Link between high‐affinity adenosine concentrative nucleoside transporter‐2 (CNT2) and energy metabolism in intestinal and liver parenchymal cells

Abstract

Concentrative nucleoside transporter 2 (CNT2) is a high-affinity adenosine transporter that may play physiological roles beyond nucleoside salvage. Previous reports relate CNT2 function to modulation of purinergic signaling and energy metabolism in intestinal and liver parenchymal cells (Duflot et al., 2004, Mol Cell Biol 24:2710-2719; Aymerich et al., 2006, J Cell Sci 119:1612-1621). In the present study, to further examine the link between CNT2 and energy metabolism, CNT2 protein partners were identified using the bacterial two-hybrid and GST pull-down approaches. The N-terminal segment of CNT2 was used as bait, since proteins lacking this domain display impaired plasma membrane insertion and intracellular retention. Glucose-regulated protein 58 (GRP58) was identified as a potential rCNT2 partner in pull-down experiments. Two-hybrid screening performed against a liver human cDNA library led to the identification of aldolase B as another hCNT2 partner. Aldolase B-RFP and endogenous GRP58 separately co-localized with CNT2 in HeLa cells transfected with YFPrCNT2. CNT2 interaction with GRP58 was validated using co-immunoprecipitation experiments. In HeLa cells, fluorescence resonance energy transfer (FRET) efficiency increased upon fructose addition, consistent with a transient interaction between aldolase B and the transporter. The physiological basis for in vivo interactions was derived from experiments in which GRP58 was inhibited or overexpressed and aldolase B activity stimulated towards glycolysis. GRP58 appeared to be a negative effector of CNT2 function, whereas aldolase B flux modulated CNT2 activity via a mechanism involving acquisition of higher affinity for its substrates. These findings support the theory that CNT2 plays roles other than salvage and establishes links with energy metabolism.

Proapoptotic Activities of Protein Disulfide Isomerase (PDI) and PDIA3 Protein, a Role of the Bcl-2 Protein Bak

“…1B). To further confirm that PDI and PDIA3 catalytic activities are vital to mediate apoptotic signaling, two other PDI and PDIA3 inhibitors, bacitracin and thiomuscimol, were investigated (18,21,28,29). In agreement with the study of securinine, both bacitracin and thiomuscimol alleviated the cytotoxic effects of different apoptotic stimuli in MEF cells (Fig.…”

“…Several small molecules have been found to inhibit PDI and PDIA3 catalytic activities and have been used to elucidate the role of the protein folding catalyst in a variety of signaling pathways (18,21,28,29). MEF cells were treated with a range of reagents known to induce apoptosis in the presence or absence of the PDI inhibitor securinine (18).…”

Structural determinants for rCNT2 sorting to the plasma membrane of polarized and non-polarized cells

“…Previous studies in our laboratory suggested that sorting signals implicated in the membrane insertion of CNTs are localized in the intracellular N-terminal tail of these proteins [13,16,26]. This is based on the evidence that an alternative splicing variant of hCNT3, which is truncated in its N-terminus tail is retained in the ER [16].…”

“…CNT2 has also been described as a key player in energy metabolism, necessary for the activation of AMPK (AMP-activated protein kinase) by extracellular adenosine [12]. Interestingly, the only proteins known so far to interact directly with rCNT2 are the glycolitic enzyme aldolase B and the glucose-dependent chaperone grp58 (glucose-regulated protein 58), both intimately implicated in energy metabolism [13]. In addition, rCNT2 expression and activity are under endocrine and paracrine control, since differentiating factors such as glucocorticoids, and proliferative agents such as EGF (epidermal growth factor) and TGFβ (transforming growth factor β) increase its expression and activity at the plasma membrane [14,15].…”

Differential response of Drosophila cell lines to extracellular adenosine

“…All equilibrative (Ent1-4 in mammals) and concentrative nucleoside transporters (Cnt1-3 in mammals) are able to effectively transport Ado and thus contribute to Ado signaling regulation. Moreover, it has recently been shown that nucleoside transporters might play important role in cellular metabolism independent of Ado receptor signaling (Huber-Ruano et al, 2010).…”