Multisite NHERF1 phosphorylation controls GRK6A regulation of hormone-sensitive phosphate transport

The type II sodium-dependent phosphate cotransporter (NPT2A) mediates renal phosphate uptake. The NPT2A is regulated by parathyroid hormone (PTH) and fibroblast growth factor 23, which requires Na⁺/H⁺ exchange regulatory factor-1 (NHERF1), a multidomain PDZ-containing phosphoprotein. Phosphocycling controls the association between NHERF1 and the NPT2A. Here, we characterize the critical involvement of G protein-coupled receptor kinase 6A (GRK6A) in mediating PTH-sensitive phosphate transport by targeted phosphorylation coupled with NHERF1 conformational rearrangement, which in turn allows phosphorylation at a secondary site. GRK6A, through its carboxy-terminal PDZ recognition motif, binds NHERF1 PDZ1 with greater affinity than PDZ2. However, the association between NHERF1 PDZ2 and GRK6A is necessary for PTH action. Ser¹⁶², a PKCα phosphorylation site in PDZ2, regulates the binding affinity between PDZ2 and GRK6A. Substitution of Ser¹⁶² with alanine (S¹⁶²A) blocks the PTH action but does not disrupt the interaction between NHERF1 and the NPT2A. Replacement of Ser¹⁶² with aspartic acid (S¹⁶²D) abrogates the interaction between NHERF1 and the NPT2A and concurrently PTH action. We used amber codon suppression to generate a phosphorylated Ser¹⁶²(pSer¹⁶²)-PDZ2 variant. K_D values determined by fluorescence anisotropy indicate that incorporation of pSer¹⁶² increased the binding affinity to the carboxy terminus of GRK6A 2-fold compared with WT PDZ2. Molecular dynamics simulations predict formation of an electrostatic network between pSer¹⁶² and Asp¹⁸³ of PDZ2 and Arg at position -1 of the GRK6A PDZ-binding motif. Our results suggest that PDZ2 plays a regulatory role in PTH-sensitive NPT2A-mediated phosphate transport and phosphorylation of Ser¹⁶² in PDZ2 modulates the interaction with GRK6A.