ABSTRACT
Change in gene functions (gene cooption) is one of the key mechanisms of molecular evolution. Genes can acquire new functions via alteration in properties of encoded proteins and/or via changes in temporal or spatial regulation of expression. Here we demonstrate radical changes in the functions of orthologous ATP1B4 genes during evolution of vertebrates. Expression of ATP1B4 genes is brain-specific in teleost fishes, whereas it is predominantly muscle-specific in tetrapods. The encoded beta m-proteins in fish, amphibian, and avian species are beta-subunits of Na,K-ATPase located in the plasma membrane. In placental mammals beta m-proteins lost their ancestral functions, accumulate in nuclear membrane of perinatal myocytes, and associate with transcriptional coregulator Ski-interacting protein (SKIP). Through interaction with SKIP, eutherian beta m acquired new functions as exemplified by regulation of TGF-beta-responsive reporters and by augmentation of mRNA levels of Smad7, an inhibitor of TGF-beta signaling. Thus, orthologous vertebrate ATP1B4 genes represent an instance of gene cooption that created fundamental changes in the functional properties of the encoded proteins.
Subject(s)
Evolution, Molecular , Protein Subunits/chemistry , Protein Subunits/physiology , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/physiology , Transcription Factors/chemistry , Transcription Factors/physiology , Amino Acid Sequence , Animals , Chick Embryo , Chickens , Humans , Mice , Molecular Sequence Data , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiology , Nuclear Proteins/physiology , Protein Subunits/biosynthesis , Protein Subunits/genetics , Rats , Sodium-Potassium-Exchanging ATPase/biosynthesis , Sodium-Potassium-Exchanging ATPase/genetics , Tetraodontiformes , Transcription Factors/genetics , Xenopus laevisABSTRACT
Cardiac steroids inhibit Na,K-ATPase and the related non-gastric H,K-ATPase, while they do not interact with gastric H,K-ATPase. Introducing an arginine, the residue present in the gastric H,K-ATPase, in the second extracellular loop at the corresponding position 334 in the human non-gastric H,K-ATPase (D334R mutation) rendered it completely resistant to 2mM ouabain. The corresponding mutation (E319R) in alpha1 Na,K-ATPase produced a approximately 2-fold increase of the ouabain IC(50) in the ouabain-resistant rat alpha1 Na,K-ATPase and a large decrease of the ouabain affinity of human alpha1 Na,K-ATPase, on the other hand this mutation had no effect on the affinity for the aglycone ouabagenin. These results provide a strong support for the orientation of ouabain in its biding site with its sugar moiety interacting directly with the second extracellular loop.
Subject(s)
Amino Acid Substitution , Cardiac Glycosides/metabolism , H(+)-K(+)-Exchanging ATPase/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Amino Acid Sequence , Animals , Aspartic Acid/genetics , Binding, Competitive/drug effects , Biological Transport/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Female , Glutamic Acid/genetics , H(+)-K(+)-Exchanging ATPase/genetics , Humans , Membrane Potentials/drug effects , Mutation , Oocytes/drug effects , Oocytes/metabolism , Oocytes/physiology , Ouabain/analogs & derivatives , Ouabain/pharmacology , Protein Subunits/antagonists & inhibitors , Protein Subunits/genetics , Protein Subunits/metabolism , Proton Pump Inhibitors , Rabbits , Rats , Rubidium Radioisotopes/pharmacokinetics , Sequence Homology, Amino Acid , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/genetics , Xenopus laevisABSTRACT
The structural organization of nongastric H-K-ATPase, unlike that of closely related Na-K-ATPase and gastric H-K-ATPase, is not well characterized. Recently, we demonstrated that nongastric H-K-ATPase alpha-subunit (alpha(ng)) is expressed in apical membranes of rodent prostate. Its highest level, as well as relative abundance, with respect to alpha(1)-isoform of Na-K-ATPase, was observed in anterior lobe. Here, we aimed to determine the subunit composition of nongastric H-K-ATPase through the detailed analysis of the expression of all known X-K-ATPase beta-subunits in rat anterior prostate (AP). RT-PCR detects transcripts of beta-subunits of Na-K-ATPase only. Measurement of absolute protein content of these three beta-subunit isoforms, with the use of quantitative Western blotting of AP membrane proteins, indicates that the abundance order is beta(1) > beta(3) >> beta(2). Immunohistochemical experiments demonstrate that beta(1) is present predominantly in apical membranes, coinciding with alpha(ng), whereas beta(3) is localized in the basolateral compartment, coinciding with alpha(1). This is the first direct demonstration of the alpha(ng)-beta(1) colocalization in situ indicating that, in rat AP, alpha(ng) associates only with beta(1). The existence of alpha(ng-)beta(1) complex has been confirmed by immunoprecipitation experiments. These results indicate that beta(1)-isoform functions as the authentic subunit of Na-K-ATPase and nongastric H-K-ATPase. Putatively, the intracellular polarization of X-K-ATPase isoforms depends on interaction with other proteins.
