Protein-Protein Interactions in Human Aquaporin Regulation

Water is an essential compoment of every living orgamism and forms a major part of the human body. Regulated water transport is crucial for proper cell functioning and body homeostasis. Cell, the smallest structural and functional unit of life, uses specialized water conducting pores made up of proteins to transport water across the plasma membrane by facilitated diffusion.The discovery of one of these proteins in red blood cells was the first evidence of cellular water conducting channels and the protein was later named aquaporin 1 (AQP1). Since then, thirteen human aquaporin isoforms have been identified and classified as orthodox AQPs, those that transport water only, and aquaglyceroporins, those that also transport glycerol, ammonia, urea or other small solutes.In this thesis, we focus on human AQP0 and AQP2, with an aim to deepen our understanding of how they are regulated by protein-protein interactions and the effect of phosphorylation, a post translational modification. Human AQP2 is found in the kidney where it plays an important role in regulating urine volume and is regulated by trafficking. The phosphorylation of the C-terminal residues in AQP2 and its interaction with lysosomal trafficking regulator interacting protein 5 (LIP5) plays a major role in AQP2 trafficking and targeting for lysosomal degradation. AQP0 has dual role in the eye lens, functioning as a water transporter as well as an adhesion protein in cell junctions in lens fibre cells. Previous studies have demonstrated that the binding of calmodulin (CaM) to the C-terminal of AQP0 in the presence of calcium inhibits water transport in a calcium dependent manner.In our studies, the interaction between interacting proteins (LIP5 and CaM) and full length (FL) AQPs and AQP peptides was measured using Micro Scale Thermophoresis (MST) and fluorescence anisotropy. While we were able to show the interaction and determine the binding affinities for both FL AQPs and peptides, we also could demonstrate that using FL proteins was advantageous compared to peptides. Interaction studies between FL AQP2 and LIP5 showed that AQP2 binds the LIP5 N-terminal domain (ND-LIP5) and that one ND-LIP5 molecule bound per AQP2 tetramer. We also showed that phosphorylation of the distal C-terminus of AQP2 allosterically regulates its interaction with LIP5. The stability of AQP2 and AQP2 phoshpho mimics as well as soluble proteins in the presence of detergents was demonstrated by Circular Dichroism (CD). Small angle X-ray scattering (SAXS) measurements provided information on AQP2 in nanodiscs. However, more experimental data would be required to characterize the structural aspects of AQP2:LIP5 interaction. Our data shows that recombinantly expressed AQP0 functions as a water channel when reconstituted into proteoliposomes. We demonstrated for the first time that direct binding of CaM reduces its water permeability in vitro and that phosphorylation of S229 and S235 in AQP0 abolishes the interaction with CaM while S231 shared similar binding affinities as FL AQP0. We further showed that CaM binds AQP0 in a cooperative manner