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E E Strehler

Publications and source records attributed to E E Strehler.

At least 37 records · Page 2Linked to original sources
PubMed1996

Primary structure of human plasma membrane Ca(2+)-ATPase isoform 3.

The complete coding sequence of the human plasma membrane calcium ATPase (PMCA) isoform 3 was determined from overlapping genomic and cDNA clones. The cDNAs for the two major alternative splice variants 3a (3CII) and 3b (3CI) code for proteins of 1173 and 1220 amino-acid residues, respectively, which show 98% identity with the corresponding rat isoforms. On a multiple human tissue Northern blot, a major PMCA3 transcript of about 7 kb was detected exclusively in the brain, demonstrating the highly restricted pattern of expression of this isoform to human neuronal tissues. With the elucidation of the human PMCA3 primary structure, complete sequence information is now available for the entire family of human PMCA isoforms.

Alternative Splicing↗
PubMed1996

Plasma membrane calcium pump isoform 4a has a longer calmodulin-binding domain than 4b.

Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes the expression of two variants, hPMCA4a and hPMCA4b, which have different downstream regulatory regions. Of the two, hPMCA4a has a lower affinity for calmodulin and a lower effective affinity for Ca2+ (Enyedi, A., Verma, A. K., Heim, R., Adamo, H. P., Filoteo, A. G., Strehler, E. E., and Penniston, J. T. (1994) J. Biol. Chem. 269, 41-43). Additional consequences of the alternate splice were studied by analyzing the characteristics of constructs (expressed in COS-1 cells) containing different portions of the carboxyl terminus of hPMCA4a. Our results show striking differences in the structure of the calmodulin-binding and autoinhibitory domains of the two variants. The calmodulin-binding region of hPMCA4b is a region of about 28 residues, whereas that of hPMCA4a is about 49 residues long and is probably interrupted by a region not involved in the binding. The autoinhibitory region of hPMCA4b (a part of the downstream region that keeps the molecule inactive in the absence of Ca2+-calmodulin) is divided between the 28-residue calmodulin-binding region and a downstream region, whereas in hPMCA4a, all of it is contained within the 49-residue calmodulin-binding region.

Adenosine Triphosphate↗
PubMed1995

Transcript distribution of plasma membrane Ca2+ pump isoforms and splice variants in the human brain.

Plasma membrane calcium pumps (PMCAs) play a major role in the maintenance and fine regulation of the intracellular Ca2+ concentration. Fourteen subregions of the normal human brain were carefully dissected and analyzed by reverse transcriptase-polymerase chain reaction for the distribution of mRNAs corresponding to the four known PMCA genes as well as their alternative splicing products at two previously defined 'hotspots' A and C. All PMCA genes were found to be expressed in every brain subregion; however, consistent differences were found in the distribution of alternative splice options. The four cortical regions and hippocampus were characterized by the relative preference of variants that include an entire exon at site C and lead to the expression of isoforms of the a-type. Inferior olive and olfactory bulb showed a relative preponderance of the b-form 'default' types of alternative splicing at site C, and a decrease or even the lack of 'differentiated' forms such as variants 1a and 1c. At the N-terminal splice site A, the default x-type variants were predominant in all brain regions for PMCA 1, 3, and 4. By contrast, the pattern of PMCA2 variants was the most variable, ranging from the presence of the entire set of 2x, 2w, and 2z forms in inferior olive to the almost exclusive presence of form 2z (excluding all alternatively spliced sequences) in the four cortical regions, caudate, and hippocampus. Regional differences in the PMCA splice type distribution in normal human brain may correlate with different demands on the regulation of the set-point resting Ca2+ levels in these areas. Changes in these patterns may correlate with altered physiological states of the affected regions and/or reflect an (early) sign of Ca2+ dyshomeostasis characteristic of many neurodegenerative diseases.

Alternative Splicing↗
PubMed1994

Structure of the human CALM1 calmodulin gene and identification of two CALM1-related pseudogenes CALM1P1 and CALM1P2.

The human CALM1 calmodulin gene has been isolated and characterized. The gene contains six exons spread over about 10 kb of genomic DNA. The exon-intron structure is identical to that of the human CALM3 and of the rat CALM1 and CALM3 genes. A cluster of transcription-start sites was identified 200 bp upstream of the ATG translation-start codon, and several putative regulatory elements were found in the 5' flanking region as well as in intron 1. Sequence comparison with the rat CALM1 gene revealed significant similarities in the promoter regions of the two genes and an even more striking degree of identity (70%) in the available intron 1 sequences. A short CAG trinucleotide repeat region was identified in the 5' untranslated region of the human CALM1 gene; this sequence is not conserved in the rat counterpart. Expression of the CALM1 gene was detected in all human tissues tested, although at varying levels. A 1.7-kb mRNA was uniformly present at comparable levels, whereas a 4.2-kb mRNA species was particularly abundant in brain and skeletal muscle. Clones for two different CALM1-related pseudogenes CALM1P1 and CALM1P2 were also isolated and characterized. Both pseudogenes are intronless and non-functional as judged from the presence of mutations abolishing the open reading frame. Genomic Southern analysis indicates that the human CALM1 gene/pseudogene subfamily comprises at least three but probably no more than four members. The entire family consists of three bona fide CALM genes, at least one expressed calmodulin-like CALML gene as well as at least five pseudogenes.

Amino Acid Sequence↗
PubMed1994

Differentiation-specific isoform mRNA expression of the calmodulin-dependent plasma membrane Ca(2+)-ATPase.

The functional significance of the isoform diversity of the calmodulin-dependent plasma membrane Ca(2+)-ATPase (PMCA) is largely unknown. To determine whether the mRNA synthesis of different isoforms of the enzyme is regulated in a differentiation-specific manner, we investigated the expression of isoform-specific mRNAs in muscle and neuronal cells during differentiation by reverse transcription PCR. In the rat, the ubiquitous PMCA splicing variants 1b and 4b formed the typical PMCA isoform pattern of L6 myoblasts, the heart-derived cell line H9c2(2-1), two different fibroblast cell lines (FR and NRK-49F), smooth muscle, and endothelial cells. In addition to these two enzymes, novel expression of the splicing variants 1c, 1d, and 4a was induced during myogenic differentiation of L6 and H9c2(2-1) cells. A similar isoform subtype switch could be detected during differentiation of the neuronal PC-12 cells induced by nerve growth factor (NGF). The isoform-specific mRNAs 1c, 1d, and 4a were not expressed in cells other than myocytes and neurons, and therefore may be specific for excitable cells. The mRNA for isoform 1d was heart- and skeletal muscle-specific. To determine whether expression of a differentiation-specific PMCA mRNA pattern is under control of a myogenic determination factor, myogenin was constitutively expressed in rat fibroblasts. These cells converted to multinucleated myotubes, which displayed the PMCA isoform-specific mRNAs 1c, 1d, and 4a, typical of differentiated muscle cells. We conclude that: 1) the distribution of the various PMCA isoform-specific mRNAs and their splicing variants is cell type- and development-specific; 2) expression of the myogenic determination factor myogenin is sufficient to direct alternative splicing generating muscle-specific PMCA mRNA species; and 3) PMCA isoforms and/or splicing variants may play a role in determining functions of terminally differentiated muscle and neuronal cells and possibly during the differentiation process itself.

Alternative Splicing↗
PubMed1994

The Ca2+ affinity of the plasma membrane Ca2+ pump is controlled by alternative splicing.

The plasma membrane Ca2+ pump is a calmodulin-regulated P-type ATPase that is an essential element in controlling intracellular Ca2+ concentration. Studies on the gene structure of this pump have revealed an alternate splice option that changes the structure of the calmodulin-binding domain. This change in the structure of the enzyme results in a reduced calmodulin affinity. Tests of the enzyme's activity in the presence of a high calmodulin concentration, approximating that found inside living cells, show that this reduced calmodulin affinity causes a reduced apparent affinity of the enzyme for Ca2+. This shift in the Ca2+ activation occurs in a Ca2+ concentration range crucial to cellular function and is probably the physiologically important consequence of the alternate splice.

Alternative Splicing↗
PubMed1994

Localization of two genes encoding plasma membrane Ca2+ ATPases isoforms 2 (ATP2B2) and 3 (ATP2B3) to human chromosomes 3p26-->p25 and Xq28, respectively.

The plasma membrane Ca2+ ATPases (PMCA) represent a highly conserved, widely dispersed, multigene family in eukaryotes consisting of at least four functional genes. The genes for PMCA isoforms 1 and 4 (ATP2B1 and ATP2B4) have been previously localized to human chromosomes 12q21-->q23 and 1q25-->q32, respectively. Based upon results of fluorescence in situ hybridization (FISH), analysis of somatic cell hybrids, and genetic linkage analyses, we now report localization of ATP2B3 (PMCA isoform 3) to human chromosome Xq28, and confirm the recent localization of ATP2B2 (PMCA isoform 2) to chromosome 3p26-->p25. In contrast to ATP2B1 and ATP2B4, recent studies have suggested tissue specific regulation of expression of both ATP2B2 and ATP2B3 particularly in the nervous system. The genes for several neurological and neuromuscular diseases have been assigned to the distal portion of Xq, and ATP2B3 is a candidate gene for these diseases.

Calcium-Transporting ATPases↗
PubMed1993

Quantitative analysis of alternative splicing options of human plasma membrane calcium pump genes.

The alternative splicing options and the quantitative tissue distribution of the transcripts of the four currently known human plasma membrane calcium pump (PMCA) genes have been analyzed in seven tissues (cerebral cortex, skeletal and heart muscle, stomach, liver, lung, and kidney) by quantitative polymerase chain reaction on reverse transcribed mRNA with glyceraldehyde-3-phosphate dehydrogenase as the internal standard. The mRNAs of genes 1 and 4 were found to be present in similar amounts in all tissues, whereas the transcripts of genes 2 and 3 were expressed in a tissue-specific manner, i.e. their amounts were highest in fetal skeletal muscle and brain. Alternative splicing was found to occur in the PMCA transcripts at two major regulatory sites (sites A and C), adjacent to the amino-terminal phospholipid-responsive region and within the carboxyl-terminal calmodulin binding domain, respectively. Novel splicing variants not described previously for human genes were detected for hPMCA3 and 4 at site A and for hPMCA1, 2, and 3 at site C. For all genes a common splice variant was found at both splice sites. The common splice variant at site A was characterized by the inclusion of a small exon (hPMCA1, 39 base pairs (bp); hPMCA2, 42 bp; hPMCA3, 42 bp; hPMCA4, 36 bp). In the common splice variant at site C, an exon (hPMCA1, 154 bp; hPMCA2, 227 bp; hPMCA3, 154 bp; hPMCA4, 178 bp) was excluded in the mRNA. All genes normally express these main splice variants in all tissues in which the corresponding isoform is present. The splicing complexity at site C was found to be augmented in the transcripts of PMCA2 and PMCA3 through the use of additional exons, and in PMCA1 and 3 through the use of additional internal splice sites in the single alternatively spliced 154-base pair exon.

Aged↗
PubMed1993

Structure of the gene encoding the human plasma membrane calcium pump isoform 1.

The complete structure of the gene for the human plasma membrane calcium ATPase isoform 1 (hPMCA1) has been elucidated. The protein is encoded by 21 exons present on overlapping clones covering more than 100 kilobases (kb) of DNA. An intron of over 35 kb separates the 5'-untranslated exon 1 from the exon containing the translational start codon. The entire putative promoter and 5'-flanking region is embedded in a CpG island and is characterized by the presence of numerous Sp1 factor-binding sequences and by the absence of a TATA box. In accordance with the ubiquitous tissue distribution of its mRNA these results suggest that the hPMCA1 gene is of the housekeeping type. No alternative splicing comparable to that identified in PMCA2 RNAs at site "A" and in PMCA3 RNAs close to site "C" seems to occur in hPMCA1 transcripts; however, a region in intron 6 shows significant resemblance to the site "A" alternatively spliced exons in PMCA2 and may represent a pseudoexon or a functional exon not yet detected in any PMCA1 mRNA. At six positions, intron interruptions in the hPMCA1 gene correlate with the boundaries of putative transmembrane domains in the protein, whereas most of the remaining intron positions do not show an obvious correlation with the proposed pump domain structure. The limited conservation of intron positions in different P-type pump genes indicates their early separation from a common ancestor.

Amino Acid Sequence↗
PubMed1993

Cation binding and conformation of human calmodulin-like protein.

The Ca(2+)-binding parameters of recombinant human calmodulin-like protein (CLP), a protein specifically expressed in mammary epithelial cells, were studied by flow dialysis in the absence and presence of 2, 10, and 30 mM MgCl2. In general, the four intrinsic binding constants (K'Ca) are about 8-fold lower than in animal and plant calmodulins. In the absence of Mg2+ the K'Ca values of the four binding steps equal 4.0 x 10(3), 3.3 x 10(4), 1.0 x 10(4), and 6.0 x 10(3) M-1, respectively. They allow us to distinguish two pairs of sites: a higher affinity pair with strong positive cooperativity and a lower affinity pair composed of non-interacting sites with different affinities. Mg2+ antagonizes Ca2+ binding by decreasing only Ca(2+)-binding steps 2 and 3, so that at high Mg2+ concentrations the positive cooperativity in the high-affinity pair has been lost and that the four K'Ca values are very similar with a mean K'Ca of 4 x 10(3) M-1. Direct Mg2+ binding studies by equilibrium gel filtration indicate that 4-5 Mg2+ bind to CLP with a mean K'Mg of 250 M-1. Conformational changes in the unique Tyr138 microenvironment, monitored by fluorimetry and near-UV difference spectrophotometry, indicate that in metal-free CLP this Tyr is shielded from the polar solvent and strongly quenched by a specific chemical group; Ca2+ binding induces a shift of Tyr to a more polar environment and removal of the quenching group, but without full exposure to the solvent.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗
PubMed1993

Functional analysis of the promoters of the human CaMIII calmodulin gene and of the intronless gene coding for a calmodulin-like protein.

More than 1 kb of the 5'-flanking DNA of the human CaMIII calmodulin gene and of the calmodulin-like protein (CLP) gene have been sequenced. Notable features are the absence of a TATA-box and the presence of AGGGA elements in both upstream regions, the presence of several sequences with homology to known regulatory elements (cAMP-, retinoic acid- and interferon-responsive elements) in the CLP gene, and a high G + C-content and several putative Sp1-factor-binding sites in the CaMIII gene. 1 kb of the CaMIII upstream region was driving high-level growth hormone (hGH) reporter gene expression in human teratoma and monkey COS cells. Promoter activity dropped to about 30% and 10% when only 252 bp and 114 bp, respectively, of the CaMIII sequence were present. In contrast to the CaMIII promoter, the CLP gene upstream region was driving hGH expression only in human teratoma, but not in monkey COS cells. Addition of retinoic acid to the transfected cells had minimal effects on both promoters, leading to a 10-30% decrease of activity. The results show that the human CaMIII gene contains a strong and ubiquitously active promoter, whereas the promoter of the intronless CLP gene appears to be regulated in a cell-specific manner.

Animals↗
PubMed1993

Localization of the human bona fide calmodulin genes CALM1, CALM2, and CALM3 to chromosomes 14q24-q31, 2p21.1-p21.3, and 19q13.2-q13.3.

The three bona fide genes coding for calmodulin CALM1, CALM2, and CALM3, were assigned to human chromosomes 14, 2, and 19, respectively, by polymerase chain reaction-based amplification of calmodulin gene-specific sequences using DNA from human-hamster cell hybrids as template. Employing calmodulin gene-specific DNA probes of mainly intronic or flanking parts of the individual genes, regional sublocalization was performed by in situ hybridization on metaphase spreads of human lymphocytes. The three calmodulin genes map to chromosomes 14q24-q31, 2p21.1-p21.3, and 19q13.2-q13.3, respectively. These results represent the first complete chromosomal localization study on a mammalian calmodulin gene family and indicate that these structurally closely related genes were most likely dispersed throughout the genome concomitantly with their generation from an ancestral precursor gene.

Animals↗
PubMed1993

Localization of the intronless gene coding for calmodulin-like protein CLP to human chromosome 10p13-ter.

The functional intronless gene coding for a calmodulin-like protein (CLP) has been localized to human chromosome 10p13-ter. Chromosomal assignment was performed by Southern blot analysis of DNA from human-rodent somatic cell hybrids and amplification of a CLP gene-specific 1090-bp DNA fragment by the polymerase chain reaction (PCR) on DNA from human-hamster cell hybrids. Chromosomal sublocalization was carried out by in situ hybridization of human chromosome metaphase spreads. The CLP gene is the first member of the human calmodulin/calmodulin-like gene family to be chromosomally sublocalized. Its presence near the telomeric end of the short arm of chromosome 10 may be of significance with respect to its highly (epithelial) cell-type restricted expression in vivo and strong downregulation upon malignant transformation. The generation of a human CLP gene-specific sequence tag site specified by the two primers used for PCR should prove useful for future linkage studies.

Animals↗
PubMed1992

Characterization of the human calmodulin-like protein expressed in Escherichia coli.

The protein-coding region of an intronless human calmodulin-like gene [Koller, M., & Strehler, E. E. (1988) FEBS Lett. 239, 121-128] has been inserted into a pKK233-2 expression vector, and the 148-residue, M(r) = 16,800 human protein was purified to apparent homogeneity by phenyl-Sepharose affinity chromatography from cultures of Escherichia coli JM105 transformed with the recombinant vector. Several milligrams of the purified protein were obtained from 1 L of bacterial culture. A number of properties of human CLP were compared to those of bacterially expressed human calmodulin (CaM) and of bovine brain CaM. CLP showed a characteristic Ca(2+)-dependent electrophoretic mobility shift on SDS-polyacrylamide gels, although the magnitude of this shift was smaller than that observed with CaM. CLP was able to activate the 3',5'-cyclic nucleotide phosphodiesterase to the same Vmax as normal CaM, albeit with a 7-fold higher Kact. In contrast, the erythrocyte plasma membrane Ca(2+)-ATPase could only be stimulated to 62% of its maximal CaM-dependent activity by CLP. CLP was found to contain four Ca(2+)-binding sites with a mean affinity constant of 10(5) M-1, a value about 10-fold lower than that for CaM under comparable conditions. The highly tissue-specifically-expressed CLP represents a novel human Ca(2+)-binding protein showing characteristics of a CaM isoform.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment.

Isoform 4b of the human plasma membrane Ca2+ pump was expressed in COS cells and in the baculovirus system (Sf9 cells). A 105-kDa pump fragment lacking the first two transmembrane domains and the so-called transduction domain was also expressed. The expression level was 2-4 times the background in COS cells and at least 7 times in the baculovirus system. Tests on membranes from both systems showed that the expressed pump was active. The expressed pump and the 105-kDa fragment were isolated from Sf9 cell membranes by calmodulin affinity chromatography. The pump had Ca(2+)-dependent ATPase activity with a calmodulin stimulation factor of 3, formed a La(3+)-stabilized phosphoenzyme, and had a KM (Ca2+) in the presence of calmodulin of about 1 microM. The 105-kDa fragment, assayed by the phosphoenzyme test on COS or Sf9 cell membranes or by ATPase measurements after isolation from Sf9 cells, proved inactive. Laser confocal microscopy on Sf9 cells showed that both the pump and the 105-kDa fragment were apparently associated with the plasma membrane. The expressed pump in COS and Sf9 cells and the endogenous pump in a number of other cell lines had a slower gel mobility (i.e. a higher apparent molecular mass) than the erythrocyte pump.

Amino Acid Sequence↗
PubMed1992

Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.

Amino Acid Sequence↗