Characterization of the Fourth α Isoform of the Na,K-ATPase

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K _D values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na⁺ and K⁺. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis. Received: 4 December 1998/Revised: 1 February 1999     

Na,K-ATPase and the role of α isoforms in behavior

The Na,K-ATPase is composed of multiple isoforms and the isoform distribution varies with the tissue and during development. The α1 isoform for example, is the major isoform in the kidney and many other tissues, while the α2 isoform is the predominate one in skeletal muscle. All three isoforms are found in the brain although in adult rodent brain, the α3 isoform is located essentially in neurons while the α2 isoform is found in astrocytes and some limited neuronal populations. Interestingly the α4 isoform is found exclusively in the mid region of the sperm tail. The distribution of the isoforms of the Na,K-ATPase has been extensively studied in many tissues and during development. The examples cited above provide some indication to the diversity of Na,K-ATPase isoform expression. In order to understand the significance of this distribution, we have developed animals which lack the α1, α2, and α3 isoforms. It is anticipated that these studies will provide insight into the role that these isoforms play in driving various biological processes in specific tissues. Here we describe some of our studies which deal with the behavioral aspects of the α1, α2, and α3 deficient mice, particularly those that are haploinsufficient in one isoform i.e. lacking one functional gene for the α1, α2, or α3 isoforms. Such studies are important as two human diseases are associated with deficiency in the α2 and α3 isoforms. These are Familial Hemiplegic Migraine type 2 and Rapid-Onset Dystonia Parkinsonism, these diseases result from α2 and α3 isoform haploinsufficiency, respectively. We find that the haploinsufficiency of both α2 and α3 isoforms result in behavioral defects.     

Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

Background  

Capping protein (CP), a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform.     

Heterogeneity of α-cardiac myosin heavy chains in a small marsupial, Antechinus flavipes, and the effect of hypothyroidism on its ventricular myosins

The effect of drug-induced hypothyroidism on ventricular myosin gene expression was explored in a small marsupial, Antechinus flavipes. Pyrophosphate gel electrophoresis, SDS-PAGE and western blotting were used to analyse changes in native myosin isoforms and myosin heavy chains (MyHCs) in response to hypothyroidism. In some animals, five instead of the normal three native myosin components were found: V_1a, V_1b,V_1c, V₂ and V₃, in order of decreasing mobility. In western blots, V_1a, V_1b, and V_1c reacted with anti-α-MyHC antibody, but not with anti-β-MyHC, whereas V₂ and V₃ reacted with anti-β-MyHC antibody. SDS-PAGE of the unusual ventricular myosins revealed three MyHC isoforms, two of which bound anti-α-MyHC antibody while the third bound anti-β-MyHC antibody. We conclude that V_1a, V_1b, V_1c are triplets arising from the dimerization of two distinct α-MyHC isoforms. Hypothyroidism, verified by metabolic studies, decreased α-MyHC content significantly (t-test, P < 0.001) from 91.6 ± 5.9% (SEM, n = 4) in control animals to 67.2 ± 5.7% (SEM, n = 4) in hypothyroid animals, with a concomitant increase in β-MyHC content. We conclude that in adult marsupials, ventricular myosins are also responsive to changes in the thyroid state as found in eutherians, and suggest that evolution of the molecular mechanisms underlying this thyroid responsiveness predate the divergence of marsupials and eutherians.     

Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines

The receptor for advanced glycation end-products (RAGE) is thought to be expressed ubiquitously as various protein isoforms. Our objective was to use Northern blotting, immunoblotting, and sensitivity to N-glycanase digestion to survey RAGE isoforms expressed in cell lines and mouse tissues in order to obtain a more comprehensive view of the RAGE expressome. Pulmonary RAGE mRNA (1.4 kb) was smaller than cell-line and tissue RAGE mRNA (6 kb-10 kb). Three anti-RAGE antibodies that recognized three distinct RAGE epitopes were used for protein studies (N-16, H-300, and αES). Lung expressed three predominant protein isoforms with apparent molecular masses of 45.1, 52.6, and 57.4 kDa (N-16/H-300) and four isoforms at 25.0, 46.9, 52.5, and 54.2 kDa (αES). These isoforms were expressed exclusively in lung. Heart, ileum, and kidney expressed a 44.0-kDa isoform (N-16), whereas aorta and pancreas expressed a 53.3-kDa isoform (αES). Each of these isoforms were absent in tissue extracts prepared from RAGE^−/− mice. Cell lines expressed a 70.0-kDa isoform, and a subset expressed a 30.0-kDa isoform (αES). Lung RAGE appeared to contain two N-linked glycans. Tissue and cell-line RAGE isoforms were completely insensitive to PNGase F digestion. Thus, numerous RAGE protein isoforms are detectable in tissues and cell lines. Canonical transmembrane and soluble RAGE appear to be expressed solely in lung (N-16/H-300). Non-pulmonary tissues and cell lines, regardless of the source tissue, both express distinct RAGE protein isoforms containing the N-terminal N-16 epitope or the αES RAGE epitope encoded by alternate exon 9, but lacking the H-300 epitope. This work was supported by NIH grants R01 GM37631 and GM68481.     

The mRNA of lipin1 and its isoforms are differently expressed in the <Emphasis Type="Italic">longissimus dorsi</Emphasis> muscle of obese and lean pigs

Lipin1 has been documented to play an important role in adipogenesis. In the present study, the mRNA expression level of lipin1 and its isoforms in longissimus dorsi muscle were determined by semi-quantification RT-PCR in lean PIC and obese Rongchang pigs. Further, we determined mRNA expression for lipin1 and its two isoforms in Rongchang obese pigs which had either a high or low intramuscular fat content. We demonstrate for the first time that porcine lipin1 has two alternative forms, lipin-α and lipin-β. Unlike mice and humans where the lipin-β has 99 more nucleotides than lipin-α, we found that in swine, lipin-β has 108 more nucleotides than lipin-α. Our results indicate that the longissimus dorsi muscle of Rongchang obese pigs have a higher level of mRNA expression for lipin1 and its isoforms than PIC lean pigs. Furthermore, Rongchang pigs with higher intramuscular fat content had a higher lipin1 and lipin-β mRNA expression in longissimus dorsisi muscle than Rongchang pigs with lower intramuscular fat content (P < 0.05), whereas no difference was seen in lipin-α mRNA expression between Rongchang pigs with high or low intramuscular fat. The ratio of lipin-β mRNA to lipin-α mRNA was also significantly different between Rongchang pigs distinguished by a high intramuscular fat content compared with those with low intramuscular fat (P < 0.05). These data suggested that the lipin1 gene may have a crucial effect on body lipid accumulation in pigs, whereas the lipin-β isoform may play an important role in intramuscular fat deposition in obese pigs.     

Substitution of only two residues of human Hsp90α causes impeded dimerization of Hsp90β

Two isoforms of the 90-kDa heat-shock protein (Hsp90), i.e., Hsp90α and Hsp90β, are expressed in the cytosol of mammalian cells. Although Hsp90 predominantly exists as a dimer, the dimer-forming potential of the β isoform of human and mouse Hsp90 is less than that of the α isoform. The 16 amino acid substitutions located in the 561–685 amino acid region of the C-terminal dimerization domain should be responsible for this impeded dimerization of Hsp90β (Nemoto T, Ohara-Nemoto Y, Ota M, Takagi T, Yokoyama K. Eur J Biochem 233: 1–8, 1995). The present study was performed to define the amino acid substitutions that cause the impeded dimerization of Hsp90β. Bacterial two-hybrid analysis revealed that among the 16 amino acids, the conversion from Ala₅₅₈ of Hsp90β to Thr₅₆₆ of Hsp90α and that from Met₆₂₁ of Hsp90β to Ala₆₂₉ of Hsp90α most efficiently reversed the dimeric interaction, and that the inverse changes from those of Hsp90α to Hsp90β primarily explained the impeded dimerization of Hsp90β We conclude that taken together, the conversion of Thr₅₆₆ and Ala₆₂₉ of Hsp90α to Ala₅₅₈ and Met₆₂₁ is primarily responsible for impeded dimerization of Hsp90β.     

Homology modeling and dynamics of the extracellular domain of rat and human neuronal nicotinic acetylcholine receptor subtypes α4β2 and α7

In recent years, it has become clear that the neuronal nicotinic acetylcholine receptor (nAChR) is a valid target in the treatment of a variety of diseases, including Alzheimer’s disease, anxiety, and nicotine addiction. As with most membrane proteins, information on the three-dimensional (3D) structure of nAChR is limited to data from electron microscopy, at a resolution that makes the application of structure-based design approaches to develop specific ligands difficult. Based on a high-resolution crystal structure of AChBP, homology models of the extracellular domain of the neuronal rat and human nAChR subtypes α4β2 and α7 (the subtypes most abundant in brain) were built, and their stability assessed with molecular dynamics (MD). All models built showed conformational stability over time, confirming the quality of the starting 3D model. Lipophilicity and electrostatic potential studies performed on the rat and human α4β2 and α7 nicotinic models were compared to AChBP, revealing the importance of the hydrophobic aromatic pocket and the critical role of the α-subunit Trp—the homolog of AChBP-Trp 143—for ligand binding. The models presented provide a valuable framework for the structure-based design of specific α4β2 nAChR subtype ligands aimed at improving therapeutic and diagnostic applications. Figure Electrostatic surface potential of the binding site cavity of the neuronal nicotinic acetylcholine receptor (nAChR). Nicotinic models performed with the MOLCAD program: a rat α7, b rat α4β2, c human α7, d human α4β2. All residues labeled are part of the α7 (a,c) or α4 (b,d) subunit with the exception of Phe 117, which belongs to subunit β2 (d). Violet Very negative, blue negative, yellow neutral, red very positive     

Differential expression of a subunit isoforms of the vacuolar-type proton pump ATPase in mouse endocrine tissues

Vacuolar-type proton ATPase (V-ATPase) is a multi-subunit enzyme that couples ATP hydrolysis to the translocation of protons across membranes. Mammalian cells express four isoforms of the a subunit of V-ATPase. Previously, we have shown that V-ATPase with the a3 isoform is highly expressed in pancreatic islets and is located in the membranes of insulin-containing granules in the β cells. The a3 isoform functions in the regulation of hormone secretion. In this study, we have examined the distribution of a subunit isoforms in endocrine tissues, including the adrenal, parathyroid, thyroid, and pituitary glands, with isoform-specific antibodies. We have found that the a3 isoform is strongly expressed in all these endocrine tissues. Our results suggest that functions of the a3 isoform are commonly involved in the process of exocytosis in regulated secretion. This research was supported in part by Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan and by the Hayashi, Takeda, and Noda Foundations.     

Na+,K+-ATPase Na+ Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na⁺/K⁺-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na⁺,K⁺-ATPase activity, and the Na⁺ affinity of Na⁺,K⁺-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na⁺ affinity was higher (K _m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na⁺,K⁺-ATPase isoform analysis implied that heterodimers containing the β₁ isoform have a higher Na⁺ affinity than heterodimers containing the β₂ isoform. Immunoprecipitation experiments demonstrated that dimers with α₁ are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α₂ isoform with ouabain suggested that heterodimers containing the α₁ isoform have a higher Na⁺ affinity than heterodimers containing the α₂ isoform. The estimated K _m values for Na⁺ are 4.0, 5.5, 7.5 and 13 mM for α₁β₁, α₂β₁, α₁β₂ and α₂β₂, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na⁺,K⁺-ATPase at physiological Na⁺ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.     

The mouse adducin gene family: alternative splicing and chromosomal localization

Mouse cDNA sequences encoding α, β, and γ adducins were cloned from a mouse reticulocyte cDNA library. The purified clones contain alternatively spliced exons from all three adducin genes. In the case of α and β, the inclusion of the alternatively spliced exons results in truncated polypeptide isoforms (called α-2 and β-2). The mouse predicted amino acid sequences are compared with published rat and human sequences. For completion of this comparison, cDNA encoding the rat β-1 carboxy terminus was cloned by PCR. The carboxy terminal region containing MARCKS homology, calmodulin-binding region-2, and spectrin-actin-binding site, is conserved among α-1, β-1, and γ-1 isoforms in mouse, rat, and humans. We also report here the localization of the gene encoding γ adducin (Add3) to murine Chr 19, in a region that shows conserved synteny with human Chr 10. Received: 1 June 1999 / Accepted: 25 August 1999     

Genomic organization and refined mapping of the mouse β-dystrobrevin gene

β-Dystrobrevin, a dystrophin-related protein that is expressed in non-muscle tissues, is highly homologous to α-dystrobrevin, a member of the dystrophin-associated protein complex (DPC). β-Dystrobrevin associates with Dp71 and syntrophin and is believed to have a role in non-muscle DPCs. Here we report the characterization and mapping of the mouse β-dystrobrevin gene. The mouse β-dystrobrevin gene is organized into 21 exons spanning over 130 kb of DNA. We provide evidence that this gene is transcribed from at least two promoter regions but appears to utilize a common translation initiation site. We show that the similarity between β-dystrobrevin and α-dystrobrevin is reflected in the conservation of their exon-intron junctions. β-Dystrobrevin has been localized to proximal mouse Chromosome (Chr) 12 by backcross mapping. A database search revealed that two mouse genetic diseases involving tissues expressing β-dystrobrevin have been mapped to this region, namely, congenital polycystic kidneys (cpk) and fatty liver dystrophy (fld). However, refined mapping analysis has excluded β-dystrobrevin as a candidate gene for either disease. Received: 1 June 1998 / Accepted: 16 July 1998     

Generation of novel chimeric LacdiNAcS by gene fusion of α-lactalbumin and β1,4-galactosyltransferase 1

Novel chimeric lacdiNAc (GalNAc(β1-4)GlcNAc) synthase (c-LacdiNAcS) was generated by gene fusion of α-lactalbumin (α-LA) and β1,4-galactosyltransferase 1 (β1,4-GalT1). c-LacdiNAcS was expressed in Lec8 Chinese hamster ovary (Lec8 CHO) cells and exhibited N-acetylgalactosaminyltransferase (GalNAcT) activity in the absence of exogenous α-LA as well as other glycosyltransferase activities including lactose synthase (LacS), and β1,4-GalT. These glycosyltransferase activities of c-LacdiNAcS were compared to those activities induced in LacS system under the co-presence of bovine β1,4-GalT1 and α-LA, indicating that each domain of α-LA and β1,4-GalT1 on c-LacdiNAcS is not only folding correctly, but also interacting together. Furthermore, c-LacdiNAcS was found to be auto-lacdiNAcylated and can synthesize lacdiNAc structures on cellular glycoproteins, demonstrating that GalNAcT activity of c-LacdiNAcS is functional in Lec8 CHO cells.     

Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2

Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2α (K _m 0.35 μM) and CK2α′ (K _m 0.18 μM) as well as CK2 holoenzyme (K _m 1.1 μM). Different K _m values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor Svf1. Reconstitution of α′₂ββ′ isoform of CK2 holoenzyme shows that β/β′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of α₂ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K¹⁹⁹EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED²⁴⁸ of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.