Galectin‐4 interacts with the drug transporter human concentrative nucleoside transporter 3 to regulate its function

Abstract

The intracellular N-terminal domain of the nucleoside and drug transporter human concentrative nucleoside transporter (hCNT)3 was used as bait in a glutathione S-transferase pull-down approach, to identify hCNT3 protein partners, using human colon homogenates as a prey source. Galectin (Gal)-4 was identified as a potential hCNT3 partner in the colon. The biochemical validation of the Gal-4-hCNT3 interaction was verified by targeted pull-down assays and coimmunoprecipitation experiments in HT-29 cells, which endogenously express hCNT3 and Gal-4. Furthermore, Gal-4 was shown to colocalize with hCNT3 in HT-29 cells. The biologic significance of this interaction was obtained from experiments in which Gal-4 was knocked down, showing that this protein is a regulator of hCNT3 trafficking and retention at the cell membrane, reducing its plasma membrane location by 70%. Conversely, the addition of Gal-4 increased hCNT3 location at the plasma membrane by 77%, thereby demonstrating that this lectin modulates hCNT3 function in colonic cells. The integrity of this partnership may be clinically relevant, because hCNT3 may be responsible for the translocation of thiopurines, such as 6-mercaptopurine, a front-line treatment in inflammatory bowel disease. The expression of Gal-4 and hCNT3 proteins is not impaired in inflamed colon from patients with Crohn's disease, thereby anticipating the integrity of this system for drug targeting.

Toward a Molecular Basis of Cellular Nucleoside Transport in Humans

“…82 It is important to note while the CNT Nterminal domain is functionally dispensable, it likely plays important roles in mediating protein−protein interactions, controlling transporter trafficking, and may contribute to transporter stability. 85,86 Although these initial studies were important advancements to the field of nucleoside research, it remained a challenge to accurately predict the transporter architecture. To gain a detailed understanding of the molecular requirements for nucleoside recognition, ion-coupling and the mechanism of nucleoside membrane translocation exhibited by CNTs, experimental transporter structures were in dire need.…”

Emerging Roles of Nucleoside Transporters

“…This regulatory function seems to be exclusively related to trafficking events resulting in the down-regulation of these proteins without any evident effect of RS1 at the transcriptional level ( Errasti-Murugarren et al, 2012 ). Interaction of hCNT3 with galectin-4 was described in colonic epithelial cells where this partnership appears to be crucial for the proper insertion and retention of this transporter at the apical membrane, determining also hCNT3 turnover ( Fernández-Calotti et al, 2016 ).…”

Adenosine in Intestinal Epithelial Barrier Function

“…In polarized epithelial cells, CNTs preferentially localize to the apical surface [ 46 , 47 , 48 ]. In colonic epithelial cells, this retention is facilitated by galectin-4, which anchors CNT3 to the apical surface [ 49 ].…”