Essential role for NHERF in cAMP-mediated inhibition of the Na+-HCO3- co-transporter in BSC-1 cells.
Prior studies have indicated a requirement for the PDZ domain-containing protein, Na(+)/H(+) Exchanger Regulatory Factor (NHERF), for protein kinase A (PKA)-mediated inhibition of the renal basolateral Na(+)-HCO(3)(-) co-transporter (NBC). The present studies explore the potential mechanisms by which NHERF transduces cAMP signals to inhibit NBC. In BSC-1 cells, cells that express NBC but lack NHERF, 8-bromo-cAMP (100 microm for 15 min) failed to inhibit transport until wild-type mNHERF-(1-355) was expressed. mNHERF-(116-355) containing PDZ II and C-terminal ezrin-binding sequences or a mutant unphosphorylated form of rabbit NHERF effectively transduced the cAMP signals that inhibited NBC. By contrast, mNHERF-(1-126) encompassing N-terminal PDZ I and mNHERF-(1-325), which lacks ezrin-binding, failed to support cAMP inhibition of NBC activity. NBC and NHERF did not associate with each other in yeast two-hybrid or co-immunoprecipitation assays, and confocal microscopy indicated distinct subcellular localization of the two proteins. NBC was phosphorylated in BSC-1 cells, but its phosphorylation was not increased by cAMP nor was immunoprecipitated NBC phosphorylated by PKA in vitro. Acute exposure of mNHERF-(1-355)-expressing BSC-1 cells to cAMP did not change cell surface expression of NBC. Although these results established an essential role for NHERF in cAMP-mediated inhibition of NBC in BSC-1 cells, they also suggest a novel mechanism for NHERF-mediated signal transduction distinct from that previously characterized from studies of other NHERF targets.
Weinman
E J
EJ
Department of Medicine, University of Maryland School of Medicine and the Medical Service, Department of Veterans Affairs Medical Center, Baltimore, Maryland 21201, USA. eweinman@medicine.umaryland.edu
Evangelista
C M
CM
Steplock
D
D
Liu
M Z
MZ
Shenolikar
S
S
Bernardo
A
A
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D051379:Mice; D010750:Phosphoproteins / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation; D011817:Rabbits; D015398:Signal Transduction; D027982:Sodium-Bicarbonate Symporters / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000502:physiology; D017923:Sodium-Hydrogen Exchangers; D013329:Structure-Activity Relationship
D010750:Phosphoproteins; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases