EWI-2 and EWI-F link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixin-moesin proteins

J Biol Chem. 2006 Jul 14;281(28):19665-75. doi: 10.1074/jbc.M602116200. Epub 2006 May 10.


EWI-2 and EWI-F, two members of a novel subfamily of Ig proteins, are direct partners of tetraspanins CD9 (Tspan29) and CD81 (Tspan28). These EWI proteins contain a stretch of basic charged amino acids in their cytoplasmic domains that may act as binding sites for actin-linking ezrin-radixin-moesin (ERM) proteins. Confocal microscopy analysis revealed that EWI-2 and EWI-F colocalized with ERM proteins at microspikes and microvilli of adherent cells and at the cellular uropod in polarized migrating leukocytes. Immunoprecipitation studies showed the association of EWI-2 and EWI-F with ERM proteins in vivo. Moreover, pulldown experiments and protein-protein binding assays with glutathione S-transferase fusion proteins containing the cytoplasmic domains of EWI proteins corroborated the strong and direct interaction between ERMs and these proteins. The active role of ERMs was further confirmed by double transfections with the N-terminal domain of moesin, which acts as a dominant negative form of ERMs, and was able to delocalize EWIs from the uropod of polarized leukocytes. In addition, direct association of EWI partner CD81 C-terminal domain with ERMs was also demonstrated. Functionally, silencing of endogenous EWI-2 expression by short interfering RNA in lymphoid CEM cells augmented cell migration, cellular polarity, and increased phosphorylation of ERMs. Hence, EWI proteins, through their direct interaction with ERM proteins, act as linkers to connect tetraspanin-associated microdomains to actin cytoskeleton regulating cell motility and polarity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / chemistry*
  • Animals
  • Antigens, CD / biosynthesis
  • Antigens, CD / physiology*
  • Cell Line, Tumor
  • Cells, Cultured
  • Cytoplasm / metabolism
  • Cytoskeleton / metabolism*
  • DNA-Binding Proteins / metabolism*
  • HeLa Cells
  • Humans
  • Membrane Glycoproteins / biosynthesis
  • Membrane Proteins / chemistry*
  • Membrane Proteins / physiology
  • Mice
  • Tetraspanin 28
  • Tetraspanin 29
  • Transcription Factors / metabolism*


  • Actins
  • Antigens, CD
  • CD81 protein, human
  • CD9 protein, human
  • Cd81 protein, mouse
  • Cd9 protein, mouse
  • DNA-Binding Proteins
  • ETV5 protein, human
  • Etv5 protein, mouse
  • IGSF8 protein, human
  • Membrane Glycoproteins
  • Membrane Proteins
  • Tetraspanin 28
  • Tetraspanin 29
  • Transcription Factors