The O-glycosylated Ectodomain of FXYD5 Impairs Adhesion by Disrupting Cell-Cell Trans-Dimerization of Na,K-ATPase β1 Subunits

J Cell Sci. 2016 Jun 15;129(12):2394-406. doi: 10.1242/jcs.186148. Epub 2016 May 3.

Abstract

FXYD5 (also known as dysadherin), a regulatory subunit of the Na,K-ATPase, impairs intercellular adhesion by a poorly understood mechanism. Here, we determined whether FXYD5 disrupts the trans-dimerization of Na,K-ATPase molecules located in neighboring cells. Mutagenesis of the Na,K-ATPase β1 subunit identified four conserved residues, including Y199, that are crucial for the intercellular Na,K-ATPase trans-dimerization and adhesion. Modulation of expression of FXYD5 or of the β1 subunit with intact or mutated β1-β1 binding sites demonstrated that the anti-adhesive effect of FXYD5 depends on the presence of Y199 in the β1 subunit. Immunodetection of the plasma membrane FXYD5 was prevented by the presence of O-glycans. Partial FXYD5 deglycosylation enabled antibody binding and showed that the protein level and the degree of O-glycosylation were greater in cancer than in normal cells. FXYD5-induced impairment of adhesion was abolished by both genetic and pharmacological inhibition of FXYD5 O-glycosylation. Therefore, the extracellular O-glycosylated domain of FXYD5 impairs adhesion by interfering with intercellular β1-β1 interactions, suggesting that the ratio between FXYD5 and α1-β1 heterodimer determines whether the Na,K-ATPase acts as a positive or negative regulator of intercellular adhesion.

Keywords: Cell adhesion; Cell–cell interaction; Epithelial cell adhesion molecule; Epithelium; FXYD5; Na,K-ATPase; O-glycosylation.

MeSH terms

  • A549 Cells
  • Amino Acids / metabolism
  • Animals
  • Antibody Specificity
  • Cell Adhesion
  • Cell Line, Tumor
  • Cell Membrane / metabolism
  • Dogs
  • Epithelial Cells / metabolism
  • Gene Knockdown Techniques
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Madin Darby Canine Kidney Cells
  • Membrane Glycoproteins / metabolism*
  • Mice
  • Neoplasm Proteins / metabolism*
  • Protein Binding
  • Protein Multimerization*
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism*
  • Rats
  • Sodium-Potassium-Exchanging ATPase / chemistry
  • Sodium-Potassium-Exchanging ATPase / metabolism*

Substances

  • Amino Acids
  • FXYD5 protein, human
  • Membrane Glycoproteins
  • Neoplasm Proteins
  • Protein Subunits
  • Sodium-Potassium-Exchanging ATPase