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Na + -H + exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser 46 , Ser 162 , Ser 181 , Ser 269 , Ser 280 , Ser 291 , Thr 293 , Ser 299 , and Ser 302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser 290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser 290 Mutating 257 VPF 259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser 290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that β-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser 290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser 290 phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport., (© 2019 Zhang et al.)

Na + /H + exchanger regulatory factor-1 (NHERF1) is a scaffolding protein containing two PSD95/discs large protein/ZO1 (PDZ) domains that modifies the signaling, trafficking, and function of the parathyroid hormone receptor (PTHR), a family B G-protein-coupled receptor. PTHR and NHERF1 bind through a PDZ-ligand-recognition mechanism. We show that PTH elicits phosphorylation of Thr591 in the canonical -ETVM binding motif of PTHR. Conservative substitution of Thr591 with Cys does not affect PTH(1-34)-induced cAMP production or binding of PTHR to NHERF1. The findings suggested the presence of additional sites upstream of the PDZ-ligand motif through which the two proteins interact. Structural determinants outside the canonical NHERF1 PDZ-PTHR interface that influence binding have not been characterized. We used molecular dynamics (MD) simulation to predict residues involved in these interactions. Simulation data demonstrate that the negatively charged Glu side chains at positions -3, -5, and -6 upstream of the PDZ binding motif are involved in PDZ-PTHR recognition. Engineered mutant peptides representing the PTHR C-terminal region were used to measure the binding affinity with NHERF1 PDZ domains. Comparable micromolar affinities for peptides of different length were confirmed by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance. Binding affinities measured for Ala variants validate MD simulations. The linear relation between the change in enthalpy and entropy following Ala substitutions at upstream positions -3, -5, and -6 of the PTHR peptide provides a clear example of the thermodynamic compensation rule. Overall, our data highlight sequences in PTHR that contribute to NHERF1 interaction and can be altered to prevent phosphorylation-mediated inhibition.

Despite numerous reports implicating NADPH oxidases (Nox) in the pathogenesis of many diseases, precise regulation of this family of professional reactive oxygen species (ROS) producers remains unclear. A unique member of this family, Nox1 oxidase, functions as either a canonical or hybrid system using Nox organizing subunit 1 (NoxO1) or p47(phox), respectively, the latter of which is functional in vascular smooth muscle cells (VSMC). In this manuscript, we identify critical requirement of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50; aka NHERF1) for Nox1 activation and downstream responses. Superoxide (O2 (•-)) production induced by angiotensin II (AngII) was absent in mouse EBP50 KO VSMC vs. WT. Moreover, ex vivo incubation of aortas with AngII showed a significant increase in O2 (•-) in WT but not EBP50 or Nox1 nulls. Similarly, lipopolysaccharide (LPS)-induced oxidative stress was attenuated in femoral arteries from EBP50 KO vs. WT. In silico analyses confirmed by confocal microscopy, immunoprecipitation, proximity ligation assay, FRET, and gain-/loss-of-function mutagenesis revealed binding of EBP50, via its PDZ domains, to a specific motif in p47(phox) Functional studies revealed AngII-induced hypertrophy was absent in EBP50 KOs, and in VSMC overexpressing EBP50, Nox1 gene silencing abolished VSMC hypertrophy. Finally, ex vivo measurement of lumen diameter in mouse resistance arteries exhibited attenuated AngII-induced vasoconstriction in EBP50 KO vs. WT. Taken together, our data identify EBP50 as a previously unidentified regulator of Nox1 and support that it promotes Nox1 activity by binding p47(phox) This interaction is pivotal for agonist-induced smooth muscle ROS, hypertrophy, and vasoconstriction and has implications for ROS-mediated physiological and pathophysiological processes., Competing Interests: The authors declare no conflict of interest.

Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD) simulations in combination with biological mutagenesis, fluorescent polarization (FP) binding assays, and isothermal titration calorimetry (ITC), we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn) decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar KDs. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development.