Abstract
Cell-surface tumor marker EpCAM plays a key role in proliferation, differentiation and adhesion processes in stem and epithelial cells. It is established as a cell-cell adhesion molecule, forming intercellular interactions through homophilic association. However, the mechanism by which such interactions arise has not yet been fully elucidated. Here, we first show that EpCAM monomers do not associate into oligomers that would resemble an inter-cellular homo-oligomer, capable of mediating cell-cell adhesion, by using SAXS, XL-MS and bead aggregation assays. Second, we also show that EpCAM forms stable dimers on the surface of a cell with pre-formed cell-cell contacts using FLIM-FRET; however, no inter-cellular homo-oligomers were detectable. Thus, our study provides clear evidence that EpCAM indeed does not function as a homophilic cell adhesion molecule and therefore calls for a significant revision of its role in both normal and cancerous tissues. In the light of this, we strongly support the previously suggested name Epithelial Cell Activating Molecule instead of the Epithelial Cell Adhesion Molecule.
Highlights
Epithelial Cell Adhesion Molecule (EpCAM) is a cell-surface type I transmembrane glycoprotein, which was initially identified as a colorectal carcinoma antigen[1,2]
First we utilized small angle X-ray scattering (SAXS) on highly concentrated EpCAM’s extracellular domain (EpEX) samples to see if high protein concentration leads to the formation of amount of higher-order oligomers which could be below the detection limit at low concentration, since the dissociation constant (Kd) of the proposed trans-tetramer is much higher than the Kd of the cis-dimer
Initial analysis of the function of EpCAM determined a role in cell-cell adhesion[19,26] through homophilic interactions[23,24]. These assigned functions led to its name as epithelial cell adhesion molecule
Summary
Epithelial Cell Adhesion Molecule (EpCAM) is a cell-surface type I transmembrane glycoprotein, which was initially identified as a colorectal carcinoma antigen[1,2]. RIP, the initial event in signaling, is believed to be triggered by trans-oligomerization of EpCAM’s extracellular domain, i.e. by binding of free EpEX to cell-bound EpCAM or via inter-cellular interaction between EpCAM molecules on adjacent cells[22], as opposed to cis-oligomerization involving EpCAM molecules on the same cell. In our previous work with soluble EpEX25 we failed to observe any evidence of higher-order oligomerization (i.e., more than dimerization) Combined, these findings encouraged us to reinvestigate the mechanism of EpCAM’s role as a cell-cell adhesion molecule (CAM) on a molecular level with an aim to provide a detailed explanation of the observed inconsistencies regarding the protein’s function. We present structural analysis of EpCAM oligomerization and its ability to form higher-order oligomers, which would be consistent with the formation of homophilic cell-cell adhesion units. Subunits in the dimer are depicted as cyan and magenta ribbons
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