Abstract
The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that binds to diverse ligands and initiates a downstream proinflammatory signaling cascade. RAGE activation has been linked to diabetic complications, Alzheimer disease, infections, and cancers. RAGE is known to mediate cell signaling and downstream proinflammatory gene transcription activation, although the precise mechanism surrounding receptor-ligand interactions is still being elucidated. Recent fluorescence resonance energy transfer evidence indicates that RAGE may form oligomers on the cell surface and that this could be related to signal transduction. To investigate whether RAGE forms oligomers, protein-protein interaction assays were carried out. Here, we demonstrate the interaction between RAGE molecules via their N-terminal V domain, which is an important region involved in ligand recognition. By protein cross-linking using water-soluble and membrane-impermeable cross-linker bis(sulfosuccinimidyl) suberate and nondenaturing gels, we show that RAGE forms homodimers at the plasma membrane, a process potentiated by S100B and advanced glycation end products. Soluble RAGE, the RAGE inhibitor, is also capable of binding to RAGE, similar to V peptide, as shown by surface plasmon resonance. Incubation of cells with soluble RAGE or RAGE V domain peptide inhibits RAGE dimerization, subsequent phosphorylation of intracellular MAPK proteins, and activation of NF-kappaB pathways. Thus, the data indicate that dimerization of RAGE represents an important component of RAGE-mediated cell signaling.
Highlights
Binds to multiple ligands, including S100 proteins, amphoterin/ high mobility group box-1, amyloid-, and Mac-1 [2,3,4,5]
Soluble receptor for advanced glycation end products (RAGE) may act as a dominant negative isoform and block RAGE signaling by functioning as an extracellular “decoy receptor” to inhibit RAGE ligand binding [14]
RAGE belongs to the immunoglobulin superfamily, and the protein consists of an N-terminal signal peptide, a V-type immunoglobulin-like domain, two tandem C-type immunoglobulin-like domains, a single transmembrane domain, and a short C-terminal intracellular cytoplasmic tail [1]
Summary
Cell Culture, Treatments, and Transfections—HEK293T cells from the American Type Culture Collection (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal calf serum (FCS) and 100 g/ml PrimocinTM and were maintained in a humidified incubator containing 5% CO2 at 37 °C. To further examine the RAGE homo-interaction, RAGE was expressed as a GST fusion protein in E. coli, purified, immobilized on glutathione-Sepharose 4B beads, and incubated with the lysates from GFP-RAGE-overexpressing HEK293T cells. Co-immunoprecipitation revealed that the deletions lacking the RAGE N-terminal V domain were incapable of binding to GFP-RAGE (Fig. 3B), indicating that the V domain is indispensable for RAGE homo-interaction Further evidence for this was obtained from fluorescent microscopy of HEK293T cells that were transfected with GFP-tagged V, C1, or C2 expression plasmid. Consistent with the previous results, S100B sRAGE or RAGE V peptide-treated cells compared with GST and AGE-BSA treatments increased GFP-RAGE dimerization control protein-treated cells (Fig. 6A).
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