Abstract: SA-PO224
C-terminal Region of the TMEM174 Protein Regulates PTH-Induced NPT2A Endocytosis
Session Information
- CKD-MBD: Basic and Translational
October 26, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Bone and Mineral Metabolism
- 501 Bone and Mineral Metabolism: Basic
Authors
- Miranda, Jose, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, Colorado, United States
- Miyazaki-anzai, Shinobu, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, Colorado, United States
- Blaine, Judith, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, Colorado, United States
- Miyazaki, Makoto, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, Colorado, United States
Background
Sodium-dependent phosphate cotransporter 2a (NPT2A or SLC34A1) is a solute-carrier (SLC) transporter located in the brush border membrane of kidney proximal tubule that reabsorbs glomerular-filtered phosphate. We recently found that a proximal tubule-specific protein TMEM174 regulates phosphate reabsorption by inducing the degradation of NPT2A (PMID: 35459732). However, the molecular mechanism by which TMEM174 facilitates the degradation of NPT2A is unknown.
Methods
To obtain clues as to how TMEM174 induces the degradation of NPT2A, we examined which region of TMEM174 protein is essential to bind with NPT2A and induce the NPT2A endocytosis by the advanced microscope techniques such as Total Internal Reflection Fluorescence (TIRF) and Förster Resonance Energy Transfer (FRET) microscopy and the immunoprecipitation (IP) assay using DNA constructs containing full length TMEM174, TMEM174 lacking N-terminal 34 amino acids (TMEM174ΔN) and TMEM174 lacking C-terminal 130 amino acids (TMEM174ΔC).
Results
OK-P cells were transiently transfected with plasmids containing TMEM174 with moxCerulean3 (mC3) at its C-terminal and/or NPT2A with eYFP also at its C-terminal in the presence or absence of TMEM174 siRNA. TIRF microscopy analysis showed that treatment with TMEM174 siRNA blocked PTH induced-NPT2A endocytosis and increased the retention of NPT2A in the apical membrane of OK-P cells. In addition, FRET analysis showed that intact TMEM174 and TMEM174ΔN proteins are associated with NPT2A protein because upon PTH treatment NPT2A degradation is no decrease in the FRET ratio. Whereas TMEM174ΔC was significantly disassociated because under PTH treatment the FRET ratio decreased over-time signifying that NPT2A-eYFP was being degraded. IP assay confirmed that intact TMEM174 and TMEM174ΔN but not TMEM174ΔC were pull down with NPT2A.
Conclusion
The C-terminal region of TMEM174 protein is critical for regulating in the NPT2A endocytosis.
Funding
- NIDDK Support