CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation

PLoS One. 2016 Mar 1;11(3):e0147489. doi: 10.1371/journal.pone.0147489. eCollection 2016.


CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC) caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Breast Neoplasms / metabolism*
  • Breast Neoplasms / pathology
  • Cell Adhesion
  • Cell Adhesion Molecules / physiology*
  • Cell Differentiation
  • Chloride Channels / metabolism*
  • Conserved Sequence
  • Epithelial Cells / physiology*
  • Epithelial-Mesenchymal Transition
  • Female
  • Homeostasis
  • Humans
  • Intercellular Junctions / metabolism
  • MCF-7 Cells
  • Mammary Glands, Human / pathology
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Transport
  • Signal Transduction
  • Zonula Occludens-1 Protein / metabolism
  • beta Catenin / metabolism


  • CLCA2 protein, human
  • CTNNB1 protein, human
  • Cell Adhesion Molecules
  • Chloride Channels
  • MPZL2 protein, human
  • TJP1 protein, human
  • Zonula Occludens-1 Protein
  • beta Catenin