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

Abstract

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.

Noncanonical Sequences Involving NHERF1 Interaction with NPT2A Govern Hormone-Regulated Phosphate Transport: Binding Outside the Box

“…GRK6A, like NPT2A, associates with NHERF1 PDZ domains through its C-terminal motif (-T −2 R −1 L 0 ). Knocking down Grk6a by siRNA blocks Npt2a-dependent phosphate uptake in response to PTH [63]. Thus, GRK6A is an essential regulatory component of NPT2A-dependent PTH-sensitive phosphate transport and corroborates previous findings that GRK6A pharmacological inhibitors abolish PTH action [44].…”

“…Thus, GRK6A is an essential regulatory component of NPT2A-dependent PTH-sensitive phosphate transport and corroborates previous findings that GRK6A pharmacological inhibitors abolish PTH action [44]. Binding affinities (3-5 µM) for the C-terminal PDZ ligand of GRK6A [63] or NPT2A (22 aa) [45] with NHERF1 are comparable and suggest that the binding mechanism is presumable identical. NPT2A and GRK6A interact with PDZ1 with a greater affinity than to PDZ2, thereby confirming that PDZ1 is naturally optimized to bind the -TRL sequence.…”

“…Ala replacement at Ser 162 (NHERF1 Ser 162 Ala) diminished PTH-inhibitable phosphate transport suggesting that Ser 162 is an essential regulator of hormone-sensitive phosphate uptake. Unexpectedly, phosphomimic Ser 162 Asp mutation in NHERF1 disrupts basal phosphate transport and blocks PTH action on phosphate uptake (Figure 8) [63]. Clearly, the discrepancy in charge and size between phosphate and carboxylate groups of phosphorylated Ser and Asp, respectively explains the observed difference.…”

“…Previously, semi-synthesis was effectively applied to generate site-specific phosphorylated PDZ domains [33]. Here, we used amber codon suppression to genetically encode pSer at position 162 [63], which in the future will allow to generate phosphorylated full-length proteins comprising both PDZ domains. Replacement of Ser 162 by pSer 162 in recombinant PDZ2 permits estimating the binding affinity between pSer 162 -PDZ2 and the C-terminal PDZ-ligand of GRK6A.…”

“…N1P2-GAGA-NHERF1 and WT-NHERF1 support PTH-inhibitable phosphate uptake, whereas N1P1-GAGA does not. Results report the mean ± SEM (n = 4, **** p < 0.0001, ANOVA)[63].…”