Mutations in an unrecognized internal NPT2A PDZ motif disrupt phosphate transport and cause congenital hypophosphatemia

The Na⁺-dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na⁺/H⁺ Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein, establishes NPT2A membrane localization and is required for hormone-inhibitable phosphate transport. NPT2A also possesses an uncharacterized internal PDZ ligand. Two recent clinical reports describe congenital hypophosphatemia in children harboring Arg⁴⁹⁵His or Arg⁴⁹⁵Cys variants within the internal PDZ motif. The wild-type internal ⁴⁹⁴TRL⁴⁹⁶ PDZ ligand binds NHERF1 PDZ2, which we consider a regulatory domain. Ablating the internal PDZ ligand with a ⁴⁹⁴AAA⁴⁹⁶ substitution blocked hormone-inhibitable phosphate transport. Complementary approaches, including CRISPR/Cas9 technology, site-directed mutagenesis, confocal microscopy, and modeling, showed that NPT2A Arg⁴⁹⁵His or Arg⁴⁹⁵Cys variants do not support PTH or FGF23 action on phosphate transport. Coimmunoprecipitation experiments indicate that both variants bind NHERF1 similarly to WT NPT2A. However, in contrast with WT NPT2A, NPT2A Arg⁴⁹⁵His, or Arg⁴⁹⁵Cys variants remain at the apical membrane and are not internalized in response to PTH. We predict that Cys or His substitution of the charged Arg⁴⁹⁵ changes the electrostatics, preventing phosphorylation of the upstream Thr⁴⁹⁴, interfering with phosphate uptake in response to hormone action, and inhibiting NPT2A trafficking. We advance a model wherein the carboxy-terminal PDZ ligand defines apical localization NPT2A, while the internal PDZ ligand is essential for hormone-triggered phosphate transport.