Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

Bin Wang,Chris K Means,Yanmei Yang,Tatyana Mamonova,Alessandro Bisello,Daniel L Altschuler,John D Scott,Peter A Friedman

doi:10.1074/jbc.m112.369405

Abstract

Congenital defects in the Na/H exchanger regulatory factor-1 (NHERF1) are linked to disordered phosphate homeostasis and skeletal abnormalities in humans. In the kidney, these mutations interrupt parathyroid hormone (PTH)-responsive sequestration of the renal phosphate transporter, Npt2a, with ensuing urinary phosphate wasting. We now report that NHERF1, a modular PDZ domain scaffolding protein, coordinates the assembly of an obligate ternary complex with Npt2a and the PKA-anchoring protein ezrin to facilitate PTH-responsive cAMP signaling events. Activation of ezrin-anchored PKA initiates NHERF1 phosphorylation to disassemble the ternary complex, release Npt2a, and thereby inhibit phosphate transport. Loss-of-function mutations stabilize an inactive NHERF1 conformation that we show is refractory to PKA phosphorylation and impairs assembly of the ternary complex. Compensatory mutations introduced in mutant NHERF1 re-establish the integrity of the ternary complex to permit phosphorylation of NHERF1 and rescue PTH action. These findings offer new insights into a novel macromolecular mechanism for the physiological action of a critical ternary complex, where anchored PKA coordinates the assembly and turnover of the Npt2a-NHERF1-ezrin complex.

Highlights

Some inherited defects in Na/H exchanger regulatory factor-1 (NHERF1) are associated with high phosphate (Pi) excretion and skeletal abnormalities
We report that NHERF1, a modular PDZ domain scaffolding protein, coordinates the assembly of an obligate ternary complex with Npt2a and the protein kinase A (PKA)-anchoring protein ezrin to facilitate parathyroid hormone (PTH)-responsive cAMP signaling events
On the basis of their location, it is unlikely that these single amino acid substitutions interfere with Npt2a interactions with NHERF1 or signaling initiated upon PTH receptor (PTHR) binding

Summary

Background

Some inherited defects in NHERF1 are associated with high phosphate (Pi) excretion and skeletal abnormalities. Congenital defects in the Na/H exchanger regulatory factor-1 (NHERF1) are linked to disordered phosphate homeostasis and skeletal abnormalities in humans In the kidney, these mutations interrupt parathyroid hormone (PTH)-responsive sequestration of the renal phosphate transporter, Npt2a, with ensuing urinary phosphate wasting. Activation of ezrin-anchored PKA initiates NHERF1 phosphorylation to disassemble the ternary complex, release Npt2a, and thereby inhibit phosphate transport. Compensatory mutations introduced in mutant NHERF1 re-establish the integrity of the ternary complex to permit phosphorylation of NHERF1 and rescue PTH action These findings offer new insights into a novel macromolecular mechanism for the physiological action of a critical ternary complex, where anchored PKA coordinates the assembly and turnover of the Npt2a-NHERF1-ezrin complex. Npt2a is tightly regulated by PTH, which inhibits Pi transport by promoting Npt2a endocytosis and lysosomal degradation [16] These functions are not shared by NHERF2 or other NHERF1-like PDZ proteins [14]. These findings have been consolidated into a new molecular mechanism that can account for diminished Pi transport

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION