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D M Fambrough

Publications and source records attributed to D M Fambrough.

At least 37 records · Page 2Linked to original sources
PubMed1993

Assembly of the extracellular domain of the Na,K-ATPase beta subunit with the alpha subunit. Analysis of beta subunit chimeras and carboxyl-terminal deletions.

The role of the extracellular domain of the Na,K-ATPase beta subunit in assembly with the alpha subunit was investigated. A chimeric protein consisting of the extracellular domain of the beta subunit fused with the transmembrane and cytoplasmic domains of dipeptidyl peptidase IV assembles with the alpha subunit. An inverse chimera consisting of the cytoplasmic and transmembrane domains of the beta subunit fused with the extracellular domain of dipeptidyl peptidase IV does not assemble with the alpha subunit. The assembly data from these chimeras demonstrate that the extracellular domain of the beta subunit is both necessary and sufficient for assembly with the alpha subunit. Deletions of up to 146 extracellular amino acids from the carboxyl terminus of the beta subunit appear to result in misfolding of the subunit, but do allow reduced assembly with the alpha subunit. Together, the assembly data from chimeras and carboxyl-terminal deletions have identified a 96-residue extracellular domain which contains sequences involved in subunit assembly. While the chimeric subunits properly localize to the plasma membrane, deletion of as few as 4 amino acids from the carboxyl terminus impairs the ability of the beta subunit to be transported to the plasma membrane.

Amino Acid Sequence↗
PubMed1993

Intramolecular fusion of Na pump subunits assures exclusive assembly of the fused alpha and beta subunit domains into a functional enzyme in cells also expressing endogenous Na pump subunits.

Experiments designed to identify Na pump structural features which tag the molecule for asymmetric cell-surface localization are inherently complex because either subunit, or both, may contain targeting information and because the cells which recognize those targeting signals and maintain asymmetric plasma membrane domains also express their own Na pumps, the subunits of which can assemble into hybrid pump molecules with pump subunits expressed from transfected cDNA clones. Cotransfecting cDNA for both subunits only complicates matters further by resulting in expression of four distinct dimeric molecular species. To eliminate the potential for cross-assembly in these and other experiments we have constructed cDNA encoding a "single-subunit" Na pump (called "alpha-beta") in which the alpha and beta subunits are joined by a linker of 17 amino acids. By all criteria tested alpha-beta functioned as a normal heterodimeric Na pump. It was expressed in a variety of mammalian cell lines as a single, high molecular weight polypeptide located primarily on the surface membrane, with the beta subunit exposed to the extracellular medium. Binding of the conformation-sensitive monoclonal antibody 24 to the beta subunit indicated that the fusion protein was folded as a properly "assembled" sodium pump. Expression of alpha-beta in ouabain-resistant mouse L cells resulted in high affinity ouabain binding and ouabain-sensitive, sodium-dependent rubidium transport. The enzyme was properly targeted to the basolateral plasma membrane in polarized epithelial cells. The functional integrity of the fusion protein renders it suitable for site-directed mutagenesis studies of targeting and enzymology where control of subunit assembly is desired. These results also support topological models in which the carboxyl terminus of the alpha subunit is cytoplasmic.

Amino Acid Sequence↗
PubMed1993

Ca(2+)-dependent and thapsigargin-inhibited phosphorylation of Na+,K(+)-ATPase catalytic domain following chimeric recombination with Ca(2+)-ATPase.

Two chimeric proteins comprising the Na,K-ATPase catalytic domain (large cytosolic loop) and the two flanking regions of the Ca-ATPase were obtained by transient or stable expression in mammalian cells transfected with recombinant DNA. In the first chimera (CpNC), a large portion (containing the nucleotide-binding site) of the cytosolic loop between putative membrane spans M4 and M5 of the sarcoendoplasmic reticulum Ca2+ (SERCA) 1 (fast muscle) ATPase was replaced by the corresponding portion of the Na,K-ATPase alpha 1 subunit. In the second chimera (CNpC), an even larger portion (containing the nucleotide-binding site and the phosphorylation site) of the analogous cytosolic loop of the SERCA2 (cardiac muscle) ATPase was replaced by the corresponding portion of the Na,K-ATPase alpha 1 subunit. Steady state Ca2+ transport and coupled ATP hydrolysis by the chimeric proteins were negligible as compared to those obtained with SERCA enzymes. Nevertheless, the chimeric proteins were able to utilize ATP to form phosphoenzyme levels equal to those formed by SERCA ATPases. Chimeric and SERCA enzymes exhibited an identical Ca2+ requirement for ATP utilization and sensitivity to thapsigargin (TG) which is a specific inhibitor of SERCA ATPase and not of Na,K-ATPase. Furthermore, both SERCA and chimeric enzymes could be phosphorylated with P(i), and this reaction required removal of Ca2+. In comparative experiments, the functional pattern of seemingly unaffected phosphoenzyme formation and inhibited Ca2+ transport was produced in the SERCA ATPase even by single mutation of Pro337 to Ala, evidently due to defective protein conformation. Retention of Ca2+ and TG sensitivity by the chimeric proteins demonstrates that the Ca(2+)- and TG-binding domains do not reside within the cytosolic loop replaced by chimeric substitution and strongly support previous studies suggesting that binding of calcium required for enzyme activation occurs within the membrane-bound region of the SERCA ATPases (Clarke et al., 1989a; Sumbilla et al., 1991).

Amino Acid Sequence↗
PubMed1993

Sequence analysis of DNA encoding an avian Na+,K(+)-ATPase beta 2-subunit.

The DNA encoding a chicken Na+,K(+)-ATPase beta 2-subunit was cloned and sequenced. The deduced amino acid sequence has structural features common to all known Na+,K(+)-ATPase beta-subunits. It is proposed to belong to the beta 2-isoform family, though the amino acid sequence has significantly diverged from mammalian beta 2-subunit sequences. Similar to other Na+,K(+)-ATPase beta 2-isoforms, the chicken beta 2-isoform mRNA is predominantly expressed in brain tissue.

Amino Acid Sequence↗
PubMed1993

Differential distribution of the alternative forms of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, SERCA2b and SERCA2a, in the avian brain.

Cellular distribution of the two forms of SERCA2 was examined in adult chicken brain. Four regions of the brain were analyzed with three immunological reagents: a monoclonal antibody that recognizes both forms of SERCA2, and two antisera which are specific for the two alternative forms, SERCA2b or SERCA2a. Cerebellar Purkinje cells express predominantly SERCA2b but also low levels of SERCA2a, as has been reported for mammals. The nucleus isthmo-opticus, nucleus magnocellularis cochlearis, and nucleus laminaris all express high levels of SERCA2 but with different ratios of SERCA2b and SERCA2a. These immunohistochemical results were supported by in situ hybridization analysis. Therefore, it appears that regions within the brain have specific requirements for the two forms of SERCA2. This suggests functional significance for the alternative forms SERCA2b and SERCA2a, and possible functions are discussed.

Animals↗
PubMed1993

Structural analysis and expression of a chromosomal gene encoding an avian Na+/K(+)-ATPase beta 1-subunit.

Chicken chromosomal DNA encoding the Na+/K(+)-ATPase beta 1-subunit was cloned and characterized. Its exon-intron structure is identical to mammalian (human and rat) beta 1-subunit genes. The transcription initiation site, TATA box, and an ATTGG (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 beta 1-subunit gene was stably incorporated into mouse L cells and C2C12 cells, the avian beta 1-subunit was expressed under the control of the its own promoter.

Amino Acid Sequence↗
PubMed1993

Mutation of a conserved proline residue in the beta-subunit ectodomain prevents Na(+)-K(+)-ATPase oligomerization.

A highly conserved sequence motif (4 tyrosines and 1 proline: YYPYY) of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) beta 1-subunit ectodomain has been mutagenized to study its possible role in alpha/beta-assembly and sodium pump function. Single as well as double tyrosine mutants (tyrosine to phenylalanine: Y to F) of Xenopus laevis beta 1-subunits are able to associate with alpha 1-subunits and form functional Na-K pumps at the plasma membrane that are indistinguishable from wild-type alpha 1, beta 1-Na-K pumps (as assessed by measurements of ouabain binding, 86Rb flux, Na-K pump current, and activation by external potassium). In contrast, a single proline mutation (proline to glycine: P244G) reduced by > 90% the proper assembly and function of Na(+)-K(+)-ATPase, despite a normal rate of synthesis and core glycosylation. Our data indicate that proline-244 plays a critical role in the proper folding of the beta-subunit and its ability to associate efficiently with the alpha 1-subunit in the endoplasmic reticulum.

Animals↗
PubMed1992

The carboxyl-terminal 161 amino acids of the Na,K-ATPase alpha-subunit are sufficient for assembly with the beta-subunit.

Chimeric cDNAs encoding regions of the Na,K-ATPase alpha-subunit and a sarcoplasmic reticulum Ca(2+)-ATPase were constructed and expressed together with the avian Na,K-ATPase beta-subunit cDNA in COS-1 cells to determine which regions of the alpha-subunit are required for assembly with the beta-subunit. Assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the avian beta-subunit. A chimera composed of the amino-terminal two-thirds of the Na,K-ATPase and carboxyl-terminal one-third of the Ca(2+)-ATPase did not assemble with the avian beta-subunit. In contrast, the reciprocal chimera, containing the carboxyl-terminal one-third of the Na,K-ATPase, assembled with the beta-subunit. A third chimera, in which 161 amino acids of the Na,K-ATPase carboxyl terminus replaced the corresponding amino acids of the Ca(2+)-ATPase carboxyl terminus, also assembled with the beta-subunit. These results suggest that the aminoacyl residues of the Na,K-ATPase alpha-subunit critical for subunit assembly lie within the carboxyl-terminal 16% of the sequence.

Animals↗
PubMed1992

The alternative carboxyl termini of avian cardiac and brain sarcoplasmic reticulum/endoplasmic reticulum Ca(2+)-ATPases are on opposite sides of the membrane.

The sarcoplasmic/endoplasmic reticulum slow-twitch or cardiac Ca(2+)-ATPase (SERCA2) is expressed as two forms (SERCA2a and SERCA2b) which vary at their extreme carboxyl termini. SERCA2a and SERCA2b are derived from alternatively spliced primary transcripts of the same gene. These two alternative carboxyl termini are highly conserved in mammals (Eggermont, J. A., Wuytack, F., De Jaegere, S., Nelles, L., and Casteels, R. (1989) Biochem. J. 260, 757-761; Lytton, J., and MacLennan, D. H. (1988) J. Biol. Chem. 263, 15024-15031) and birds (Campbell, A. M., Kessler, P. D., Sagara, Y., Inesi, G., and Fambrough, D. M. (1991) J. Biol. Chem. 266, 16050-16055). The topology of SERCA2a is believed to be identical to the fast-twitch Ca(2+)-ATPase (SERCA1) with 10 membrane-spanning domains. Based on hydropathy analysis, the extended carboxyl terminus of SERCA2b is predicted to span the endoplasmic reticulum (ER) membrane an additional (i.e. 11th) time. We have added the human c-myc epitope, a 10-amino acid sequence recognized by monoclonal antibody 9E10, onto the carboxyl termini of SERCA2a and SERCA2b to test whether or not their carboxyl termini are on the same side of the ER membrane. The added epitopes do not appear to disrupt topology as judged from unaltered Ca2+ transport. Immunocytochemical studies demonstrate that SERCA2a and SERCA2b have their carboxyl termini on opposite sides of the ER membrane; SERCA2a's is in the cytosol and SERCA2b's is in the ER lumen.

Amino Acid Sequence↗
PubMed1992

Molecular dissection of functional domains of the E1E2-ATPase using sodium and calcium pump chimeric molecules.

Proposed models for the catalytic subunit of the E1E2-ATPases (ion pumps) predict that the first four transmembrane domains (M1 - M4) reside in the NH2 terminal one-third of the molecule, and the remainder (M5 - M10) in the COOH terminal one-third. The amino-acid sequences for the 5'-(p-fluorosulfonyl)-benzoyl-adenosine (FSBA) binding region residing just before M5 segment are very well conserved among distinct ion pumps. Taking advantage of these models, we have constructed a set of chicken chimeric ion pumps between the (Na++ K+)-ATPase alpha-subunit and the Ca(2+)-ATPase using the FSBA-binding site as an exchange junction, thereby preserving overall topological structure as E1E2 ATPases. From various functional assays on these chimeric ion pumps, including ouabain-inhibitable ATPase activity, Ca2+ binding, Ca2+ uptake, and subunit assembly based on immuno-coprecipitation, the following conclusions were obtained: (a) A (Na++ K+)-ATPase inhibitor, ouabain, binds to the regions before M4 in the alpha-subunit and exerts its inhibitory effect. (b) The regions after M5 of the (Na++ K+)-ATPase alpha-subunit bind the beta-subunit, even when these regions are incorporated into the corresponding domains in the Ca(2+)-ATPase. (c) The corresponding domains of the Ca(2+)-ATPase, the regions after M5, bind 45Ca even when it is incorporated into the corresponding position of the (Na++ K+)-ATPase alpha-subunit.

Adenosine Triphosphatases↗
PubMed1992

The pathway and targeting signal for delivery of the integral membrane glycoprotein LEP100 to lysosomes.

A complete set of chimeras was made between the lysosomal membrane glycoprotein LEP100 and the plasma membrane-directed vesicular stomatitis virus G protein, combining a glycosylated lumenal or ectodomain, a single transmembrane domain, and a cytosolic carboxyl-terminal domain. These chimeras, the parent molecules, and a truncated form of LEP100 lacking the transmembrane and cytosolic domains were expressed in mouse L cells. Only LEP100 and chimeras that included the cytosolic 11 amino acid carboxyl terminus of LEP100 were targeted to lysosomes. The other chimeras accumulated in the plasma membrane, and truncated LEP100 was secreted. Chimeras that included the extracellular domain of vesicular stomatitis G protein and the carboxyl terminus of LEP100 were targeted to lysosomes and very rapidly degraded. Therefore, in chimera-expressing cells, virtually all the chimeric molecules were newly synthesized and still in the biosynthesis and lysosomal targeting pathways. The behavior of one of these chimeras was studied in detail. After its processing in the Golgi apparatus, the chimera entered the plasma membrane/endosome compartment and rapidly cycled between the plasma membrane and endosomes before going to lysosomes. In pulse-expression experiments, a large population of chimeric molecules was observed to appear transiently in the plasma membrane by immunofluorescence microscopy. Soon after protein synthesis was inhibited, this surface population disappeared. When lysosomal proteolysis was inhibited, chimeric molecules accumulated in lysosomes. These data suggest that the plasma membrane/early endosome compartment is on the pathway to the lysosomal membrane. This explains why mutations that block endocytosis result in the accumulation of lysosomal membrane proteins in the plasma membrane.

Amino Acid Sequence↗
PubMed1991

Cytoplasmic and transmembrane domain deletions of Na,K-ATPase beta-subunit. Effects on subunit assembly and intracellular transport.

cDNAs encoding Na,K-ATPase beta-subunits containing deletions in the cytoplasmic domain or in the single membrane-spanning domain of the molecule were constructed and expressed in mouse L cells to determine the effect(s) of deletions in these domains on alpha/beta-subunit assembly and intracellular targeting. Avian beta-subunits lacking some or all of the cytoplasmic domain (endodomain) assemble with the endogenous mouse alpha-subunit and are correctly transported to the plasma membrane. Mutants containing deletions in the transmembrane domain were constructed by fusing portions of cDNAs encoding the amino-terminal one-third of human beta-subunit deletion mutants with avian beta-subunit cDNA encoding the carboxyl two-thirds of the molecule. A deletion of 3 amino acids in transmembrane domain resulted in correct alpha/beta-subunit assembly and localization to the plasma membrane. In contrast, deletions of 5 or more amino acids in the transmembrane domain prevented expression of the beta-subunit at the cell surface and resulted in the accumulation of these molecules in the ER. In spite of these targeting differences, all beta-subunit mutants capable of membrane insertion were also able to assemble with the alpha-subunit. These results suggest that the specificity for alpha/beta assembly resides in the ectodomains of the subunits.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequences of avian cardiac and brain SR/ER Ca(2+)-ATPases and functional comparisons with fast twitch Ca(2+)-ATPase. Calcium affinities and inhibitor effects.

Two similar forms of the cardiac/slow Ca(2+)-ATPase (SERCA2a and SERCA2b), differing in sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility, are expressed in chicken heart and brain (Kaprielian, Z., Campbell, A. M., and Fambrough, D. M. (1989) Mol. Brain Res. 6, 55-60). In the current study, cDNAs encoding each form were cloned and sequenced. Chicken SERCA2a is 94% identical to its rabbit homologue, while SERCA2b has an extended carboxyl terminus with 38 of 49 amino acids identical to mammalian homologues. SERCA2b mRNA contains the SERCA2a encoding sequence within its 3'-untranslated region. Chicken genomic DNA sequence reveals that the alternate RNA splicing used to produced SERCA2a and SERCA2b subtypes involves a splice site within an exon. Tissue culture cells expressing the avian SERCA2a, SERCA2b, and SERCA1, each targetting to the endoplasmic reticulum, were used to measure Ca2+ affinities and inhibitor effects; no differences among the three pumps were detected.

Amino Acid Sequence↗
PubMed1991

Expression of Ca2(+)-ATPase isoforms in denervated, regenerating, and dystrophic chicken skeletal muscle.

The expression of fast and slow isoforms of the sarcoplasmic reticulum Ca2(+)-ATPase was studied in denervated, regenerating, and dystrophic fast and slow avian skeletal muscles. We found that both fast and slow Ca2(+)-ATPase isoforms were expressed in most myofibers following denervation of adult fast-twitch muscle, but only the slow Ca2(+)-ATPase isoform was found in slow-tonic muscle which had been denervated. Regenerating myotubes in normally innervated and previously denervated adult fast-twitch or slow-tonic muscle expressed both Ca2(+)-ATPase isoforms. Expression of the slow Ca2(+)-ATPase isoform was found to persist in dystrophic fast-twitch muscle, long after it had disappeared from normal fast-twitch muscle. However, the fast Ca2(+)-ATPase isoform disappeared from slow-tonic muscle similarly in normal and dystrophic birds. These results demonstrate that the appearance of myosin heavy chain isoforms characteristic of developing muscle is correlated with similar changes in the expression of sarcoplasmic reticulum Ca2(+)-ATPases.

Animals↗
PubMed1990

Structure of a gene for a lysosomal membrane glycoprotein (LEP100). Housekeeping gene with unexpected exon organization.

Members of a recently described family of glycoproteins constitute the major protein components of the lysosomal membrane. Changes in glycosylation patterns, redistribution to the cell surface, and increased levels of expression of these proteins are associated, in at least some cases, with cell differentiation, transformation, and metastasis. To understand further the regulation of expression of these proteins and their relationships to each other, we have isolated and characterized the gene for one of these proteins, LEP100, from chicken. Two overlapping clones were isolated which contain the nine exons and eight introns of the 17-kilobase gene for LEP 100. The 5'-flanking region of the gene contains CAAT and TATAAA sequences, but these do not appear to be used as promoter elements. Further downstream are three CCAAT boxes, with no corresponding TATA boxes, which represent putative promoter elements. Multiple CCAAT boxes, the apparent lack of a TATA box, and the GC-rich composition of the 5' region support the classification of the LEP100 gene as a TATA box-lacking housekeeping gene. As further evidence of the housekeeping nature of the LEP100 gene, Northern blots of RNA from several adult and embryonic tissues (skeletal muscle, kidney, liver, heart, gizzard, and brain) revealed a single message for LEP100 of the same size (about 3 kilobases) in each tissue. The gene's introns range in size from 104 to 7200 base pairs. Exons do not represent the four disulfide-bonded loops of the protein, but instead each cysteine of each disulfide-linked pair is encoded by a separate exon. The existence of the same sized mRNA in all tissues indicates that no alternate splicing occurs. The exon organization of the LEP100 gene suggests that it may have evolved from a primordial cysteine-containing exon by gene duplication events. It is likely that the genes of the other members of this family diverged from the same ancestral gene and have a gene organization similar to that of LEP100.

Amino Acid Sequence↗
PubMed1990

Pre-translational regulation of the (Na+ + K+)-ATPase in response to demand for ion transport in cultured chicken skeletal muscle.

The expression of the (Na+ + K+)-ATPase in cultured chicken skeletal muscle can be altered by varying the demand for ion transport. Veratridine, an activator of voltage-sensitive Na+ channels, causes a specific transient increase in biosynthesis of the sodium pump that accounts for the doubling of the number of (Na+ + K+)-ATPase molecules in the sarcolemma (Wolitzky, B.A., and Fambrough, D.M. (1986) J. Biol. Chem. 261, 9990-9999). Here we report a study of veratridine-induced up-regulation, focusing upon alpha- and beta-mRNA levels and transcription rates. Myotubes normally express the alpha 1-isoform mRNA and the beta-mRNA at a molar ratio of 0.6 +/- 0.1 (S.D.). In the presence of veratridine, the beta-mRNA is transiently up-regulated approximately 3-fold. The kinetics of this increase parallel the rate of beta-subunit protein synthesis. The increase in beta-mRNA during up-regulation is accomplished by an increase in the transcription rate of the beta gene. The veratridine-induced increase in beta-mRNA is not blocked by cycloheximide. The alpha-mRNA also increases during exposure to veratridine, but this increase is very modest and occurs very late in the up-regulation process. The increased beta-mRNA results in over-production of beta-subunits, which we postulate drives more efficient assembly of alpha beta complexes, i.e. sodium pump molecules. As the up-regulated state is achieved the level of beta-mRNA falls abruptly, reflecting a marked decrease in beta-mRNA stability. Treatment of up-regulated myotubes with tetrodotoxin, a veratridine antagonist, results in rapid down-regulation of the sodium pump, while having little or no effect on the levels of alpha 1- and beta-mRNAs.

Animals↗
PubMed1990

Stability of Na(+)-K(+)-ATPase alpha-subunit isoforms in evolution.

Encoding DNA for alpha 2- and alpha 3-isoforms of the alpha-subunit of the chicken Na(+)-K(+)-ATPase have been cloned, and their nucleotide sequences and deduced amino acid sequences are reported. Comparisons between these data and comparable data for the rat alpha-subunit isoforms make possible an assessment of alpha-subunit isoform diversity among vertebrates. There is approximately twice as much amino acid sequence difference between alpha-isoforms within a single species as there is difference between corresponding alpha-isoforms of bird and mammal. These data are consistent with triplication of the alpha-subunit gene and evolution of substantially different alpha-subunit isoforms before the separation of avian and mammalian lineages over 200 million years ago and then retention of the majority of these structural differences through subsequent evolution. The implications of this conversation of isoform-specific structural features are discussed in terms of transport functions and bioregulation of the Na(+)-K(+)-ATPase.

Amino Acid Sequence↗