Essential Role for NHERF in cAMP-mediated Inhibition of the Na+-HCO3-Co-transporter in BSC-1 Cells

Edward J. Weinman, Christine M. Evangelista, Deborah Steplock, Min-Zhi Liu, Shirish Shenolikar, Angelito Bernardo
2001 Journal of Biological Chemistry  
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 M for 15 min) failed to inhibit transport until wild-type mNHERF-(1-355) was
more » ... sed. 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 twohybrid 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. EXPERIMENTAL PROCEDURES Construction and Use of NHERF Expression Plasmids-Studies were performed using BSC-1 cells that express endogenous NBC activity but
doi:10.1074/jbc.m106153200 pmid:11535598 fatcat:3essbngw4jbr3j6tmfpwyxp64m