Sneddon W Bruce, Ramineni Suneela, Van Doorn G Emme, Hepler John R, Friedman Peter A
Laboratory for GPCR Biology, Departments of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, U.S.A.
Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, GA, U.S.A.
Biochem J. 2025 Feb 5;482(3):135-146. doi: 10.1042/BCJ20240122.
The sodium phosphate cotransporter-2A (NPT2A) mediates basal and parathyroid hormone (PTH)- and fibroblast growth factor-23 (FGF23)-regulated phosphate transport in proximal tubule cells of the kidney. Both basal and hormone-sensitive transport require sodium hydrogen exchanger regulatory factor-1 (NHERF1), a scaffold protein with tandem PDZ domains, PDZ1 and PDZ2. NPT2A binds to PDZ1. RGS14 persistently represses hormone action by binding to PDZ2. The RGS14 canonical RGS domain, Ras/Rap-binding domains, and G protein regulatory motif 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 the fibroblast growth factor receptor-1, a receptor tyrosine kinase that is not governed by G protein activity. Here, we identify the structural elements of RGS14 that mutually control the action of PTH and FGF23. RGS14 truncation constructs lacking upstream sequence and the RGS domain were fully functional. Removing the linker sequence between the RGS and RBD1 domains abolished RGS14 action. Examination of the α-helical linker region suggested candidate serine residues that might facilitate regulatory activities. RGS14 Ser266 and Ser269 are phosphorylated in response to PTH and FGF23, and replacement of these residues by Ala eliminated the actions of RGS14 on hormone-sensitive phosphate transport. PTH and FGF23 stimulated the phosphorylation of a peptide construct harboring the sites of purported phosphorylation and full-length RGS14. Mutating Ser266Ala and Ser269Ala abolished phosphorylation. The results establish that RGS14 regulation of phosphate transport requires targeted phosphorylation within the linker and an intact PDZ ligand.
磷酸钠共转运体2A（NPT2A）介导肾脏近端小管细胞中基础状态以及甲状旁腺激素（PTH）和成纤维细胞生长因子23（FGF23）调节的磷酸盐转运。基础转运和激素敏感性转运均需要钠氢交换调节因子1（NHERF1），这是一种具有串联PDZ结构域（PDZ1和PDZ2）的支架蛋白。NPT2A与PDZ1结合。RGS14通过与PDZ2结合持续抑制激素作用。RGS14的典型RGS结构域、Ras/Rap结合结构域和G蛋白调节基序无法解释其对激素敏感性磷酸盐转运的调节作用，因为这些作用不仅由G蛋白偶联受体（GPCR）甲状旁腺激素受体介导，还由不受G蛋白活性调控的受体酪氨酸激酶成纤维细胞生长因子受体1介导。在此，我们确定了相互控制PTH和FGF23作用的RGS14的结构元件。缺乏上游序列和RGS结构域的RGS14截短构建体具有完全功能。去除RGS和RBD1结构域之间的连接序列消除了RGS14的作用。对α螺旋连接区域的研究表明，可能促进调节活性的候选丝氨酸残基。RGS14的Ser266和Ser269在PTH和FGF23作用下发生磷酸化，用丙氨酸取代这些残基消除了RGS14对激素敏感性磷酸盐转运的作用。PTH和FGF23刺激了包含假定磷酸化位点的肽构建体和全长RGS14的磷酸化。将Ser266突变为Ala和Ser269突变为Ala消除了磷酸化。结果表明，RGS14对磷酸盐转运的调节需要连接区内的靶向磷酸化和完整的PDZ配体。
