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Abstract: PO1416

Critical Role of Threonine Residue in PDZ-1 Binding Motif of Type 2a Sodium-Phosphate Cotransporter (Npt2a)

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

  • 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Gagnon, Kenneth, University of Louisville Health Sciences Center, Louisville, Kentucky, United States
  • Barati, Michelle T., University of Louisville Health Sciences Center, Louisville, Kentucky, United States
  • Kitterman, Kathleen, University of Louisville Health Sciences Center, Louisville, Kentucky, United States
  • Bushau-Sprinkle, Adrienne M., University of Louisville Health Sciences Center, Louisville, Kentucky, United States
  • Clark, Barbara, University of Louisville Health Sciences Center, Louisville, Kentucky, United States
  • Lederer, Eleanor D., University of Texas Southwestern Department of Internal Medicine, Dallas, Texas, United States
Background

Npt2a brush border membrane (BBM) expression is the major determinant of proximal tubule phosphate reabsorption. Binding of the Npt2a carboxy terminus Class I PDZ binding motif (S/T-x-Φ-COOH) to NHERF1 (sodium-hydrogen exchange regulatory factor 1), a PDZ protein, is critical for Npt2a BBM expression. We have shown that NHERF1 deficiency results in increased binding of Npt2a to 14-3-3 epsilon, a chaperone protein with a Class III PDZ-binding domain. Phosphorylation of the Class I PDZ binding motif Thr residue converts it into a Class III binding motif (pT-x-x-COOH). We hypothesize that phosphorylation of the Thr residue prevents NHERF1 interaction, promotes 14-3-3 epsilon interaction, and inhibits BBM expression of Npt2a.

Methods

We generated cDNA constructs modifying the Thr residue at position -2 (T635) in the Npt2a PDZ binding motif to phosphomimic (T635E) or phosphonull (T635A) status. We sub-cloned WT and mutant Npt2a cDNAs alone, with NHERF1, or with 14-3-3 epsilon in an IRES-containing bicistronic mammalian expression vector. We transiently transfected these cDNA constructs in NHERF1-deficient opossum kidney cells, assessed membrane expression by confocal microscopy, and Npt2a function by (32P) phosphate uptake.

Results

Npt2a (T635), Npt2a (E635), or Npt2a (A635) alone showed dense cytosolic expression and negligible 32P uptake. Npt2a (T635) with NHERF1 colocalized at the plasma membrane and increased 32P uptake seven-fold. Npt2a (E635) and Npt2a (A635) appeared at the plasma membrane, but neither co-localized with NHERF1 nor showed 32P uptake. Each Npt2a plus14-3-3 cDNA construct exhibited apparent membrane localization, but none co-localized with 14-3-3 epsilon or exhibited significant 32P uptake.

Conclusion

We conclude that the -2 Thr of the Class I PDZ binding motif of Npt2a is essential for interaction with NHERF1 and functional activity of the cotransporter. 14-3-3 promotes Npt2a membrane localization but not function in NHERF1 deficient states and may not be dependent on the phosphorylation status of the -2 Thr.

Funding

  • Veterans Affairs Support