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

Abstract

Sodium-dependent phosphate transport in NHERF-1−/− proximal tubule cells does not increase when grown in a low phosphate media and is resistant to the normal inhibitory effects of parathyroid hormone (PTH). The current experiments employ adenovirus-mediated gene transfer in primary cultures of mouse proximal tubule cells from NHERF-1 null mice to explore the specific role of NHERF-1 on regulated Npt2a trafficking and sodium-dependent phosphate transport. NHERF-1 null cells have decreased sodium-dependent phosphate transport compared with wild-type cells. Infection of NHERF-1 null cells with adenovirus-GFP-NHERF-1 increased phosphate transport and plasma membrane abundance of Npt2a. Adenovirus-GFP-NHERF-1 infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP demonstrated increased phosphate transport and Npt2a abundance in the plasma membrane when grown in low phosphate (0.1 mM) compared with high phosphate media (1.9 mM). PTH inhibited phosphate transport and decreased Npt2a abundance in the plasma membrane of adenovirus-GFP-NHERF-1-infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP. Interestingly, phosphate transport is inhibited by activation of protein kinase A and protein kinase C in wild-type proximal tubule cells but not in NHERF-1−/− cells. Together, these results highlight the requirement for NHERF-1 for physiological control of Npt2a trafficking and suggest that the Npt2a/NHERF-1 complex represents a unique PTH-responsive pool of Npt2a in renal microvilli.

Parathyroid hormone inhibits renal phosphate transport by phosphorylation of serine 77 of sodium-hydrogen exchanger regulatory factor–1

“…Acting through the activation of PKC and PKA, PTH facilitates the internalization of Npt2a from the apical membrane of renal proximal tubule cells and, as a result, decreases the reabsorption of phosphate (1,3,6). The biochemical pathways mediating these responses, however, have not been well defined.…”

“…This hypothesis derives from the observations that phosphate transport in renal proximal tubule cells from NHERF-1 -/-mice are resistant to the inhibitory effect of PTH as well as activators of PKC and PKA, the second messenger pathways of the PTH1 receptor (2, 3). Rescue of these cells using viral-mediated gene transfer of NHERF-1 restores the inhibitory response to PTH and to second messenger agonists of PKC and PKA (6). Of the 4 potential phosphorylation sites in PDZ I of NHERF-1, S77 was of particular interest given its location near the binding groove of PDZ I domain and the fact that it is phosphorylated in vivo (15).…”

“…An insight into this process was provided by the observations that Npt2a binds to the PDZ domain adaptor protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) and that NHERF-1 -/-mice demonstrate phosphaturia and mistargeting of Npt2a (4,5). Subsequent experiments demonstrated that NHERF-1 functions as a membrane retention signal for Npt2a and that sodium-dependent phosphate transport in renal proximal tubule cells from NHERF-1 mice was resistant to the inhibitory effect of PTH (3,6,7). NHERF-1 -/-cells were also resistant to the inhibitory effect of activators of PKC and PKA, the 2 major signaling pathways of the PTH1 receptor, indicating that the resistance to PTH derived from the interaction between NHERF-1 and Npt2a (6).…”

“…Subsequent experiments demonstrated that NHERF-1 functions as a membrane retention signal for Npt2a and that sodium-dependent phosphate transport in renal proximal tubule cells from NHERF-1 mice was resistant to the inhibitory effect of PTH (3,6,7). NHERF-1 -/-cells were also resistant to the inhibitory effect of activators of PKC and PKA, the 2 major signaling pathways of the PTH1 receptor, indicating that the resistance to PTH derived from the interaction between NHERF-1 and Npt2a (6). It was originally hypothesized that the regulation of Npt2a involved the phosphorylation of the transporter itself, but extensive mutagenesis studies by Murer and colleagues failed to identify modifiable residues that accounted for the effect of PTH on the apical membrane abundance of Npt2a (8,9).…”

Dynamic Na+-H+ Exchanger Regulatory Factor-1 Association and Dissociation Regulate Parathyroid Hormone Receptor Trafficking at Membrane Microdomains

“…Membrane-targeted CFP (myristoylation-palmitoylation CFP) (26), HA-PTHR (15), GFP NHERF (27), mCerulean 3-C1 (28),…”