ARL15 Regulates CNNM2-dependent Mg2+ Transport by Modulating its N-linked Glycosylation

Background A large Genome‐wide association study identified that ARL15, a small GTP‐binding protein, is associated with urinary Mg 2+ excretion. Within the kidney, ARL15 is highly expressed in the thick ascending limb (TAL) and distal convoluted tubule (DCT), where Mg 2+ reabsorption is tightly regulated. However, the exact function of ARL15 and the mechanism by which ARL15 regulates renal Mg 2+ handling are still unknown. Method To identify protein‐interaction between ARL15 and Cyclin M (CNNM) proteins, proximity‐dependent biotin identification (BioID) and co‐immunoprecipitation were performed. Immunohistochemistry were used to investigate co‐localization in mouse kidney and human embryonic kidney (HEK293) cells. Furthermore, cell surface biotinylation and 25 Mg 2+ uptake assays were used to assess cell surface expression of CNNM2 and Mg 2+ transport activity. The glycosylation pattern of CNNMs was determined by far lectin Western blot and glycosidase assays. Results We identified members of the CNNM family as direct interaction partners of ARL15 by BioID. Immunoprecipitation with truncated CNNM2 proteins indicated that ARL15 interacts with CNNM2 at its carboxyl‐(C)‐terminal conserved CBS domain. CNNM2 and ARL15 co‐localize in the basolateral membrane of DCT. Interestingly, overexpression of ARL15 in HEK293 cells showed subcellular localization in the Golgi‐apparatus and resulted in an increased N‐glycosylation of CNNM proteins. This ARL15‐mediated glycosylation was Mg 2+ ‐sensitive and encompassed hybrid and complex glycosylation. The functional consequences of ARL15‐dependent glycosylation were examined by 25 Mg 2+ uptake experiments. ARL15 increased 25 Mg 2+ uptake via CNNM2 by increasing its cell surface expression. Conclusion ARL15 increases CNNM2 plasma membrane expression by regulating its N‐glycosylation pattern. Altogether, our results establish ARL15 as novel regulatory mechanism of Mg 2+ transport within the DCT. Support or Funding Information This work was financially supported by grants from the Netherlands Organization for Scientific Research NWO Veni 016.186.012 and VICI 016.130.668).