Purpose: The Receptor for Advanced Glycation Endproducts (RAGE) is a pattern-recognition, cell-surface receptor of the immunoglobulin-like receptor superfamily. RAGE is an actively investigated therapeutic target for Acute Respiratory Syndrome (clinical trial identifiers: NCT01270295), Alzheimer's disease (clinical trial identifiers: NCT00566397 and NCT02916056) and is under intense pre-clinical investigation for several cancers (Curr. Mol. Med.; 7(8), 777-789) and vascular disease (Mol. Med.; 13(11-12), 625-635). RAGE activation by binding of its soluble ligands has been documented to promote pro-inflammatory signaling, cytokine release, and phenotypic changes. While the pathophysiological significance of RAGE as a signaling receptor is undisputed, it is not clear whether RAGE may also have critical roles in cell-cell and cell-matrix adhesion. Based on computational sequence and structure analysis, it has been suggested that RAGE is a cell-adhesion molecule with similarity to ALCAM, BCAM, and MCAM. A more refined understanding of the biochemical and biophysical properties of RAGE will be of tremendous value to rationalize its biological function in disease development and progression. Identifying the biological functions of individual protein domains of the RAGE receptor molecule will accelerate the rational discovery of drugs targeting RAGE.
Methods: For this study, a protein engineering approach is used to express full length RAGE (F-RAGE) and a panel of domain deletion constructs (ΔV, ΔC1, ΔC2, DN-RAGE, TmCyto RAGE) of the receptor. The necessary expression constructs were assembled in the pcDNA 3 vector and the RAGE variants were expressed in Hek293 cells. The expression and cellular localization in Hek 293 cells were analyzed using western blot, immunofluorescence, and flow cytometry techniques. The contribution of individual domains of RAGE towards cell adherence and spreading were evaluated by cell adhesion and cell spreading assays for all domain deletion constructs. Differences in cell adhesion and expression of cell adhesion molecules were investigated.
Results: Confocal fluorescence microscopy and flow cytometry was used to determine the cellular localization of the RAGE constructs. All constructs localized to the plasma membrane, except for the TmCyto and ΔV-RAGE constructs, which had an intercellular or nuclear localization. Deletion of the V-domain appears to impair translocation of RAGE to the cell surface. Cell adhesion experiments using 96-well plate assays demonstrated a 2-fold increased adhesion in RAGE transfected HEK 293 cells compared to the mock transfected cells. Cell adhesion measurements to collagen IV using a Xcelligence RTCA DP system revealed that deletion of individual domains gradually reduced cell adhesion compared to full-length RAGE. Surprisingly, the DN-RAGE construct, which differs from F-RAGE only in the deletion of the intracellular tail, showed complete loss of RAGE–mediated cell adhesion. This suggests that the cytoplasmic tail is required to modulate cell adhesion via RAGE. Interestingly, expression of the membrane anchored cytoplasmic tail (TmCyto), restored cell adhesion to level exceeding those observed for F-RAGE expressing cells. Cell spreading data showed a similar pattern as the cell adhesion results in which F-RAGE and TmCyto transfected cells had a dense, almost neuronal-like, spread pattern with low circularity of <0.4 (a circularity value of 1 indicates perfect circle) compared to mock and DN-RAGE which were clearly rounded in shape with circularity values of > 0.8.
These data suggest that RAGE expression modulates cell adhesion through two mechanisms. Direct physical binding of the extracellular portion of RAGE to matrix molecules and RAGE-mediated signaling that leads to changes in cell adhesion molecule expression. In support of this hypothesis is the observation that RAGE expression led to significant changes in the expression of certain cell adhesion molecules as demonstrated by qPCR and Western analysis.
Conclusion: RAGE does modulate cell adhesion and cell spreading properties through direct interaction of the extracellular domains of RAGE with matrix proteins, as well as through RAGE-signaling, which does require the intracellular tail of RAGE. Drugs targeting the intracellular cytoplasmic domain of RAGE might become useful to modulate tumor cell adhesion, spreading, and migration in cancers with high levels of RAGE expression.