Abstract: FR-PO551
mTORC1-Mediated Megalin Phosphorylation Acts as a Switch between Endocytosis and Cell Proliferation
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolytes, and Acid-Base Disorders
- 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Dahlke, Eileen, CAU Kiel Institute of Anatomy, Kiel, Germany
- Kunke, Madlen, CAU Kiel Institute of Anatomy, Kiel, Germany
- Knöfler, Hannah, CAU Kiel Institute of Anatomy, Kiel, Germany
- Shen, Yuanhao, CAU Kiel Institute of Anatomy, Kiel, Germany
- Anan, Yaman, CAU Kiel Institute of Anatomy, Kiel, Germany
- Theilig, Franziska, CAU Kiel Institute of Anatomy, Kiel, Germany
Background
The importance of proper renal proximal tubular (PT) function is displayed by its loss of function in Fanconi syndrome which can be induced by mTORC1 inhibition. Protein endocytosis is one of the main PT function and involves the scavanger receptor megalin (LRP2) to internalize a variety of proteins from the ultrafiltrate. mTORC1 induces PT endocytosis and we discovered a specific mTORC1 phosphorylation site on megalin S4577 which function we aimed to identify.
Methods
Transient transfection of megalin mini-construct 2 (MMR2) with mutations in S4577 (MMR S->A mimics permanent non-phosphorylated state, MMR2 S->D mimics permanent phosphorylated state) in MDCK2 or EBNA/HEK cells, followed by functional endocytosis assays, immunocytological stainings and super resolution STED microscopy, western blotting, surface expression analysis, and co-immunoprecipitations were performed.
Results
Megalin phosphorylation by mTORC1 in S4577 was confirmed. MMR2 S4577 phosphorylation showed no impact on MMR2 surface expression level, but demonstrated a reduced endocytosis rate for albumin. The subcellular distribution of mutants showed minor alterations in co-localization with clathrin vesicles and early endosomes and no difference in co-localization with Rab35-positive vesicles. Surprisingly, S4577 phosphorylation leads to stronger binding affinity of MMR2 to the endocytosis adaptor protein ARH. In culture conditions, MMR2 was occassionally observed to be colocalized with alpha-tubulin in mitotic spindles and S4577 phosphorylation reduced cell proliferation rate.
Conclusion
mTORC1 mediated phosphorylation of megalin C-terminus at S4577 lowers endocytosis rate to allow increased cell proliferation and may therefore be encountered as cellular switch between endocytotic function and cell proliferation.