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. Ser(162), a PKCα phosphorylation site in PDZ2, regulates the binding affinity between PDZ2 and GRK6A. Substitution of Ser(162) with alanine (S(162)A) blocks the PTH action but does not disrupt the interaction between NHERF1 and the NPT2A. Replacement of Ser(162) with aspartic acid (S(162)D) abrogates the interaction between NHERF1 and the NPT2A and concurrently PTH action. We used amber codon suppression to generate a phosphorylated Ser(162)(pSer(162))-PDZ2 variant. K(D) values determined by fluorescence anisotropy indicate that incorporation of pSer(162) 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 pSer(162) and Asp(183) 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 Ser(162) in PDZ2 modulates the interaction with GRK6A.