Adenosine A1 receptor activation modulates human equilibrative nucleoside transporter 1 (hENT1) activity via PKC-mediated phosphorylation of serine-281.
Equilibrative nucleoside transporter subtype 1 (ENT1) is critical for the regulation of the biological activities of endogenous nucleosides such as adenosine, and for the cellular uptake of chemotherapeutic nucleoside analogs. Previous studies have implicated protein kinase C (PKC) in the regulation of ENT1 expression/function. It was hypothesized that hENT1 activity at the plasma membrane is regulated by PKC-mediated phosphorylation of Ser281. WT (wild-type)-hENT1 or S281A-hENT1 was stably transfected into a PK15 cell variant that is deficient in nucleoside transport. Using [(3)H]nitrobenzylthioinosine (NBMPR) binding and [(3)H]2-chloroadenosine uptake analyses, it was determined that S281A-hENT1 exhibited functional characteristics similar to WT-hENT1. Direct activation of PKC with PMA or indirect activation with the adenosine A1 receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) led to significant increases in [(3)H]NBMPR binding and [(3)H]2-chloroadenosine uptake in WT-hENT1 transfected cells. The PKC inhibitor Gö6983 blocked these effects of both PMA and CCPA, and the CCPA-mediated increase was also blocked by the A1 adenosine receptor antagonist DPCPX. In contrast, neither PMA nor CCPA affected [(3)H]NBMPR binding or [(3)H]2-chloroadenosine uptake in cells transfected with S281A-hENT1. shRNAi silencing studies implicated PKCδ in this regulation of hENT1 activity. Immunocytochemical analysis and cell surface biotinylation assays showed that activation of PKC with PMA, but not CCPA, led to a significant increase in the plasma membrane localization of hENT1. These data suggest that phosphorylation of hENT1 by PKC has effects on both the function and subcellular trafficking of hENT1. This signaling pathway represents a feedback loop whereby adenosine receptor signaling can lead to increased adenosine reuptake into cells via hENT1.
Department of Physiology and Pharmacology, Western University, London N6A 5C1, Canada. Electronic address: scottj.hughes@mail.utoronto.ca.
Scott J
SJ
Hughes
Department of Pharmacology, University of Alberta, Edmonton T6G 2H7, Canada.
Xenia
X
Cravetchi
Department of Pharmacology, University of Alberta, Edmonton T6G 2H7, Canada.
Gonzalo
G
Vilas
Department of Pharmacology, University of Alberta, Edmonton T6G 2H7, Canada; Department of Physiology and Pharmacology, Western University, London N6A 5C1, Canada. Electronic address: james.hammond@ualberta.ca.
James R
JR
Hammond
D002460:Cell Line; D033721:Equilibrative Nucleoside Transporter 1 / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D043682:Receptor, Adenosine A1 / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
D033721:Equilibrative Nucleoside Transporter 1; D043682:Receptor, Adenosine A1; C438649:SLC29A1 protein, human; D012694:Serine; D011493:Protein Kinase C
Kinase; Membranes; Mutagenesis; Nucleoside; Regulation; Transport