Post-transcriptional Control of Na,K-ATPase Activity and Cell Growth by a Splice Variant of FXYD2 Protein with Modified mRNA

Abstract

In kidney, FXYD proteins regulate Na,K-ATPase in a nephron segment-specific way. FXYD2 is the most abundant renal FXYD but is not expressed in most renal cell lines unless induced by hypertonicity. Expression by transfection of FXYD2a or FXYD2b splice variants in NRK-52E cells reduces the apparent Na ؉ affinity of the Na,K-ATPase and slows the cell proliferation rate. Based on RT-PCR, mRNAs for both splice variants were expressed in wild type NRK-52E cells as low abundance species. DNA sequencing of the PCR products revealed a base alteration from C to T in FXYD2b but not FXYD2a from both untreated and hypertonicity-treated NRK-52E cells. The 172C3 T sequence change exposed a cryptic KKXX endoplasmic reticulum retrieval signal via a premature stop codon. The truncation affected trafficking of FXYD2b and its association with Na,KATPase and blocked its effect on enzyme kinetics and cell growth. The data may be explained by altered splicing or selective RNA editing of FXYD2b, a supplementary process that would ensure that it was inactive even if transcribed and translated, in these cells that normally express only FXYD2a. 172C3 T mutation was also identified after mutagenesis of FXYD2b by error-prone PCR coupled with a selection for cell proliferation. Furthermore, the error-prone PCR alone introduced the mutation with high frequency, implying a structural peculiarity. The data confirm truncation of FXYD2b as a potential mechanism to regulate the amount of FXYD2 at the cell surface to control activity of Na,K-ATPase and cell growth. Na,K-ATPase is an enzyme in eukaryotic cells that provides non-equilibrium distribution of Na ϩ and K ϩ ions across the plasma membrane. Besides its obligatory ␣ and ␤ subunits, the complex contains a so-called "FXYD" subunit (1), a short single span membrane protein involved in regulation of the kinetic properties of Na,K-ATPase. In mammals, there are seven different FXYD genes that are expressed in a tissue-and cell-specific manner. Remarkably, association of the Na,K-ATPase with each of them leads to specific changes in kinetic parameters of the pump either at the level of K 0.5 for the substrates or V max (for a review, see Ref.2).FXYD2, also called the ␥ subunit, was the first accessory FXYD protein discovered in connection with the Na,K-ATPase (3). It was identified as a proteolipid in membranes from dog kidney that was labeled with a photoaffinity derivative of the specific inhibitor of the pump, ouabain, along with the ␣ and ␤ subunits (4). Expression of the FXYD2 protein is mostly in kidney (5), although the expressed sequence tag database suggests some other tissues as potential sources for FXYD2 (for instance, pancreas and mammary glands). There are two splice variants of FXYD2, FXYD2a and FXYD2b, which are identical in structure except for the N-terminal segment comprising the first exon (6, 7). Based on results from different heterologous expression systems (Xenopus oocytes or mammalian cell transfectants), FXYD2 reduces the activity of the pump by increasing the K 0.5 for ...

Expression of the Na+/K+-transporting ATPase gamma subunit FXYD2 in renal tumors

“…Previous studies have shown that FXYD2 is upregulated in cultured rat renal NRK-52E cells in response to hyperosmotic stress. 25 Interestingly, upregulation of full-length FXYD2 had a negative effect on cell growth in cultured NRK-52E cells. Expression of a mutant FXYD2 resulting in loss of the C-terminus, however, was associated with loss of inhibition of cell growth in culture.…”

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

“…This alternative use of first exons has been suggested to play a role in posttranscriptional regulation of Fxyd2b transcripts. 14 Figure 3B shows two glutaminase (Gls) isoforms with alternative carboxyl-terminal amino acid sequences and 39-untranslated regions. The short isoform predominates in segments downstream from the proximal tubule, but the long isoform is more abundant in proximal tubule.…”

Structure of the Na,K-ATPase regulatory protein FXYD2b in micelles: Implications for membrane–water interfacial arginines

“…Its function is modulated by post-translational modification [16, 18], as well as by gene splicing and RNA editing, which respectively govern the expression of two splice variants, FXYD2a and FXYD2b, and of a truncated form of FXYD2b that is not exported to the plasma membrane [5, 13, 14, 21]. The two splice variants of FXYD2 have identical amino acid sequences, except in their N-terminal segments, encoded by the first exon [22, 23].…”