The Physiological Role of Kidney Anion Exchanger 1 Protein Interaction with Adaptor Protein 1 A and B

  • Author / Creator
    Almomani, Ensaf Y
  • Distal Renal Tubular Acidosis (dRTA) is a disease characterized by metabolic acidosis, an inability to acidify urine, development of kidney stones and renal failure if untreated. dRTA patients can carry a mutation in the SLC4A1 gene encoding the kidney anion exchanger 1 (kAE1), a Cl-/HCO3- exchanger basolaterally expressed in kidney acid-secreting alpha intercalated cells. Three reported SLC4A1 mutations that cause dRTA, including the kAE1 R901X mutation, affect the interaction site of kAE1 with adaptor protein 1 (AP-1). AP-1 is a cytosolic tetrameric protein complex, which has two forms, A and B. It is involved in protein trafficking in polarized cells from the trans Golgi network or recycling endosomes to the plasma membrane. The general hypothesis of my thesis was AP-1A and/or B interaction with kAE1 is crucial for kAE1 residency at the basolateral membrane in polarized epithelial cell. I proposed that lack of proper interaction between kAE1 and AP-1A and/or B affects kAE1 trafficking and surface expression and may cause dRTA disease. Data presented here confirmed the interaction between kAE1 and the AP-1A and B protein complexes in vitro by reciprocal coimmunoprecipitation or the protein is heterologously expressed in epithelial cells and in vivo by coimmunoprecipitation from mouse kidney extract. When endogenous μ1A and μ1B subunits from AP-1A and B, respectively, were knocked down using short interfering RNA, kAE1 protein was unable to traffic to the plasma membrane and was rapidly degraded via a lysosomal pathway. Expression of siRNA-resistant μ1A or μ1B rescued kAE1 trafficking to the cell surface and increased its stability. Furthermore, I found that newly synthesized kAE1 does not traffic through recycling endosomes to the plasma membrane, suggesting that AP-1B, located in these endosomes, is not primarily involved in trafficking of newly synthesized kAE1 when AP-1A is present in the cells. The physiological role of AP-1B was investigated in LLC-PK1 cells, a renal epithelial cell line that lacks the endogenous AP-1B. Surprisingly, μ1B expression in these cells reduced the amount of cell surface kAE1-WT at steady state. This decrease was due to μ1B expression increasing the endocytosis rate and decreasing the recycling rate of kAE1-WT. Although kAE1-R901X mutant, which lacks a canonical AP-1 binding site, was endocytosed slightly faster than kAE1-WT, its recycling rate was significantly decreased compared to that of kAE1-WT. Unlike kAE1-WT, kAE1-R901X recycling was independent from μ1B expression. Importantly, kAE1 endocytosis was dynamin and clathrin dependent. Finally, this thesis showed that heterologous expression of μ1A or μ1B displaces the physical interaction of endogenous glyceraldehyde-3-phosphate dehydrogenase with kAE1-WT, supporting that AP-1A or B and glyceraldehyde-3-phosphate dehydrogenase bind to an overlapping site on kAE1. This work also revealed that phosphorylation of tyrosine 904 within the canonical AP interaction motif does not alter the interaction between kAE1 and AP-1A/B. My results suggest that AP-1A regulates the processing of the newly synthesized kAE1 to the basolateral surface while AP-1B plays a role in kAE1 trafficking in absence of AP-1A. Further, kAE1 recycling and endocytosis are dependent on AP-1B expression. In conclusion, both AP-1A and B adaptors are required for normal kAE1 trafficking, and we propose that the apically mis-targeted kAE1- R901X dRTA mutant may fail to recycle back to the basolateral membrane due to its inability to properly interact with AP-1B.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Physiology
  • Supervisor / co-supervisor and their department(s)
    • Cordat, Emmanuelle (Physiology)
  • Examining committee members and their departments
    • Simmen, Thomas (Cell Biology)
    • Young, James (Physiology)
    • Hanrahan, John (Physiology-McGill University)
    • Casey, Joesph R (Biochemistry)