NHERF associations with sodium-hydrogen exchanger isoform 3 (NHE3) and ezrin are essential for cAMP-mediated phosphorylation and inhibition of NHE3

Biochemistry. 2000 May 23;39(20):6123-9. doi: 10.1021/bi000064m.


The sodium-hydrogen exchanger regulatory factor (NHERF) is an essential cofactor for cAMP-mediated inhibition of the Na(+)/H(+) exchanger isoform, NHE3, in renal brush border membranes. NHERF is also an ezrin-binding protein. To define the functional importance of ezrin binding for NHERF's function as a NHE3 regulator, we transfected stable PS120 cells expressing NHE3 with plasmids encoding WT and truncated mouse NHERF proteins. Co-immunoprecipitation established that in PS120 cells, NHE3 bound to full-length NHERF(1-355), the C-terminal domain, NHERF(147-355), and NHERF(1-325), which lacks the proposed ezrin-binding domain. The N-terminal domain, NHERF(1-146), failed to bind the antiporter. Ezrin was also co-immunoprecipitated with NHERF(1-355) but not with NHERF(1-325). 8Br-cAMP inhibited NHE3 activity in cells that expressed NHERF(1-355) or NHERF(147-355) but had no effect on the formation of NHE3-NHERF or NHERF-ezrin complexes. Na(+)/H(+) exchange was unaffected by 8Br-cAMP in cells that expressed NHERF(1-146) or NHERF(1-325). NHE3 phosphorylation in vivo was enhanced by 8Br-cAMP only in cells where NHERF bound to both NHE3 and ezrin. The data suggest that NHERF functions as a scaffold to link NHE3 with ezrin and that this multiprotein complex is essential for cAMP-mediated phosphorylation of NHE3 and the inhibition of Na(+)/H(+) exchange.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Cells, Cultured
  • Cyclic AMP / physiology*
  • Cytoskeletal Proteins
  • Humans
  • Peptide Fragments / metabolism
  • Peptide Fragments / physiology
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Precipitin Tests
  • Protein Isoforms / antagonists & inhibitors
  • Protein Isoforms / metabolism
  • Rabbits
  • Signal Transduction
  • Sodium-Hydrogen Exchanger 3
  • Sodium-Hydrogen Exchangers / antagonists & inhibitors*
  • Sodium-Hydrogen Exchangers / metabolism*
  • Transfection


  • Cytoskeletal Proteins
  • Peptide Fragments
  • Phosphoproteins
  • Protein Isoforms
  • SLC9A3 protein, human
  • Slc9a3 protein, mouse
  • Sodium-Hydrogen Exchanger 3
  • Sodium-Hydrogen Exchangers
  • ezrin
  • sodium-hydrogen exchanger regulatory factor
  • Cyclic AMP