Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport

J Biol Chem. 2019 Mar 22;294(12):4546-4571. doi: 10.1074/jbc.RA119.007421. Epub 2019 Jan 29.


Na+-H+ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser46, Ser162, Ser181, Ser269, Ser280, Ser291, Thr293, Ser299, and Ser302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser290 Mutating 257VPF259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that β-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser290 phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.

Keywords: NHERF1; NPT2A; PDZ domain; hydrogen–deuterium exchange; phosphate transport; phosphoprotein phosphatase 1 (PP1); protein phosphorylation; protein–protein interaction; structural biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Crystallography, X-Ray
  • Furans / pharmacology
  • HEK293 Cells
  • Humans
  • Ion Transport / physiology
  • Lipids / pharmacology
  • Parathyroid Hormone / physiology*
  • Phosphates / metabolism*
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Conformation
  • Receptors, Neuropeptide Y / antagonists & inhibitors
  • Receptors, Neuropeptide Y / metabolism
  • Serine / metabolism
  • Sodium-Hydrogen Exchangers / chemistry
  • Sodium-Hydrogen Exchangers / metabolism*
  • Sodium-Phosphate Cotransporter Proteins, Type IIa / physiology*


  • Furans
  • Lipids
  • Parathyroid Hormone
  • Phosphates
  • Phosphoproteins
  • Receptors, Neuropeptide Y
  • SLC34A1 protein, human
  • Sodium-Hydrogen Exchangers
  • Sodium-Phosphate Cotransporter Proteins, Type IIa
  • sodium-hydrogen exchanger regulatory factor
  • tautomycetin
  • Serine
  • neuropeptide Y4 receptor

Associated data

  • PDB/1I92
  • PDB/4Q3H
  • PDB/5IOH
  • PDB/4LMM
  • PDB/2KRG