The cytoplasmic domain of RAGE (Receptor for Advanced Glycation Endproduct) is selfsufficient to contribute cell adhesion in the presence of extracellular matrix

Abstract

PurposeThe Receptor for Advanced Glycation Endproducts (RAGE) is a pattern‐recognition, cell‐surface receptor of the immunoglobulins‐like receptor superfamily. 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. A more defined 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.MethodsFor 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 Hek 293 cells. 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.ResultsConfocal 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. 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 a 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 compared to mock and DN‐RAGE which were clearly rounded in shape.ConclusionRAGE 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.Support or Funding InformationThis research was supported by the NDSU College of Health Professions, the NDSU Department of Pharmaceutical Sciences, NIH Grant GM103332 and XCELLigence Research grant. Cartoon representation of the domain organization of full‐length RAGE (F‐RAGE) and the domain deletion variants used in this study.imageCartoon representation of the domain organization of full‐length RAGE (F‐RAGE) and the domain deletion variants used in this study.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.