentrifugation in a 5– 40% sucrose gradient and CD40 was detected by Western Blotting of the distinct sucrose fractions. A small amount of CD40 was found to translocate to the lipid raft fractions after 15 min. of stimulation whereas no CD40 was found in lipid rafts without stimulation. We evaluated the functional relevance of lipid raft integrity for CD40 signaling by investigating activation of ERK1/2 and p38 after CD40 stimulation when lipid rafts were disrupted by methyl-beta cyclodextrin (5 mM) or nystatin (50 μg/ml). We found CD40 stimulation to induce activation of ERK1/2 and p38 even when lipid rafts were disrupted, indicating that CD40 signaling in macrophages does not require intact lipid rafts. In conclusion, we identified a novel interaction of CD40 with the actin-binding protein filamin, which has a role in signaling by binding and clustering of membrane receptors, probably/potentially within lipid rafts. In macrophages, however, we showed CD40 signaling to be independent of lipid rafts. NHS grant 2003B226 & 2005R015