Structural analysis and expression of a chromosomal gene encoding an avian Na+K+-ATPase β1-subunit

212

Biochimica et Biophysica Acta, 1172 (1993) 212-216 Elsevier Science Publishers B.V.

BBAEXP 90468

Short Sequence-Paper

Structural analysis and expression of a chromosomal gene encoding an avian Na+/K+-ATPase/31-subunit Kunio Takeyasu a, Maura Hamrick b, Andrew M. Barnstein c and Douglas M. Fambrough b u Department of Medical Biochemistry and Biotechnology Center, The Ohio State University, Columbus, OH (USA), t, Department of Biology, The Johns Hopkins Unit,ersi~. , Baltimore, MD (USA) and " Vanderbilt Unit,ersit.v School of Medicine, Nashcille, TN (USA) (Received 23 November 1992)

Key words: ATPase, Na+/K+-;/31-Subunit; 5' Flanking and exon sequence; (Chicken)

Chicken chromosomal DNA encoding the Na+/K+-ATPase/31-subunit was cloned and characterized. Its exon-intron structurc is identical to mammalian (human and rat) /31-subunit genes. The transcription initiation site, TATA box, and an ATI'GG (antisense CCAAT) sequence follow approximately 1 kilobase of GC-rich 5' upstream sequence that contains many consensus sequences for transcription factors whose relative positions are conserved between human and chicken genes. When this ¢tl-subunit gene was stably incorporated into mouse L cells and C2C12 cells, the avian /31-subunit was expressed under the control of the its own promoter.

The N a + / K + - A T P a s e (sodium-pump) plays a central role in many essential cellular functions, including cell volume regulation and setting of m e m b r a n e potential. Recent studies have demonstrated that selective regulation of transcription of the /31-subunit gene underlies up-regulation of the sodium-pump in response to increased demand for Na + transport in cultured muscle cells [15,16] and LLC-PK1 cells [6]. To understand the mechanisms regulating N a + / K + - A T P a s e expression, some of the mammalian a- and /3-subunit genes and an avian c~-subunit gene have been isolated and characterized [3-5,7,8,10,11,14]. In this study we isolated about 15 kilobases of D N A that include the chicken sodium-pump /31-subunit gene, and we analyzed the promoter region and the exon-intron structure and demonstrated its competence for expression. Genomic chicken D N A libraries were constructed in AEMBL4 and five million recombinant phages were screened with 32p-labeled c D N A encoding the ~31-subunit [13]. Five overlapping clones encoding the /31-subunit were obtained. We analyzed the phage clone,

Correspondence to: D.M. Fambrough, Department of Biology, The Johns Hopkins University, 34th and Charles Streets, Baltimore 21218, MD, USA. The nucleotide sequences reported appear in the EMBL, GenBank and DDBJ Data Bases under the accession numbers M75030 and M75031.

CGb-1, that was found to contain the longest 5' upstream region and the entire fll-subunit protein coding and 3' untranslated sequences. Fig. 1 contains a map of the /31-subunit gene, showing restriction enzyme sites for EcoRI, HindIII, BamHI, Smal, BglII and SpeI. D N A sequencing was used to determine all the exon-intron boundaries precisely, and these exactly matched those found in the human fll gene (Table I). The first exon encodes the 5' untranslated sequence and the N-terminal 33 amino acids that form the cytoplasmic domain of the/~-subunit (reviewed in Ref. 9). The second exon encodes the single membrane spanning domain. The remaining four exons encode the extracellular domain and ~ 1.4 kilobase 3' untranslated region with four potential poly-adenylation signal sequences, A A T A A A . The nucleotide sequences of the 5' untranslated and flanking regions and for the exons of the fil-subunit gene are shown in Fig. 2. The major site of transcription initiation was found at - 2 2 5 from the start-translation codon by primer extension experiments (data not shown). R N A blot analysis showed that the chicken /31-subunit gene is transcribed into a single size ( ~ 2.3 kb) of m R N A [12], consistent with the primer-extension data. A consensus T A T A box sequence is located 26 bp 5' upstream of the cap site, and a consensus C C A A T box sequence ( A T T G G ) is located on the noncoding D N A strand 64 bp 5' upstream of the cap site.

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TABLE I Nucleotide sequences at exon-intron boundaries in chicken and human ~-subunit genes The chicken sequences were taken from Fig. 2 in this study and the h u m a n sequences were taken from Lane et al. [11].

Chicken

GC~AGC I E 4 3 T / g t g a g t g c g g g

Exon 1

ttctcttgcag/ TT AAG ATC Exon 2

Intron 1

Human

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GCA GCC CCA G/ gtaactggatg

gccttccctag/ GA CTG ACT

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GCC CCG CCA G/ gtaaaatcca

Gly Pro Pro G Chicken Exon 3 Human

Chicken Exon 4

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Lys Pro Lys Chicken

GTT GCC AAG /

Exon 5 Human

gcctggtctag/ GA TTA ACA ly Leu Thr

GAC TGC GGA G/ gtaagtcctgg ccctgtgatag/ AC Intron 3 GAT TGT GGC G/ gtaagtagact tttatttttag/ AT Asp Cys Gly A sp AAA OCT AAG /

Human

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gtttcttccag/C<~CCAGAA Ala Glu Intron 4 Exon 5 gcaagtaatat tttatttttag/OCTCCCAAG Pro Pro Lys gtaagtgagat ccccatctcag/AGG GAC GAG Intron 5 Exon 6 gtaaagacaaa ctggatttcag/CGA GAT GAA Arg Glu Glu

214 The 5' upstream regions are extremely GC-rich; e.g., 13 GC boxes (GGGCGG and CCGCCC) are found in the 5' flanking (11 GC boxes) and untranslated (2 GC boxes) regions, and are potential recognition sites for the SP1 transcription factor [2]. These characteristics have also been found in rat and human /31-subunit genes [5,7]. There are several common features in the 5' regions of the avian and mammalian /31-subunit genes. These features include the relative

positions of GC-boxes before and after the TATA box, a GCCCCGC(G/C)C sequence repeated twice between the first and second potential SP1 binding sites before the CCAAT sequence, and CCAAT box sequences located on the noncoding strand. There are several CANNTG sites (E-boxes or mef-1 sites) located within about 1.0 kilobase of the 5' flanking regions of both avian and mammalian/31-genes. A series of DNA binding proteins (including a myogenic gene product, 5 ' . ....... G A ~ T A G C C A T A A T G

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tttgccttccctaG

GA CTG ACT CAA GTC CCC CAG GTA CAA AAG ly Leu Thr Gln Val Pro Gln Val Gln Lys

A C A G A A A'I~ TCC TTT A C T GTC AAC GAT CCC A A A AGC TAC GAC CCG TAT GTG AAG AAT CTG GAG GGG TTC TTA AAC AAG TAC AGC GCT GGT GAG CAG ACT GAC Thr Glu Ile Set Phe Thr Va/ Ash Asp Pro Lys Set Tyr Asp Pro q~r Va/ Lys Asn Lau Glu Gly Phe Leu ASh Lys Tyr Ser A l a Gly Glu Gin ~ r ^,p AAC ATC GTA ~FP CAG GAC T~C GGA G gtaa~tcctg~ttatttcct ............ Asn Ile V~l Pha Gln Asp Cys Gly A

ggtt~ttttttcgttcctcccctgtgatagAC

ATA CCT ACG GAT TAC A A A GAG A G A GGA Lys Glu Ar~ G1y sp Ile Pro Thr Asp ~

GGG TAC AAG GAT CCA TAC AAT GAT GCC C A A GGT CAG AAG AAG GTC TGC AAG TTC AAA CGT GAG TOG CTG GAG AAC TGC TCT GGG CTG CAG GAT AAC ACC ~ Pro Tyr Asn Asp A l a Gln Gly Gln Lys Lys Val Cys Lys Phe Lys Ar~ G1u Trp Leu G1u Ash Cys Set Gly Leu Gln Asp Ash Thr Phe Gly Tlrr Lys Asp GGC A A A CCA TGC A T T C T C G T C A A G C T C A A ~ A G A A T T A T C G G C T T C A A A C C T A A G g c a m 4 ~ t a t t t c c t c c c t t t c t a Gly Lys Pro Cys Ile I~u w l Lys Leu Asn Ar~ Ile ile Gly ~he Lys Fro Lys

............ cattaacattgccctgttgtttcttcca~

GCAGGAAAATACAAC CCC TAT C T C A T C CCT GTC CAC TGT GTT GCC AAG gtaagtgagatacaacaaca~agaggt GCG CCA GAA AAT GAG AOC CTC CCC TCA GAT ~ Ala Pro Glu ASh GIu Set Leu Pro Set Asp Leu Ala Gly Lys Tlrr Ash Pro Tyr Leu I1e Pro Va/ His Cys Val Ala Lys ggg~at ctgt aggca~a~gca~gcaQa~t cct tgt c cgatggt aa ............................................ g~tGttttcatcttctgttcttttccccatctca~

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TAC TAC CCG TAC TAC GGC AGG CTC CTG CAG CCG CA~ TAC CTG CA~ CCA CTG GTG GCA GTG CAG TTC ACC AAC CTG ACC TAC GAC GTG G A A GTG CGC GTG GAA Tyr Tyr Pro Tyr Tyr Gly Arg Leu Leu Gln Pro Gln Tyr Leu Gln Pro Leu Val Ala Val Gln Phe Thr Ash Leu Thr Tyr Asp Val Glu Val Ar~ Val Glu TGC RAG GCC TAT GGG CAG AAC ATC CAG TAC AGC GAC AAA GAC CGC ~fC CAG GGA CGC TTT GAT ATT AAA TTT GAC ATA A A A AGC AGC TGA TTGTAAGCACAACTC Cys Lys A l a Tyr G l y Gln ASh Ile Gln Tlrr Ser Asp Lys Asp Arg Phe Gln Gly Arg Phe A~p Ile Lys Pha Asp Ila Lys Ser Ser * AGCCA~AAAAAAGGq~

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215 MyoD) which contain a basic helix-loop-helix structure have been known to recognize this C,KNNTG motif [1]. A major difference between the avian and mammalian /31-genes is that there are relatively long non-homologous sequences at several positions in the mammalian genes. These sequences may play mammalian specific roles, if any, while conserved sequences might be involved in gene regulation common to all vertebrate classes, including low K--induced up-regulation of the sodium-pump. One purpose of our gene analysis study is to use DNA sequence information as a basis for understanding gene-level regulation of Na+/K+-ATPase expression. Since clone CGb-1 appears to contain the chicken promoter and the entire chicken protein coding sequences, we introduced this DNA into mammalian cells to verify the ability of this gene to regulate its expression. Mouse Ltk ceils and C2C12 ceils were co-transfected with CGb-1 DNA together with a plasmid containing either the herpes tk gene or the neomycin resistance gene. Transient expression of avian

/31-subunit was observed; stable cell lines expressing the avian /31-subunit were selected by drug resistance. Fig. 3 shows two of these cell lines (C2G/31 and LG~31) that express the chicken/31-subunit on the cell surface. Southern blot analysis on HindlII-digested genomic DNA, probed with 32p-labeled EcoRI-P~ulI fragment of the chicken /31-cDNA, indicated that both cell lines bear a single or very few copies of the chicken/31-subunit gene in their genome (data not shown). Immunofluorescence microscopy of live cells (no fixation or permeabilization) demonstrated that the chicken /31subunit was expressed well in mouse cells and transported to the cell surface (Fig. 3). In conclusion, the sodium-pump /31-subunit gene was found to be conserved through avian/human lineage in terms of its exon-intron structure and tissue specific expression. The promoter structure shows many common features between different vertebrate classes, consistent with the known similarities in tissue and cell-type expression [12,17]. By using a heterologous gene expression system such as presented in this study,

Fig. 3. Immunofluorescence micrograph of stable expression of the chicken /31-subunit gene in mouse cells. Transfected mouse L cells (left), and C2H12 cells (right) were selected for integration of one or a few copies of the chicken gene into mouse genome by Southern blot analysis and for expression of the chicken sodium-pump/31-subunit under control of the chicken promoter. For immunofluorescence microscopy, live cells grown on coverslips were labeled with monoclonal IgG 24, specific for the avian /31-subunit, and tetramethylrhodamine-labeled goat anti-mouse IgG, demonstrating that the chicken /31-subunits synthesized in mouse cells are transported to the cell surface. Untransfected and mock-transfected cells showed no specific binding of IgG 24 and virtually no fluorescence.

216 we hope to identify the regulatory sequences involved in response to demand for ion transport. We thank Delores Somerville and Atsushi Mizushima for their technical help. M.H. is a recipient of NSF Predoctoral Fellowship, and K.T. is an Established Investigator of the American Heart Association. This work was supported by grants from the American Heart Association National Center (901107 to K.T.) and the National Institutes of Health (GM44373 to K.T. and NS23241 to D.M.F.). References 1 Blackwell, T.K. and Weintraub, H. (1990) Science 250, 1104-1110. 2 Kadonaga, J.T., Jones, K.A. and Tjian, R. (1986) Trends Biochem. Sci. 11, 20-23. 3 Kano, I., Nagai, F., Satoh, K., Ushiyama, K., Nakao, T. and Kano, K. (1989) FEBS Lett. 250, 91-98. 4 Kawakami, K., Suzuki-Yagawa, Y., Watanabe, Y. and Nagano, K. (1992) J. Biochem. 111,515-522. 5 Lane, L.K., Shull, M.M., Whitmer, K.P. and Lingrel, J.B. (1989) Genomics 5, 445-453. 6 Lescale-Matys, L., Hensley, C.B., Crnkovic-Markovic, R., Putnam, D.S. and McDonough, A.A. (1990) J. Biol. Chem. 265, 17935-17940.

7 Liu, B. and Gick, G. (1992) Biochim. Biophys. Acta 1130, 336-338. 80vchinnikov, Y.A., Monastyrskaya, G.S., Broude, N.E,, Ushkaryov, Y.A., Melkov, A.M., Smirnov, Y.V., Malyshev, I.V.~ Allikmets, R.L., Kostina, M.B., Dulubova, I.E., Kiyatkin, N.I., Grishin, A.V., Modyanov, N.N. and Sverdlov, E.D. (1988) FEBS Lett. 233, 87-94. 9 Pedemonte, C.H. and Kaplan, J.H. (1990) Am. J. Physiol. 258, C1-23. 10 Shull, M.M. and Lingrel, J.B. (1987) Proc. Natl. Acad, Sci. USA 84, 4039-4043. 11 Shull, M.M., Pugh, D.G. and Lingrel, J.B. (1989) J. Biol. Chem. 264, 17532-17543. 12 Takeyasu, K., Renaud, K.J., Taormino, J., Wolitzky, B.A., Barnstein, A., Tamkun, M.M. and Fambrough, D.M. (1989) Current Topics Membr. Transp. 34, 143-165. 13 Takeyasu, K., Tamkun, M.M., Siegel, N.R. and Fambrough, D.M. (1987) J Biol. Chem. 262, 10733-10740. 14 Takeyasu, K., Mizushima, A., Barnstein, A.M., Hamrick, M.H. and Fambrough, D.M. (1991) In The Sodium Pump: Recent Developments (Kaplan, J.H. and DeWeer, P., eds.), pp 11-17, Rockefeller University Press, New York. 15 Taormino, J.P. and Fambrough, D.M. (1990) J. Biol. Chem. 265, 4116-4123. 16 Wolitzky, B.A. and Fambrough, D.M. (1986) J. Biol. Chem. 261, 9990-9999. 17 Young, R.M. and Lingrel, J.B. (1987) Biochem. Biophys. Res. Commun. 145, 52-58.