title: 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 172C→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,K-ATPase 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. 172C→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.
authors: Laboratory of Membrane Biology, Department of Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
authors: Kathleen J
authors: KJ
authors: Sweadner
authors: Jennifer L
authors: JL
authors: Pascoa
authors: Cynthia A
authors: CA
authors: Salazar
authors: Elena
authors: E
authors: Arystarkhova
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation; D021382:Protein Sorting Signals / Q000502:physiology*; D017393:RNA Editing / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D007527:Isoenzymes; D021382:Protein Sorting Signals; D012333:RNA, Messenger; C498817:Fxyd2 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase