Xβm, chicken βm (chβm), and pufferfish βm (pβm), but not hβm, associate with Na,K-ATPase α-subunits and form functional Na,K-ATPase at the cell surface. (a–c) Expression in Xenopus oocytes and coimmunoprecipitation of ³⁵S-metabolically labeled βm of different species with Na,K-ATPase α-subunits. Shown are SDS/PAGE (a, lanes 1–6) and immunoprecipitations with Na,K-ATPase α-subunit antibody (a, lanes 7–18, and b and c). Indicated are the positions of the α-subunit (α), nonglycosylated β (ng), coreglycosylated β (cg), hybrid glycosylated β (hβ), and fully glycosylated β (fg) subunits. endo H, endoglycosidase H. (d) Na,K-pump currents of α1 plus human βm (bar 1) or human β1 (bar 2); chicken α1 plus chicken βm (bar 3) or chicken β1 (bar 4); Xenopus α1 plus Xenopus βm (bar 5) or Xenopus β1 (bar 6); Xenopus α1 plus puffer fish βm (bar 7); Xenopus βm alone (bar 8); Xenopus β1 alone (bar 9). Shown are means ± SE of 21 oocytes from four different batches. All βm vs. β1, P < 0.01. (e) [³H]Ouabain binding. Injection of cRNAs are the same as in d. Shown are means ± SE of 21 oocytes from four different batches.

Changes in functional properties of βm-proteins encoded by orthologous vertebrate ATP1B4 genes during evolution. Direct β,β association in Na,K-ATPase (37) and results of present yeast two-hybrid assays indicate that βm may exist as a dimer in vivo. DNA binding components of transcriptional complex are denoted by X.

Evolutionary changes in the structure of βm N-terminal domains and tissue specificity of expression. (a) Sequence alignment of βm-proteins: human (Hum); mouse (Mus); chicken (Chi); clawed toad, Xenopus laevis (Xen); and pufferfish, Tetraodon nigroviridis (Puf). Identical residues in two or more sequences are highlighted in black, and conserved substitutions are highlighted in gray. Gaps (represented by dashed lines) were introduced to maintain alignment. Arrows mark positions of the exon boundaries, except for the exon encoding the first Met in the chicken βm (represented by ∗). (b) Tissue-specific expression of ATP1B4 genes in chicken, Xenopus, pufferfish, and zebrafish (Danio rerio). RT-PCR analysis of βm-transcripts was done with total RNA extracted from tissues by using specific primers (SI Materials and Methods). The band intensities were normalized using GAPDH RT-PCR products.

βm functions as a transcriptional coregulator in mammalian cells. (a) Interaction of βm and SKIP coexpressed in C2C12 cells and endogenous βm and SKIP in rat neonatal skeletal muscle. Controls were as follows: input, C2C12 (10%), muscle (5%) (lane 1), no cell extract (lane 2), no antibodies (lane 3). Immunoprecipitated (IP) sample is shown in lane 4. (b) Effect of βm–SKIP interaction on the activity of TGF-β-responsive 3TP-luciferase construct in the absence (open bars) and presence of TGF-β, 2 ng/ml (filled bars). (c) βm induces the Smad7 luciferase reporter gene in a dose-dependent manner. Na,K-ATPase β1 subunit was used as a control. (d) Effect of βm-SKIP interaction on Smad7 luciferase reporter gene expression. (b–d) The basal activities of reporter plasmids were set to 1, and all results are shown as means ± SD; n = 3. Transfection efficiencies were normalized by measuring the activities of β-galactosidase (c and d) or Renilla luciferase (b). (e) Effect of βm on endogenous mRNA levels of Smad7, SKIP, and GAPDH. C2C12 cells were transfected with increasing amounts of βm in ng/ml (lane 1, 0; lane 2, 10; lane 3, 20; lane 4, 40; lane 5, 60; lane 6, 100; lane 7, 150; lane 8, 200; lane 9, 250; lane 10, 500; lane 11, 1,000; lane 12, 1,500) and analyzed by RT-PCR using specific primers in ng/ml (SI Materials and Methods). (f) Up-regulation of endogenous Smad7 protein by increasing amounts of transfected βm in C2C12 cells. Shown are the Western blot analyses of equal amounts of proteins and densitometry results of three experiments (means ± SE). (g) ChIP and re-ChIP assays in neonatal rat skeletal muscle and C2C12 cells cotransfected with βm and SKIP. Controls were as follows: chromatin alone (lane 1); IgG (lane 2); and input, 0.05% of total (lane 3). ChIP with antibodies against βm (lane 4) and SKIP (lane 5). For re-ChIP, βm antibody was used first followed by SKIP antibody (lane 4).