Abstract: TH-PO380

Multiple Roles for NHERF1 in Forward Trafficking and Apical Membrane Anchoring of NPT2a

Session Information

Category: Cell Biology

  • 201 Cell Signaling, Oxidative Stress

Authors

  • Gagnon, Kenneth, University of Louisville, Louisville, Kentucky, United States
  • Barati, Michelle T., University of Louisville, Louisville, Kentucky, United States
  • Merchant, Michael, University of Louisville Medicine, Louisville, Kentucky, United States
  • Clark, Barbara, University of Louisville, Louisville, Kentucky, United States
  • Lederer, Eleanor D., University of Louisville; Robley Rex VA Medical Center, Louisville, Kentucky, United States
Background

Loss of NHERF1 (Na-H Exchanger Regulatory Factor Isoform 1), a PDZ domain scaffolding protein, results in phosphate wasting and stone formation in animals and humans. NHERF1 knockout mice show diminished proximal tubule apical membrane expression of the sodium-phosphate cotransporter IIa (NPT2a). We have previously demonstrated in cell culture that NHERF1 and NPT2a exhibit a dynamic association between the Golgi and apical membrane, leading to the hypothesis that NHERF1 regulates trafficking and apical membrane anchoring of NPT2a through assembly of accessory protein partners.

Methods

To test this hypothesis, we immunoprecipitated (IP) NPT2a from kidney cortex lysate of wild-type (Wt) and NHERF1 KO (KO) littermates, compared the expression of associated proteins by mass spectrometry (MS), and performed western blot (WB) of proteins in cortex lysates. We compared mouse NPT2a localization and glycosylation status in HEK293 cells transfected with either a GFP-tagged full-length mouse NPT2a construct (NPT2a-FL) or a GFP-tagged mouse NPT2a construct lacking the PDZ binding motif required for interaction with NHERF1 (NPT2a-TRL).

Results

We identified 164 proteins having greater and 8 proteins having lower expression levels in Wt vs KO IPs. Expression of the F-actin binding protein ezrin decreased 75% in KO vs Wt, while total ezrin expression was similar in both pre-IP cortex lysates. WB’s revealed a 2-fold increase in PDZK1 (a.k.a. NHERF3) and a 4-fold decrease in the SNARE accessory protein Munc18-2 in KO versus Wt. WB’s for NPT2a in Wt revealed multiple bands at 35, 75-100, and 150-200 kDa and a 3-fold decrease in expression of the largest band in KO. WB from the NPT2a-TRL transfected cells showed a 41% decrease in expression of the larger band compared to the NPT2a-FL. F-glycosidase treatment of the NPT2a-FL and NPT2a-TRL lysates reduced the expression of the larger protein band by 68% and 44%, respectively. Confocal microscopy of transfected HEK293 cells showed NPT2a-FL localized at the plasma membrane, while NPT2a-TRL expression was cytosolic.

Conclusion

We conclude that NHERF1 is essential for the forward trafficking of NPT2a through direct PDZ domain interaction and through coordinated assembly of accessory proteins responsible for glycosylation, vesicle fusion, and membrane anchoring.

Funding

  • Veterans Affairs Support