BetaPix up-regulates Na+/H+ exchanger 3 through a Shank2-mediated protein-protein interaction

J Biol Chem. 2010 Mar 12;285(11):8104-13. doi: 10.1074/jbc.M109.055079. Epub 2010 Jan 14.


Na(+)/H(+) exchanger 3 (NHE3) plays an important role in neutral Na(+) transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of betaPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and betaPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, betaPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of betaPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in betaPix-mediated NHE3 regulation. Knockdown of endogenous betaPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that betaPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Polarity / physiology
  • Cells, Cultured
  • Cricetinae
  • Endothelial Cells / cytology
  • Endothelial Cells / enzymology*
  • Fibroblasts / cytology
  • Fibroblasts / enzymology
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Humans
  • Kidney / cytology
  • Membrane Proteins / metabolism
  • Nerve Tissue Proteins / metabolism*
  • Protein Binding / physiology
  • Protons
  • Rats
  • Rats, Sprague-Dawley
  • Rho Guanine Nucleotide Exchange Factors
  • Sodium / metabolism
  • Sodium-Hydrogen Exchanger 3
  • Sodium-Hydrogen Exchangers / metabolism*
  • Up-Regulation / physiology
  • rho GTP-Binding Proteins / metabolism


  • Guanine Nucleotide Exchange Factors
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Protons
  • Rho Guanine Nucleotide Exchange Factors
  • SHANK2 protein, human
  • SLC9A3 protein, human
  • Shank2 protein, rat
  • Slc9a3 protein, rat
  • Sodium-Hydrogen Exchanger 3
  • Sodium-Hydrogen Exchangers
  • Sodium
  • rho GTP-Binding Proteins