Sodium-hydrogen exchanger regulatory factor 1 (NHERF-1) transduces signals that mediate dopamine inhibition of sodium-phosphate co-transport in mouse kidney

Abstract

Dopamine inhibited phosphate transport in isolated renal brush border membrane vesicles and in cultured renal proximal tubule cells from wild-type but not from NHERF-1 null mice. Co-immunoprecipitation experiments established that NHERF-1 associated with D1-like receptors. In wild-type mice, dopamine stimulated cAMP accumulation and protein kinase C (PKC) activity in renal proximal tubule cells, an effect that was abolished by SCH-23390, a D1-like receptor antagonist. In NHERF-1 null kidney tissue; however, dopamine failed to stimulate either cAMP accumulation or PKC activity. Infection of proximal tubule cells from NHERF-1 null mice with adenovirus-green fluorescent protein-NHERF-1 restored the ability of dopamine to stimulate cAMP and PKC. Finally, in (32)P-labeled wild-type proximal tubule cells and in opossum kidney cells, dopamine increased NHERF-1 phosphorylation at serine 77 of the PDZ I domain of NHERF-1, a site previously shown to attenuate binding of cellular targets including the Npt2a (sodium-dependent phosphate transporter 2a). Together, these studies establish that NHERF-1 plays a key role in dopamine signaling and is also a downstream target of D1-like receptors in the mouse kidney. These studies suggest a novel role for the PDZ adapter protein NHERF-1 in coordinating dopamine signals that inhibit renal phosphate transport.

FIGURE 1.

Sodium-dependent phosphate transport was measured in the absence (−D) or presence (+D) of dopamine in cultured NHERF-1 null proximal tubule cells infected with adenovirus GFP or adenovirus GFP-NHERF-1. *, p < 0.05.

FIGURE 2.

Representative Western immunoblots of brush border membrane vesicles from wild-type (WT) and NHERF-1^−/− mice using an antibody to the dopamine D1-like receptor (upper panel) and alkaline phosphatase (lower panel).

FIGURE 3.

D1-like receptors or NHERF-1 was immunoprecipitated (IP) from kidney slices from wild-type animals. The precipitates were immunoblotted for D1-like receptors (DR1) and NHERF-1. A representative experiment is shown.

FIGURE 4.

The accumulation of cAMP was determined in control (−D) or dopamine-treated (+D) kidney slices (A) and cultured proximal tubule cells (B) from wild-type or NHERF-1 null (NHERF-1 KO) animals. The results are expressed as fold increases ± S.E. relative to controls (−D) (shown as = 1). *, p < 0.05.

FIGURE 5.

PKC activation was determined in control (−D) or dopamine-treated (+D) kidney slices (A) and cultured proximal tubule cells (B) from wild-type or NHERF-1 null (NHERF-1 KO) animals. The results are expressed as fold increases ± S.E. relative to controls (−D) (shown as = 1). *, p < 0.05.

FIGURE 6.

cAMP accumulation (A) and PKC activation (B) was measured in control (−D) or dopamine-treated (+D) cultured proximal tubule cells from NHERF-1 null mice infected with adenovirus GFP or adenovirus GFP-NHERF-1. *, p < 0.05.

FIGURE 7.

The accumulation of cAMP was determined in control (−D) or dopamine-treated (+D) kidney slices (A) and cultured proximal tubule cells (B) from wild-type mice in the absence (Control) or presence of SCH-23390, a D1-like receptor antagonist. The results are expressed as fold increases ± S.E. relative to controls (−D) (shown as = 1). *, p < 0.05.

FIGURE 8.

PKC activation was determined in control (−D) or dopamine-treated (+D) kidney slices (A) and cultured proximal tubule cells (B) from wild-type mice in the absence (Control) or presence of SCH-23390, a D1-like receptor antagonist. The results are expressed as fold increases ± S.E. relative to controls (−D) (shown as = 1). *, p < 0.05.

FIGURE 9.

A, NHERF-1 was immunoprecipitated from [³²P]orthophosphate metabolically labeled wild-type proximal tubule cells in culture under control conditions (−Dopa) or treatment with dopamine (+Dopa). The proteins were resolved by SDS-PAGE, and a representative autoradiograph is shown. After the isotope counts dissipated, the gel was immunoblotted for NHERF-1. B, NHERF-1 cDNAs representing wild-type PDZ I, PDZ I in which all four serine and threonine residues were mutated to alanine residues (Mutation 1), or PDZ I in which all serine and threonine residues except serine 77 were mutated to alanine residues (Mutation 2) were expressed in [³²P]orthophosphate metabolically labeled wild-type proximal tubule cells in culture under control conditions (−D) or treatment with dopamine (+D). The PDZ I polypeptides were recovered using nickel chromatography. Representative autoradiographs are shown.

FIGURE 10.

Plasma membranes were prepared from kidney slices from wild-type animals under control conditions (−D) or after dopamine treatment (+D). The proteins were separated by SDS-PAGE and immunoblotted for Npt2a and NHERF-1.