NKCC2 Surface Expression in Mammalian Cells - Université Pierre et Marie Curie Accéder directement au contenu
Article Dans Une Revue Journal of Biological Chemistry Année : 2007

NKCC2 Surface Expression in Mammalian Cells

Résumé

Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at under-standing the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.
Fichier principal
Vignette du fichier
1-s2.0-S0021925820717187-main.pdf (1.33 Mo) Télécharger le fichier
Origine : Fichiers éditeurs autorisés sur une archive ouverte
licence : CC BY - Paternité

Dates et versions

hal-03160997 , version 1 (07-06-2023)

Identifiants

Citer

Boubacar Benziane, Sylvie Demaretz, Nadia Defontaine, Nancy Zaarour, Lydie L Cheval, et al.. NKCC2 Surface Expression in Mammalian Cells. Journal of Biological Chemistry, 2007, 282 (46), pp.33817-33830. ⟨10.1074/jbc.M700195200⟩. ⟨hal-03160997⟩
21 Consultations
1 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More