Reciprocal protein kinase A regulatory interactions between cystic fibrosis transmembrane conductance regulator and Na+/H+ exchanger isoform 3 in a renal polarized epithelial cell model.
Although Cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to regulate the activity of NHE3, the potential reciprocal interaction of NHE3 to modulate the protein kinase A (PKA)-dependent regulation of CFTR in epithelial cells is still unknown. In the present work, we describe experiments to define the interactions between CFTR and NHE3 with the regulatory, scaffolding protein, NHERF that organize their PKA-dependent regulation in a renal epithelial cell line that expresses endogenous CFTR. The expression of rat NHE3 significantly decreased PKA-dependent activation of CFTR without altering CFTR expression, and this decrease was prevented by mutation of either of the two rat NHE3 PKA target serines to alanine (S552A or S605A). Inhibition of CFTR expression by antisense treatment resulted in an acute decrease in PKA-dependent regulation of NHE3 activity. CFTR, NHE3, and ezrin were recognized by NHERF-2 but not NHERF-1 in glutathione S-transferase pull-down experiments. Ezrin may function as a protein kinase A anchoring protein (AKAP) in this signaling complex, because blocking the binding of PKA to an AKAP by incubation with the S-Ht31 peptide inhibited the PKA-dependent regulation of CFTR in the absence of NHE3. In the A6-NHE3 cells S-Ht31 blocked the PKA regulation of NHE3 whereas it now failed to affect the regulation of CFTR. We conclude that CFTR and NHE3 reciprocally interact via a shared regulatory complex comprised of NHERF-2, ezrin, and PKA.
Bagorda
Anna
A
Department of General and Environmental Physiology, University of Bari, Bari 70126, Italy.
Guerra
Lorenzo
L
Di Sole
Francesca
F
Hemle-Kolb
Corinna
C
Cardone
Rosa A
RA
Fanelli
Teresa
T
Reshkin
Stephan J
SJ
Gisler
Serge M
SM
Murer
Heini
H
Casavola
Valeria
V
D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D002713:Chlorine / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008856:Microscopy, Fluorescence; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D000099149:Ezrin
D002713:Chlorine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003598:Cytoskeletal Proteins; D005982:Glutathione Transferase; D007477:Ions; D016376:Oligonucleotides, Antisense; D010455:Peptides; D010750:Phosphoproteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D000099149:Ezrin; C093376:sodium-hydrogen exchanger regulatory factor