β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

Abstract

Clifford RJ, Kaplan JH. ␤-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells. Am J Physiol Renal Physiol 295: F1314 -F1323, 2008. First published August 13, 2008 doi:10.1152/ajprenal.90406.2008In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the Na-K-ATPase ␣-and ␤-subunits led us to question whether this was alterable and thus if some form of regulation was involved. We have examined the consequences of overexpressing Na-K-ATPase ␤ 1-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged ␤ 1-subunits (␤1flag) or Myc-tagged ␤1-subunits (␤1myc) under the control of a tetracycline-dependent promoter. The induction of ␤ 1flag subunit synthesis in MDCK cells, which increases ␤1-subunit expression at the plasma membrane by more than twofold, while maintaining stable ␣ 1 expression levels, revealed that all mature ␤ 1-subunits associate with ␣1-subunits, and no evidence of "free" ␤ 1-subunits was obtained. Consequently, the ratio of assembled ␤1-to ␣1-subunits is significantly increased when "extra" ␤-subunits are expressed. An increased ␤ 1/␣1 stoicheometry is also observed in cells treated with tunicamycin, suggesting that the protein-protein interactions involved in these complexes are not dependent on glycosylation. Confocal images of cocultured ␤ 1myc-expressing and ␤1flag-expressing MDCK cells show colocalization of ␤1myc and ␤1flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the ␤-subunits. Immunoprecipitation using MDCK cells constitutively expressing ␤1myc and tetracycline-regulated ␤1flag subunits confirmed ␤-␤-subunit interactions. These results demonstrate that the equimolar ratio of assembled ␤1/

Subunit Isoform Selectivity in Assembly of Na,K-ATPase α-β Heterodimers

“…5B and Fig. 8, C and D) (34,40). These results are consistent with the previous finding showing that both YFP-␤ 1 and YFP-␤ 2 compete with the endogenous ␤ 1 subunits for binding to the limited amount of the endogenous ␣ 1 subunits (34).…”

Dynamic internalization and recycling of a metal ion transporter: Cu homeostasis and CTR1, the human Cu+ uptake system

“…Protein samples were subject to SDS-PAGE and western blot analysis (Clifford and Kaplan, 2008). To obtain total cell lysates, cells were washed twice with PBS, harvested by scraping, and whole cells pelleted at 1000 g for 5 min.…”

The O-glycosylated ectodomain of FXYD5 impairs adhesion by disrupting cell–cell trans-dimerization of Na,K-ATPase β1 subunits

“…It acts as a cell adhesion molecule by undergoing both intercellular trans-dimerization through its β 1 subunit and intracellular association with the cytoskeleton through its α 1 subunit (Clifford and Kaplan, 2008;Shoshani et al, 2005). N-glycosylation of the β 1 subunit is important for normal cell adhesion because MDCK cells (a cell line with characteristics of the distal renal tubule) expressing the unglycosylated β 1 subunit form cell-cell contacts more slowly than non-transfected MDCK cells (Vagin et al, 2006(Vagin et al, , 2008.…”