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 pr otein, Na+/H+ Exchanger Regulatory Factor (NHERF), for protein kinase A (PK A)mediated inhibition of the renal basolateral Na+-HCO3- co-transporter (NB C). The present studies explore the potential mechanisms by which NHERF tra nsduces cAMP signals to inhibit NBC. In BSC-1 cells, cells that express NBC but lack NHERF, 8-bromo-cAMP (100 mum for 15 min) failed to inhibit transp ort until wild-type mNHERF-(1-355) was expressed. mNHERF-(116-355) containi ng PDZ II and C-terminal ezrin-binding sequences or a mutant unphosphorylat ed form of rabbit NHERF effectively transduced the cAMP signals that inhibi ted NBC. By contrast, mNHERF-(1-126) encompassing N-terminal PDZ I and mNDE RF-(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 phosphoryla ted in BSC-1 cells, but its phosphorylation was not increased by cAMP nor w as 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 tha t previously characterized from studies of other NHERF targets.