Article

Multiplicity of expression of FXYD proteins in mammalian cells: dynamic exchange of phospholemman and -subunit in response to stress

Laboratory of Membrane Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
AJP Cell Physiology (Impact Factor: 3.67). 04/2007; 292(3):C1179-91. DOI: 10.1152/ajpcell.00328.2006
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ABSTRACT Functional properties of Na-K-ATPase can be modified by association with FXYD proteins, expressed in a tissue-specific manner. Here we show that expression of FXYDs in cell lines does not necessarily parallel the expression pattern of FXYDs in the tissue(s) from which the cells originate. While being expressed only in lacis cells in the juxtaglomerular apparatus and in blood vessels in kidney, FXYD1 was abundant in renal cell lines of proximal tubule origin (NRK-52E, LLC-PK1, and OK cells). Authenticity of FXYD1 as a part of Na-K-ATPase in NRK-52E cells was demonstrated by co-purification, co-immunoprecipitation, and co-localization. Induction of FXYD2 by hypertonicity (500 mosmol/kgH(2)O with NaCl for 48 h or adaptation to 700 mosmol/kgH(2)O) correlated with downregulation of FXYD1 at mRNA and protein levels. The response to hypertonicity was influenced by serum factors and entailed, first, dephosphorylation of FXYD1 at Ser(68) (1-5 h) and, second, induction of FXYD2a and a decrease in FXYD1 with longer exposure. FXYD1 was completely replaced with FXYD2a in cells adapted to 700 mosmol/kgH(2)O and showed a significantly decreased sodium affinity. Thus dephosphorylation of FXYD1 followed by exchange of regulatory subunits is utilized to make a smooth transition of properties of Na-K-ATPase. We also observed expression of mRNA for multiple FXYDs in various cell lines. The expression was dynamic and responsive to physiological stimuli. Moreover, we demonstrated expression of FXYD5 protein in HEK-293 and HeLa cells. The data imply that FXYDs are obligatory rather than auxiliary components of Na-K-ATPase, and their interchangeability underlies responses of Na-K-ATPase to cellular stress.

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    • "In the kidney, several FXYD proteins (i.e., FXYD1, FXYD2, FXYD4, and FXYD5) have been identified (Arystarkhova et al. 2007; Béguin et al. 2001; Bogaev et al. 2001; Lubarski et al. 2005; Mercer et al. 1993; Pihakaski-Maunsbach et al. 2008). FXYDs were suggested to be obligatory rather than auxiliary components of NKA, and their interchangeability underlies responses of NKA to cellular stress (Arystarkhova et al. 2007). Among them, FXYD2 (c subunit of NKA) is the most important regulator modulating the properties of NKA in renal cells Fig. 6 Functional inhibition of FXYD2a/2b/2c or FXYD2a/2b with shRNA-A or shRNA-B abrogated the mRNA level of fxyd2c under isotonic or hypertonic conditions. "
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    ABSTRACT: Na(+)/K(+)-ATPase (NKA) is a widely found and important transporter in mammals. The kidney is a major osmoregulatory organ of which the proximal tubules play a crucial role in the maintenance of ionic homeostasis functioning via salt and water reabsorption. FXYD (FXYD domain-containing protein) 2, the γ-subunit of NKA, is the first identified and the most abundant member of FXYD family, affecting the sodium/potassium affinity of NKA in the kidney. Based on DNA microarray analysis, the expression levels of fxyd2 gene are markedly increased upon hypertonic challenge. Combined with bioinformatic analysis using the NCBI database, we identified an unnamed protein with 145 amino acids, of which the N-terminus involved the FXYD sequence similar to FXYD2a and FXYD2b, and thus, named as FXYD2c. However, the role of FXYD2c protein in the regulation of NKA expression in the kidney has not been elucidated. In this study, we found that the mRNA and protein levels of FXYD2c were significantly increased upon hypertonic challenge. Immunoprecipitation data revealed that FXYD2c interacts with the NKA α1 subunit. Subsequently, the functional inhibition of fxyd2c using short hairpin RNA abrogated NKA activity. Taken together, our study offers novel insight into the potential function of FXYD2c in promoting NKA activity upon hypertonic challenge in HK-2 cells.
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    • "The kinetic properties of the enzyme also may be modulated via physical association with the so-called FXYD proteins, a family of small, homologous, single-span membrane proteins . To date, six members of this family are known to be associated with the (Na, K)-ATPase in a cell-or tissue-specific manner [7]. According to Axelsen and Palmgren's classification [8], the (Na, K)-ATPase is a P 2c -type ATPase, that is, an ATPpowered ion pump that translocates cations across plasma membranes. "
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    • "The kinetic properties of the enzyme also may be modulated via physical association with the so-called FXYD proteins, a family of small, homologous, single-span membrane proteins . To date, six members of this family are known to be associated with the (Na, K)-ATPase in a cell-or tissue-specific manner [7]. According to Axelsen and Palmgren's classification [8], the (Na, K)-ATPase is a P 2c -type ATPase, that is, an ATPpowered ion pump that translocates cations across plasma membranes. "
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