Activity and PI3-kinase dependent trafficking of the intestinal anion exchanger downregulated in adenoma depend on its PDZ interaction and on lipid rafts

Abstract

The Cl/HCO(3) exchanger downregulated in adenoma (DRA) mediates electroneutral NaCl absorption in the intestine together with the apical Na/H exchanger NHE3. Lipid rafts (LR) modulate transport activity and are involved in phosphatidylinositol 3-kinase (PI3-kinase)-dependent trafficking of NHE3. Although DRA and NHE3 interact via PDZ adaptor proteins of the NHERF family, the role of LR and PDZ proteins in the regulation of DRA is unknown. We examined the association of DRA with LR using the nonionic detergent Triton X-100. DRA cofractionated with LR independently of its PDZ binding motif. Furthermore, DRA interacts with PDZK1, E3KARP, and IKEPP in LR, although their localization within lipid rafts is independent of DRA. Disruption of LR integrity resulted in the disappearance of DRA from LR, in a decrease of its surface expression and in a reduction of its activity. In HEK cells the inhibition of DRA by LR disruption was entirely dependent on the presence of the PDZ interaction motif. In addition, in Caco-2/BBE cells the inhibition by LR disruption was more pronounced in wild-type DRA than in mutated DRA (DRA-ETKFminus; lacking the PDZ binding motif)-expressing cells. Inhibition of PI3-kinase decreased the activity and the cell surface expression of wild-type DRA but not of DRA-ETKFminus; the partitioning into LR was unaffected. Furthermore, simultaneous inhibition of PI3-kinase and disruption of LR did not further decrease DRA activity and cell surface expression compared with LR disruption only. These results suggest that the activity of DRA depends on its LR association, on its PDZ interaction, and on PI3-kinase activity.