Regulation and Quaternary Structure of Human Equilibrative Nucleoside Transporter 1

• Author / Creator

  Cravetchi, Xenia

• Equilibrative nucleoside transporter 1 (ENT1) is an ubiquitously expressed membrane transporter in mammalian cells responsible for the transmembrane flux of endogenous nucleosides such as adenosine, as well as chemotherapeutic, anti-viral, and anti-parasitic nucleoside analogues. The present studies were designed to: 1) assess whether changes in ENT1 trafficking to the plasma membrane are responsible for enhanced uptake function observed in response to stimulation at adenosine receptor 1 (A1AR) or protein kinase C (PKC) activation, and 2) determine whether ENT1 forms a multimeric protein complex. Immunofluorescence co-localization and cell surface biotinylation studies were conducted to quantify changes in plasma-membrane localized ENT1 in response to phorbol 12-myristate 13-acetate (PMA), a PKC activator, and 2-chloro-N(6)-cyclopentyladenosine (CCPA), an A1AR stimulator. It was found that an increase in ENT1 trafficking to the plasma membrane in response to PKC stimulation partially accounts for enhanced uptake function; however, intermediate factors may be involved as in PK15-S281A-hENT1 cells, an increase in plasma membrane expression is not reflected by an increase in uptake function. Stimulation of A1AR did not have an effect on ENT1 localization at the plasma membrane; therefore, the CCPA-induced increase in uptake activity is achieved by changes to ENT1 already at the plasma membrane. These findings provide some insight into regulation of ENT1 activity. Co-immunoprecipitation and proximity ligation assays showed that, in intact cells, ENT1 proteins interact. Moreover, mass spectrometry and proximity ligation studies identified Na+/K+-ATPase as a potential binding partner of ENT1. We conclude that hENT1 exists as a dimer in intact cells and potentially interacts with other membrane proteins. The findings of this thesis open new avenues of research relating to regulation of transporter activity, including the influence of quaternary structure and binding partners on transport function and ligand binding.

• Subjects / Keywords

  • ENT1
  • Nucleoside transport
  • Trafficking
  • Complex
  • Oligomerization

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3KH0F916

• 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.