NHERF associations with sodium-hydrogen exchanger isoform 3 (NHE3) and ezrin are essential for cAMP-mediated phosphorylation and inhibition of NHE3.
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.
Weinman
E J
EJ
Department of Medicine, University of Maryland School of Medicine and Medical Service, Department of Veterans Affairs Medical Center, Baltimore, Maryland 21201, USA.
Steplock
D
D
Donowitz
M
M
Shenolikar
S
S
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D003598:Cytoskeletal Proteins; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism / Q000502:physiology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000378:metabolism; D011817:Rabbits; D015398:Signal Transduction; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D014162:Transfection; D000099149:Ezrin
D000242:Cyclic AMP; D003598:Cytoskeletal Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D020033:Protein Isoforms; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D000099149:Ezrin; C000613385:SLC9A3 protein, human; C000613386:Slc9a3 protein, mouse; C093376:sodium-hydrogen exchanger regulatory factor