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

Abstract The type II sodium-dependent phosphate cotransporter (NPT2A) mediates renal phosphate uptake. NPT2A is regulated by parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23), which requires Na+/H+ exchange regulatory factor-1 (NHERF1), a multidomain PDZ-containing phosphoprotein. Phosphocycling controls the association between NHERF1 and 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 compared to PDZ2. However, 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 PTH action, but does not disrupt the interaction between NHERF1 and NPT2A. Replacement of Ser162 with aspartic acid (S162D) abrogates the interaction between NHERF1 and NPT2A and concurrently PTH action. We used amber codon suppression to generate a phosphorylated pSer162-PDZ2 variant. KD values determined by fluorescence anisotropy indicate that incorporation of pSer162 increased the binding affinity to the carboxy-terminus of GRK6A 2-fold compared to 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.