Article

Multiplicity of expression of FXYD proteins in mammalian cells: dynamic exchange of phospholemman and gamma-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
Source: PubMed

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.

1 Bookmark
 · 
62 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Restoration of the functional potency of pancreatic islets, either through enhanced proliferation (hyperplasia) or increase in size (hypertrophy) of beta cells is a major objective for intervention in diabetes. We have obtained experimental evidence that global knockout of a small, single-span regulatory subunit of Na,K-ATPase, FXYD2, alters glucose control. Adult Fxyd2-/- mice showed significantly lower blood glucose level, no signs of peripheral insulin resistance, and improved glucose tolerance compared to their littermate controls. Strikingly, there was a substantial hyperplasia in pancreatic beta cells from the Fxyd2-/- mice compared to the wild type littermates, compatible with an observed increase in the level of circulating insulin. No changes were seen in the exocrine compartment of the pancreas, and the mice had only a mild, well-adapted renal phenotype. Morphometric analysis revealed an increase in beta cell mass in KO compared to WT mice. This appears to explain a phenotype of hyperinsulinemia. By RT-PCR, Western blot and immunocytochemistry we showed the FXYD2b splice variant in pancreatic beta cells from wild type mice. Phosphorylation of Akt kinase was significantly higher under basal conditions in freshly isolated islets from Fxyd2-/- mice compared to their WT littermates. Inducible expression of FXYD2 in INS 832/13 cells produced a reduction in the phosphorylation level of Akt, and phosphorylation was restored in parallel with degradation of FXYD2. Thus we suggest that in pancreatic beta cells FXYD2 plays a role in Akt signaling pathways associated with cell growth and proliferation.
    Journal of Biological Chemistry 01/2013; · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: FXYD5 (dysadherin or RIC) is a transmembrane auxiliary subunit of the Na(+)/K(+) ATPase shown to increase its Vmax. FXYD5 has also been identified as a cancer associated protein whose expression in tumor derived cell lines impairs cytoskeletal organization and increases cell motility. Previously, we have demonstrated that the expression of FXYD5 in M1 cells derived from mouse kidney collecting duct, impairs the formation of tight and adherence junctions. The current study aimed to further explore effects of FXYD5 at a single cell level. It was found that in M1 as well as three other cell lines, FXYD5 inhibits transformation of adhered single cells from the initial radial shape to a flattened, elongated shape, in the first stage of monolayer formation. This is also correlated to less ordered actin cables and fewer focal points. Structure-function analysis has demonstrated that the transmembrane domain of FXYD5, and not its unique extracellular segment, mediates the inhibition of change in cell shape. This domain has been shown before to be involved in the association of FXYD5 with the Na(+)/K(+) ATPase which leads to the increase in Vmax. Furthermore, specific transmembrane point mutations in FXYD5 that either increase or decrease its effect on cell elongation had a corresponding effect on the co-immunoprecipitation of FXYD5 with αNa(+)/K(+) ATPase . These findings lend support to the possibility that FXYD5 affects cell polarization through its transmembrane domain interaction with the Na(+)/K(+) ATPase. Yet interaction of FXYD5 with other proteins can not be excluded.
    AJP Cell Physiology 04/2014; 306(11). · 3.71 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    Journal of Membrane Biology 11/2013; 247(1). · 2.48 Impact Factor

Full-text (2 Sources)

Download
13 Downloads
Available from
May 22, 2014