Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1

ABSTRACT Equilibrative nucleoside transporters (ENTs) translocate nucleosides and nucleobases across plasma membranes, as well as a variety of anti‐cancer, ‐viral, and ‐parasite nucleoside analogs. They are also key members of the purinome complex and regulate the protective and anti‐inflammatory effects of adenosine. Despite their important role, little is known about the mechanisms involved in their regulation. We conducted membrane yeast 2‐hybrid and coimmunoprecipitation studies and identified, for the first time to our knowledge, the existence of protein‐protein interactions between human ENT1 and ENT2 (hENT1 and hENT2) proteins in human cells and the formation of hetero‐ and homo‐oligomers at the plasma membrane and the submembrane region. The use of NanoLuc Binary Technology allowed us to analyze changes in the oligomeric status of hENT1 and hENT2 and how they rapidly modify the uptake profile for nucleosides and nucleobases and allow cells to respond promptly to external signals or changes in the extracellular environment. These changes in hENTs oligomerization are triggered by PKC activation and subsequent action of protein phosphatase 1.—Grañe‐Boladeras, N., Williams, D., Tarmakova, Z., Stevanovic, K., Villani, L. A., Mehrabi, P., Siu, K. W. M., Pastor‐Anglada, M., Coe, I. R. Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1. FASEB J. 33, 3841–3850 (2019). www.fasebj.org