Phosphorylation-dependent interaction between AQP2 and the Ezrin FERM domain

Abstract

The topic of this study is the phosphorylation dependent interaction between aquaporin 2 (AQP2) and the FERM domain of ezrin.
AQP2 is an important water channel for finetuning the water content of the urine based on the needs of the body. The main way of regulating how much water is reabsorbed is by regulating the amount of AQP2 present in the apical membrane. This regulation is controlled through the hormone vasopressin which through a pathway causes phosphorylation of four sites in AQP2, S256, S261, S264 and T269. Phosphorylation of these different sites signals for different responses regarding the localisation of AQP2.
Ezrin is a crosslinking protein between the plasma membrane and the cytoskeleton underneath and has previously been found to play a part in the endocytosis of AQP2 from the apical membrane. The aim of this study is to investigate how the phosphorylation status of the four phosphorylation sites affects the affinity to the binding domain FERM of ezrin.
Phospho-mimicking mutants were used to investigate the effect of phosphorylation of the different sites. AQP2 was expressed in Pichia pastoris and purified using IMAC and SEC. FERM was expressed in Escherichia coli and purified with IMAC before labelling with Alexa-488. The difference in affinity between the different mutants was investigated using MST, however the results obtained had too much variation to be considered reproducible. There was however indication of binding for all mutants as well as a difference in behaviour for wild type AQP2 and S261E compared to S264 and S256E/T269E which indicates a conformational change depending on phosphorylation status. To get more reproducible data, suggestions such as change of fluorescent label, phosphatase treatment of AQP2 and an additional purification step of FERM is discussed.

Popular Abstract

The maintenance of the water balance within the body is essential for good health and life. Aquaporins are water channels located in the membrane of cells that lets water move in or out of the cell to even out the concentration of ions on either side. This study focuses on aquaporin 2 (AQP2) which can be found in the kidneys and is tasked with controlling the urine concentration based on whether the body is dehydrated or not. When the body is dehydrated, the hormone vasopressin is released which triggers a series of events ending with a phosphate group getting attached to an amino acid in AQP2 that is located in storage vesicles in the cell. This is called phosphorylation and signals for AQP2 to be moved to the cell membrane facing the collecting duct of the kidney. AQP2 contains 4 amino acids in the protein chain (S256, S261, S264 and T269) that can undergo this phosphorylation, and the phosphorylation of these different sites has been found to have different effects on the location of AQP2 in the cell.
Ezrin is a protein that has been found to be involved in removing AQP2 from the cell membrane but how is not known yet. Previous studies have shown that a part of ezrin called the FERM domain is what binds to AQP2. In this study, it was investigated how phosphorylation of each of the 4 phosphorylation sites in AQP2 effects the binding to the FERM domain using a method called microscale thermophoresis.
To investigate the effect of the different phosphorylation sites, so called phospho-mimicking mutants were used. In these mutants, the amino acid of the site had been exchanged to an amino acid that resembles a phosphorylation.
The results from this study indicated some difference in binding when different sites were phosphorylated but the data collected had too much variation for a conclusion to be drawn regarding how the binding changes. The results also indicate that there is a difference in the kind of binding when the sites S264 or T269 is phosphorylated compared to when S261 or no site was phosphorylated but more investigation is needed.
Possible improvements that could give more reproducible results are discussed, such as using a different fluorescent label for FERM and treating the AQP2 to remove any phosphorylation.

• author: Edström, Elin
• supervisor: Susanna Horsefield
• organization: Department of Chemistry
• course: KEMR45 20251
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Aquaporin 2, FERM, Microscale thermophoresis, Phosphorylation, protein-protein interaction, biochemistry
• language: English
• id: 9194576
• date added to LUP: 2025-06-11 10:38:01
• date last changed: 2025-06-11 10:38:01