Abstract 260: Interruption of Protein-Protein Interaction of CD36 With Other Proteins Alters CD36 Biological Functions
CD36 is a transmembrane glycoprotein belonging to the type 2 scavenger receptor family. On macrophages and platelets CD36 recognizes oxidized LDL and other endogenous danger signals and thereby transmits signals that promote athero-thrombotic processes. CD36 has been shown to associate physically with other transmembrane proteins, including integrins, tetraspanins, and toll-like receptors, and CD36-mediated cell signaling is modulated by these partner molecules. The CD36 N-terminal transmembrane domain (nTMD) contains a GXXXG sequence motif that in several membrane proteins has been shown to mediate protein-protein interactions. We thus hypothesized that the nTMD is involved in CD36 protein-protein interactions. We detected CD36 interactions with specific macrophage surface proteins with an immunofluorescence-based cross linking assay (Proximity Ligation Assay®/PLA) and confirmed them by immunoprecipitation/immunoblot (IP/IB). Prior to these assays cells were incubated with a synthetic 29 amino acid peptide containing the 22 amino acid of CD36 nTMD or a control peptide in which the glycine residues in GXXXG motif were replaced by valine. In functional experiments, macrophages were pre-incubated with peptides and then treated with oxLDL or native LDL prior to assessing LDL uptake at 1hr, foam cell formation after 24hr by oil red O staining, ROS generation by fluorescence probes, and cell migration in a modified Boyden Chamber Assay. CD36 nTMD peptide treated cells compared to untreated or control peptide treated cells had decreased CD36 surface associations with both CD9 and CD18 as detected by both PLA and IP/IB. Functional assays revealed that CD36 nTMD peptide significantly decreased DiI-labeled oxLDL uptake and decreased foam cell formation by 44% (p <0.0001). This was associated with a 32% decrease in oxLDL-mediated ROS generation (p < 0.001). OxLDL-induced inhibition of macrophage migration was inhibited by 84% (p < 0.0001) by TMD treatment compared to the control peptide. In summary, these data support the hypothesis that CD36 nTMD mediates interaction of CD36 with other proteins such as CD9 and CD18, and that altering these interactions in macrophages ameliorates pathologically important CD36 mediated responses to oxLDL.
Author Disclosures: W. Huang: None. R. Li: None. D. Ramakrishnan: None. R.L. Silverstein: None.
- © 2014 by American Heart Association, Inc.