ABSTRACT
Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, ß-, γ-, and ε-sites to generate soluble ectodomains, soluble Aß-like-, and intracellular fragments termed mEpEX, mEp-ß, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and ß-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aß-like fragments mEp-ß and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.
Subject(s)
Antigens, Neoplasm/metabolism , Cell Adhesion Molecules/metabolism , Cell Membrane/metabolism , Amino Acid Sequence , Amyloid Precursor Protein Secretases/metabolism , Animals , Antigens, Neoplasm/chemistry , Cell Adhesion Molecules/chemistry , Cell Line , Conserved Sequence , Epithelial Cell Adhesion Molecule , Humans , Mice , Molecular Sequence Data , Proteasome Endopeptidase Complex/metabolism , Protein Interaction Domains and Motifs , Proteolysis , VertebratesABSTRACT
The molecular association between APP and its mammalian homologs has hardly been explored. In systematically addressing this issue, we show by live cell imaging that APLP1 mainly localizes to the cell surface, whereas APP and APLP2 are mostly found in intracellular compartments. Homo- and heterotypic cis interactions of APP family members could be detected by FRET and co-immunoprecipitation analysis and occur in a modular mode. Only APLP1 formed trans interactions, supporting the argument for a putative specific role of APLP1 in cell adhesion. Deletion mutants of APP family members revealed two highly conserved regions as important for the protein crosstalk. In particular, the N-terminal half of the ectodomain was crucial for APP and APLP2 interactions. By contrast, multimerization of APLP1 was only partially dependent on this domain but strongly on the C-terminal half of the ectodomain. We further observed that coexpression of APP with APLP1 or APLP2 leads to diminished generation of Abeta42. The current data suggest that this is due to the formation of heteromeric complexes, opening the way for novel therapeutic strategies targeting these complexes.
