Abstract
[Background] Sodium-coupled monocarboxylate transporter 1, (SMCT1), mediates monocarboxylates reabsorption on the apical side of proximal tubule in the kidney. Previously, utilizing a yeast two hybrid screening system, we have identified that PDZK1 and PDZ domain-containing ring finger 3 (PDZRN3) as potent binding partners of SMCT1 through its C-terminal sequence, where the conserved PDZ motif (Thr-Arg-Leu) exists. While we have then clarified that PDZK1 enhances the transporter activity of SMCT1, interaction between SMCT1 and PDZRN3 has yet to be fully characterized, and thus roles played by PDZRN3 in functional regulation of SMCT1 remains unclear. In this study, we aimed to characterize this interaction as well as to clarify how PDZRN3 regulates SMCT1 function.
[Methods] We performed co-immunoprecipitation assays to examine the interaction between SMCT1 and PDZRN3, transport activity assays to examine the functional change of SMCT1, and immunocytochemical assays to examine localization of SMCT1, using HEK293 cells transiently transfected with SMCT1, PDZK1, and/or PDZRN3.
[Results] Co-immunoprecipitation assay showed that SMCT1 was immunoprecipitated with anti-FLAG antibody, and that PDZRN3-FLAG was immunoprecipitated with anti-SMCT1 antibody. This interaction was abolished when the PDZ motif was removed from C the terminus of SMCT1. [3H]-Nicotinate uptake via SMCT1 increased by 1.2-fold in the presence of PDZK1, but not in the presence of PDZRN3. However, enhance effect of PDZK1 on nicotinate uptake was abolished when the PDZRN3 was co-expressed. Consistently, immunocytochemical assay showed that, compared with the case of SMCT1 alone, its expression level on the cell membrane appeared to be increased in the presence of PDZK1, and that this augmented effect of PDZK1 was abolished when PDZRN3 was co-transfected.
[Conclusions] Our results showed that PDZRN3 can bind to SMCT1 through its C-terminal PDZ motif, which may play a role in regulation of SMCT1 function by interfering the interaction between SMCT1 and PDZK1.
