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

Abstract

Prior studies have indicated a requirement for the PDZ domain-containing protein, Na Inhibition of the renal apical membrane Na ϩ /H ϩ exchanger 3 (NHE3) 1 by cAMP-dependent protein kinase (PKA) requires the presence of an additional protein co-factor called the Na ϩ /H ϩ Exchanger Regulatory Factor (NHERF) (1). NHERF contains two tandem PSD-95/Dlg/ZO-1 (PDZ) domains and functions in a signal-complex of proteins, including ezrin, PKA, and NHE3 (1-3). This multiprotein complex facilitates the phosphorylation of NHE3 and the acute down-regulation of its activity (1-3). Subsequent to the elucidation of the role of NHERF in the regulation of NHE3, a role for this protein in the regulation of the activity of other transporters has been proposed, and it has been suggested that the signal-complex model of regulation of renal transport proteins may be more common than currently appreciated (4). In the renal proximal tubule, there is an intimate relation between the apical membrane Na ϩ /H ϩ exchanger and the basolateral membrane sodium bicarbonate co-transporter (NBC). These two transporters are regulated in parallel in response to a variety of experimental maneuvers, hormones, and second messengers (5-9). The coordinated regulation of NHE3 and NBC appears to be the result of specific regulatory mechanisms and not merely the consequence of the availability of ion substrates for the transporters. NBC, like NHE3, is regulated by stimuli that raise intracellular cAMP (8, 9). In association with Bernardo et al. (10), we have previously provided evidence that PKA-mediated inhibition of renal NBC requires NHERF. It is not known, however, if NHERF functions to form a signal-complex that regulates NBC activity or if NBC itself is phosphorylated by PKA. To study the relation between NBC and NHERF, wild-type, truncated, and mutated forms of NHERF were expressed in BSC-1 cells to determine if NHERF binds NBC, if NHERF is required for the phosphorylation of NBC, if the phosphorylation of NHERF is required for NBC regulation, and if NBC requires tethering to the actin cytoskeleton to be regulated by cAMP. The results indicate that the interaction between NHERF and NBC differs from the interactions between NHERF and NHE3. Although the ezrin-radixin-moesin (ERM) binding domain of NHERF is required for cAMP regulation of NBC, NHERF does not bind to NBC or facilitate cAMP-mediated phosphorylation of NBC. Moreover, 15-min treatment of the cells with cAMP does not affect the cell surface expression NBC. It is suggested that cAMP-mediated inhibition of NBC is the result of a biochemical modification of the NBC transporter by a process that requires NHERF but is not associated with phosphorylation of the transporter itself. EXPERIMENTAL PROCEDURESConstruction and Use of NHERF Expression Plasmids-Studies were performed using BSC-1 cells that express endogenous NBC activity but

Adenoviral expression of NHERF-1 in NHERF-1 null mouse renal proximal tubule cells restores Npt2a regulation by low phosphate media and parathyroid hormone

“…In cultured proximal tubule cells infected with GFP-NHERF-1 grown in low phosphate media, the increase in the plasma membrane abundance of Npt2a was not associated with a significant change in the abundance of GFP-NHERF-1. This finding is in accord with our prior observations in intact animals where feeding of a low-phosphate diet increased brush-border membrane abundance of Npt2a but not NHERF-1 (24). Taken together, these results indicate that within the sensitivity of these measurements, the recruitment of Npt2a to the plasma membrane is not obligatorily linked to the redistribution of NHERF-1.…”

“…For confocal microscopy, cells were fixed in paraformaldehyde as previously described (21,24). To obtain membrane preparations from the cultured cells, the cells were washed with sterile ice-cold phosphate-buffered saline, detached by scraping, and centrifuged for 5 min at 800 g. The supernatant was discarded and the pellet was resuspended in 1.5 ml of buffer containing 50 mM Tris (pH 7.4), 0.1 mM EDTA, 0.1% beta-mercapto-ethanol, and Complete Protease Inhibitor Cocktail (Roche Applied Science).…”

Effect of complete protein 4.1R deficiency on ion transport properties of murine erythrocytes

“…The NHE regulatory factor (NHERF) is an essential element in PKA-mediated inhibition of the predominantly renal NHE3 isoform (35)(36)(37). The COOH terminus of NHERF specifically interacts with protein of the family of membrane-cytoskeletal adapters ERM (ezrin-radixin-moesin) (33).…”

Activation-independent Parathyroid Hormone Receptor Internalization Is Regulated by NHERF1 (EBP50)

“…The role of NHERF1 in determining sensitivity to PTH-(7-34) was further and independently established by expressing a dominant negative form of NHERF1 (NHERH-(1-326), NHERF1⌬ERM) (35,36) in PCT cells that endogenously express NHERF1. Cells transfected with NHERF1⌬ERM now displayed PTH1R internalization in response to PTH-(7-34) (Fig.…”