Biology subjects

Sneddon, W. B.

Publications and source records attributed to Sneddon, W. B..

3 recordsLinked to original sources

bioRxiv2024

Structural factors of RGS14 regulating hormone-dependent phosphate transport mediated by NPT2A

The NPT2A sodium phosphate cotransporter-2a mediates basal and parathyroid hormone (PTH)- and fibroblast growth factor-23 (FGF23)-regulated phosphate transport in kidney proximal tubule cells. Both basal and hormone-sensitive transport require sodium hydrogen exchanger regulatory factor-1 (NHERF1), a scaffold protein with 2 PDZ domains. NPT2A binds to PDZ1. RGS14 persistently represses hormone action by binding to PDZ2. RGS14 contains an RGS domain, two Ras/Rap-binding domains (RBD1, RBD2), a G protein regulatory (GPR) motif, and a carboxy-terminal PDZ ligand. The intrinsic RGS14 domains cannot explain its regulatory effects on hormone-sensitive phosphate transport because these actions are mediated not only by the PTH receptor, a G protein-coupled receptor (GPCR) but also by fibroblast growth factor receptor-1 (FGFR1), a receptor tyrosine kinase that is not governed by G protein activity. Here, we sought to identify the RGS14 structural elements mutually controlling PTH and FGF23 action. RGS14 truncation constructs lacking upstream sequence and the RGS domain were fully functional. Removing the linker sequence between RGS and RBD1 domains was sufficient to abrogate RGS14 action. Examination of the alpha-helical linker region suggested candidate serine residues that might facilitate regulatory activities. Ser266 and Ser269 replacement by Ala abolished RGS14 regulatory actions on hormone-sensitive phosphate transport and binding to NHERF1. The results establish that RGS14 actions require targeted phosphorylation within the linker and an intact PDZ ligand.

cell biology↗

bioRxiv2023

Mutations in an unrecognized internal NPT2A PDZ motif disrupt phosphate transport causing 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-sensitive 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-sensitive 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 to 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 carboxyterminal PDZ ligand defines apical localization NPT2A, while the internal PDZ ligand is essential for hormone-triggered phosphate transport.

cell biology↗

bioRxiv2019

Genetic variants disrupt human RGS14 binding to NHERF1 and regulation of NPT2A-mediated phosphate transport

RGS14 is a multifunctional scaffolding protein that integrates G protein, MAPK, and Ca++/CaM signaling pathways. RGS14 contains an RGS domain, tandem Ras/Rap-binding domains, and a G protein regulatory motif. Human/primate RGS14 differ from rodent proteins by possessing a carboxy-terminal extension encoding a Type I PDZ ligand. RGS14 has been implicated in disordered phosphate metabolism. The human RGS14 gene is adjacent to SLC34A1 that encodes the NPT2A sodium-phosphate cotransporter. Hormone-regulated NPT2A requires the PDZ protein NHERF1 which contains two PDZ domains (PDZ1 and PDZ2). NHERF1 binds the PDZ ligand carboxy tail of NPT2A to regulate phosphate uptake, and this NPT2A:NHERF1 complex is inhibited by parathyroid hormone (PTH). Studies here define roles for RGS14 in NHERF1-dependent, PTH-sensitive phosphate transport. We found that RGS14 binds to NHERF1 via the PDZ2 domain. PTH inhibits NPT2A-mediated phosphate transport and RGS14 blocked this action. Several rare human mutations have been reported in the RGS14 PDZ ligand located at residues 563 (D563N, D563G) and 565 (A565S, A565V). D563N disrupted RGS14 binding to NHERF1 and did not interfere with PTH action, whereas D563G, A565S, and A565V bound NHERF1 and were functionally equivalent to wild-type RGS14. Computational analysis and molecular dynamics modeling of NHERF1 PDZ2 binding to the RGS14 C-terminal PDZ ligands refined the structural determinants of this interaction. Additional studies demonstrated that RGS14 is expressed in human kidney proximal and distal tubule cells. Together, our findings are consistent with the view that RGS14 contributes to PTH-sensitive phosphate transport in humans. RGS14 coding variants may cause disordered phosphate metabolism.

pharmacology and toxicology↗