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

Abstract 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. Competing Interest Statement The authors have declared no competing interest. The abbreviations used are - PTH - parathyroid hormone - PTHR - PTH receptor - NPT2A - Sodium-phosphate cotransport protein 2A - RGS14 - regulator of G protein signaling 14 - PDZ - postsynaptic density protein 95 (PSD95), drosophila disc large tumor suppressor (DlgA), and zonula occludens 1 protein (ZO1) - GAP - GTPase activating protein - RBD - Ras-binding domain - GPR - G protein regulatory motif - HEK293 - Human Embryonic Kidney 293 cells.