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PubMed2004

Quantification of alpha-subunit isoforms of Na,K-ATPase in rat resistance vessels.

AIM: Rat mesenteric resistance vessels (RV) were characterized with respect to concentration of individual alpha-subunit isoforms of Na,K-ATPase. METHODS: Total vessel homogenates were used to avoid any loss or subfractionation of membranes. They were applied to sodium dodecyl sulphate gels and, for calibration, in parallel lanes were run purified rat Na,K-ATPase preparations with known isoform distribution and content. The capacity per mg protein for Na+-dependent 32P-phosphorylation of Na,K-ATPase isolated from rat kidney was used for alpha1 calibration and that for high-affinity (3H)ouabain binding of Na,K-ATPase isolated from rat brain was used for (alpha2 + alpha3) calibration. Western blots containing homogenate proteins and reference enzyme were incubated with isoform-specific antibodies and radiolabelled secondary antibodies. The signals from adjacent alpha spots were used for qualitative and quantitative characterization of rat vessels. RESULTS: A concentration of 100.7 +/- 14.4 pmol (n = 11) per g wet weight of the alpha1-isoform containing Na,K-ATPase was found in RV from 12-14-week rats. A much lower and more unreliable content of alpha2- and alpha3-isoforms was found. These ouabain-sensitive isoforms seem to represent a maximum of 5-10% each compared with the ouabain-insensitive rat alpha1-isoform. CONCLUSIONS: The isoform pattern in RV, in which the isoform with high/intermediate Na+-affinity is the absolutely dominating one representing nearly all sodium pumps in this tissue, is very different from that seen in rat skeletal muscles. Due to the high content of the ouabain-insensitive alpha1-isoform in rat RV this species would seem a less relevant model in studies addressing a role of cardiac glycosides and putative endogenous ouabain-like factors in hypertension.

Animals↗
PubMed1996

FSH isoforms: bio and immuno-activities in post-menopausal and normal menstruating women.

OBJECTIVE: The full expression of gonadotrophin biological activity depends on the gonadotrophin carbohydrate component. Our aim was to study serum FSH isoforms present in the follicular phase (FPS) and in the menopause (PMS) since the endocrine status may influence the structure of incorporated oligosaccharides. SUBJECTS: Ten healthy post-menopausal women (age range 53-68) not receiving any hormonal treatment and 10 healthy women (age range 20-28) in the follicular phase of their menstrual cycle were studied. MEASUREMENTS: Bio and immuno FSH-activities (Sertoli cell aromatase induction assay and RIA, respectively) were determined in separated isoforms after concanavalin A chromatography. Isolated isoforms were: UB, unbound; WB, weakly bound and FB, firmly bound to the lectin. RESULTS: PMS showed two groups of immuno and bio-active FSH isoforms: WB, bearing biantennary galactosylated type and FB, bearing high mannose or hybrid type oligosaccharides. Immuno and bio-active FSH were not detected in the UB fractions. WB isoforms represented 82 +/- 6% of the total bioactivity recovered in samples analysed individually; their B/I ratio was 0.85 +/- 0.20. FB isoforms were 18 +/- 6%; their B/I ratio was 3.27 +/- 0.60. Whole serum B/I ratio was 1.20 +/- 0.30. Similar results were obtained when pooled sera was analysed: WB: 77%, B/I: 0.82; FB: 23%, B/I: 3.75. Whole serum B/I in pooled samples was 1.10. FPS showed a different pattern. UB isoforms, bearing triantennary or bisecting oligosaccharides, were 41 +/- 3% of the total bioactivity recovered in samples analysed individually. Their B/I ratio was 0.61 +/- 0.23. WB isoforms were 59 +/- 3% and their B/I 0.76 +/- 0.14. FB FSH isoforms were not detected. The whole serum B/I ratio was 0.60 +/- 0.30. Similar results were obtained when pooled sera was analysed: WB 43%, B/I 0.42; FB 57%, B/I 0.62. Whole serum B/I in pooled samples was 0.70. CONCLUSIONS: These results show that, in normal women, circulating FSH bioactivity is associated with isoforms with different oligosaccharide [correction of oligosacharide] structures according to hormonal status. FSH in the follicular phase has a higher degree of branching and a more complete carbohydrate chain than the FSH secreted during the menopause.

Adult↗
PubMed1997

Identical expression of CD45R isoforms by CD45RC+ 'revertant' memory and CD45RC+ naive CD4 T cells.

Naive and memory CD4 T cells are frequently defined by exon-specific monoclonal antibodies (mAb) which stain (or not) high- or low-molecular-weight (MW) isoforms of the leucocyte common antigen CD45. The link between isoform and the naive/memory designation is complicated by the fact that CD4 T cells with a 'memory' phenotype (CD45RA-, RB-, RC-, or CD45RO+) may revert ('revertants') and re-express the high mw isoform (CD45RA+, RB+, RC+). Isoform expression also changes during normal T-cell development. Furthermore, the picture may be incomplete since an exon-specific mAb will not detect all possible isoforms on a cell. We have used molecular techniques to determine whether revertant CD4 memory T cells were different from naive T cells with respect to CD45R isoform expression. Using the anti-CD45RC mAb OX22 to purify rat lymphocyte subsets, CD45R isoform expression was examined at the mRNA level in CD4 T cells at different stages of development and compared with that of B cells and unseparated lymphocytes. B cells contained abundant message for the highest MW 3-exon isoform ABC, the 2-exon isoforms AB and BC, and the null isoform O. Both immature CD45RC- (i.e. CD4+8- 'single positive' thymocytes, and peripheral Thy-1+ recent thymic emigrants) and mature CD45RC- 'antigen-experienced' CD4 T cells had message for single-exons B, possibly C and for the O exon. In contrast, CD45RC+ CD4 T cells contained mRNA coding for ABC (low level), AB, BC, B, C (low level) and O (low level). Importantly, there was no difference between CD45RC+ T cells that had not seen antigen ('truly native') and CD45RC+ antigen-experienced revertant memory T cells. This observation has implications for understanding long-term immunological memory.

Animals↗
PubMed1997

Axonal contact regulates expression of alpha2 and beta2 isoforms of Na+, K+-ATPase in Schwann cells: adhesion molecules and nerve regeneration.

Three isoforms of catalytic alpha subunits and two isoforms of beta subunits of Na+,K+-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na+,K+-ATPase was highly resistant to ouabain. The ouabain-resistant alpha1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na+,K+-ATPase activity. After sciatic nerve injury, the alpha3 and beta1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, alpha2 and beta2 isoform expression and Na+,K+-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the alpha2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that alpha3 and beta1 isoforms are exclusive for the axon and alpha2 and beta2 isoforms are exclusive for the Schwann cell, although axonal contact regulates alpha2 and beta2 isoform expressions. Because the beta2 isoform of Na+,K+-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/beta2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/beta2 may act as an adhesion molecule in peripheral nerve regeneration.

Animals↗
PubMed2000

Identification of the isoforms of Ca(2+)/Calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization.

Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) occurs in astrocytes as well as in neurons in brain. We have reported that CaM kinase II is involved in the regulation of cytoskeletal proteins and gene expression in astrocytes. In this study, we identified all isoforms of CaM kinase II in astrocytes and examined their subcellular localization. When we amplified the isoforms of four subunits by RT-PCR followed by the "nested" PCR, totally 10 isoforms were obtained. Immunoblot analyses with five types of antibodies against CaM kinase II indicated that the most abundant isoform was delta2. Immunostaining suggested that the delta2 isoform was localized predominantly at the Golgi apparatus. The localization of the delta2 isoform at the Golgi apparatus was also observed in NG108-15 cells. We overexpressed all isoforms that contained the nuclear localization signal to examine their nuclear targeting in NG108-15 cells. In contrast to the alphaB and delta3 isoforms that entered the nucleus, as reported, the gammaA isoform was excluded from the nucleus in the transfected NG108-15 cells. These results suggest that the 15-amino acid insertion following the nuclear localization signal inhibits the nuclear targeting of the gammaA isoform.

Amino Acid Sequence↗
PubMed2002

Species differences in the expression of multiple tyrosine hydroxylase protein isoforms.

In subprimates, a single form of tyrosine hydroxylase (TH) is expressed, whereas two TH protein isoforms have been identified in monkeys and four isoforms have been demonstrated in humans. In order to establish the evolutionary pattern/emergence of these multiple TH isoforms, adrenal medullae from different mammalian species were analyzed by blot immunolabeling using pan-specific TH antibodies and antibodies specific to each of the four human TH isoforms. The expression of multiple TH isoforms was primate specific and restricted to anthropoids: only a single TH isoform was detected in adrenal medullae from several subprimate and prosimian species (six species from four families), while two TH isoforms were found in all of the anthropoid species studied. The presence of four TH isoforms could only be demonstrated in human specimens. Contrary to previous suggestions, only one TH protein isoform was found in rats and only four TH protein isoforms were found in humans.

Adrenal Medulla↗
PubMed1990

Detection of P-glycoprotein isoforms by gene-specific monoclonal antibodies.

P-glycoprotein is a highly conserved membrane protein shown to be overexpressed in many multidrug-resistant tumor cell lines. P-glycoprotein is encoded by a small gene family in mammalian cells. Class I and II isoforms cause multidrug resistance, whereas class III does not. In this report, we have characterized three P-glycoprotein-specific monoclonal antibodies (mAbs) by high-resolution epitope mapping with a series of hexapeptides. mAb C494 is gene specific, binding to a sequence present only in the class I isoform of hamster and human. The mAb C32 recognizes a sequence conserved in hamster class I and II isoforms but not in class III isoforms. In contrast, the mAb C219 recognizes a highly conserved amino acid sequence found in all P-glycoprotein isoforms characterized to date. These mAbs were used to reveal differential expression and specific localization of the three P-glycoprotein isoforms in hamster tissues by immunohistochemical staining and competition with epitope-specific peptides. Colonic epithelial cells expressed predominantly the class I isoform in a polarized manner, adrenal cortical cells expressed predominantly the class II isoform, whereas a small percentage of skeletal muscle fibers expressed the class III isoform of P-glycoprotein. These findings suggest that the P-glycoprotein isoforms have distinct physiological roles associated with specialized cell functions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Neu differentiation factor/neuregulin isoforms activate distinct receptor combinations.

The multiple isoforms of Neu differentiation factor (NDF/neuregulin) induce a pleiotropic cellular response that is isoform-specific and cell type-dependent. The molecular basis of this heterogeneity was addressed by comparing the two major groups of isoforms, alpha and beta. Both groups bind to the catalytically impaired receptor tyrosine kinase ErbB-3, whose mitogenic stimulation by NDF requires transactivation by other ErbB proteins, either ErbB-1 or ErbB-2. By expressing each pair of receptors in interleukin 3-dependent myeloid cells, we found that both isoforms induced mitogenic signals in cells co-expressing the combination of ErbB-3 with ErbB-2. However, only the beta isoform stimulated cells that expressed both ErbB-3 and ErbB-1, and neither isoform was active on cells expressing ErbB-3 alone. Both isoforms bind to all ErbB-3-expressing cells, albeit with different affinities, but the co-stimulatory mitogenic effect is correlated with the ability of each auxiliary receptor to transphosphorylate ErbB-3. These results imply that NDF isoforms differ in their ability to induce receptor heterodimers; whereas both types of isoforms signal through ErbB-3/ErbB-2 heterodimers, only beta isoforms are able to stabilize ErbB-3/ErbB-1 heterodimers.

Animals↗
PubMed1996

Expression of novel secreted isoforms of human immunoglobulin E proteins.

Four human IgE isoforms produced by alternative splicing of the epsilon primary transcript were expressed as chimeric mouse/human anti 5-dimethylamino-1-naphthalenesulfonyl antibodies in the murine myeloma cell line Sp2/0. The four isoforms include the classic secreted form and three novel isoforms with altered carboxyl termini. All of these isoforms lack the transmembrane region encoded by the M1/M1' exon and are therefore predicted to be secreted proteins. When expressed in Sp2/0 cells, three of the IgE isoforms are assembled into complete molecules of two Ig heavy chains and two Ig light chains, whereas the fourth isoform is predominately assembled into half-molecules of one Ig heavy chain and one Ig light chain. All four isoforms are secreted with similar kinetics. In contrast, when the isoform containing the C epsilon4 domain joined directly to the M2 exon (IgE grandé) is expressed in the J558L cell line, it is degraded intracellularly, suggesting a cell line-dependent regulation of secretion. These data show that these novel isoforms of human IgE, predicted to occur from in vivo and in vitro mRNA analysis, can be produced and secreted by mammalian cells. The different forms of IgE may have physiologically relevant but distinct roles in human IgE-mediated immune inflammation. The availability of purified recombinant human IgE isoforms makes it possible to analyze the functional differences among them.

Alternative Splicing↗
PubMed2000

Molecular cloning and expression of three isoforms of the 100-kDa a subunit of the mouse vacuolar proton-translocating ATPase.

We have identified cDNAs encoding three isoforms (a1, a2, and a3) of the 100-kDa a subunit of the mouse vacuolar proton-translocating ATPase (V-ATPase). The predicted protein sequences of the three isoforms are 838, 856, and 834 amino acids, respectively, and they display approximately 50% identity between isoforms. Northern blot analysis demonstrated that all three isoforms are expressed in most tissues examined. However, the a1 isoform is expressed most heavily in brain and heart, a2 in liver and kidney, and a3 in liver, lung, heart, brain, spleen, and kidney. We also identified multiple alternatively spliced variants for each isoform. Reverse transcriptase-mediated polymerase chain reaction revealed that one splicing variant of the a1 isoform (a1-I) was expressed only in brain, whereas two other variants (a1-II and a1-III) were expressed in tissues other than brain. These alternatively spliced forms differ in the presence or absence of 6-7 amino acid residues near the amino and carboxyl termini of the proteins encoded. The a3 isoform is also encoded by three alternatively spliced variants, two of which are predicted to encode a protein that is truncated near the border of the amino- and carboxyl-terminal domains of the a subunit and therefore lacks the integral transmembrane-spanning helices thought to participate in proton translocation. Expression of each isoform (with the exception of a1-I) was detectable at all developmental stages investigated, with a1-I absent only in day 7 embryos. The results obtained suggest that isoforms of the 100-kDa a subunit may contribute to tissue-specific functions of the V-ATPase.

Alternative Splicing↗
PubMed2002

Constitutive phosphorylation of human endothelin-converting enzyme-1 isoforms.

To investigate the phosphorylation of human endothelin-converting enzyme-1 (hECE-1) and identify potential residues involved, both in vivo and in vitro phosphorylation labeling assays of hECE-1 isoforms were performed in combination with site-directed mutagenesis and mass spectrometric analyses. Initial studies found that endogenous hECE-1 was constitutively phosphorylated in a primary endothelial cell line. The four known isoforms of hECE-1 expressed in this cell line (1a, 1b, 1c, and 1d) were then cloned by reverse transcription-PCR to determine which isoform(s) may be phosphorylated. The isoforms differ only in the first portion of their short amino-terminal cytoplasmic domains whereas their transmembrane domains and ectodomains of the proteins are identical. Isoforms 1b, 1c, and 1d but not 1a, were constitutively phosphorylated in vivo when expressed in Chinese hamster ovary cells and casein kinase I readily phosphorylated the immunopurified isoforms in vitro. Site-directed mutagenesis established that two conserved serine residues, Ser(18) and Ser(20), (numbering based on isoform 1c) form at least one phosphorylation site in these three isoforms. Mutant forms of 1b, 1c, and 1d were constructed in which a single alanine was introduced at either serine residue and a double mutant for each isoform was constructed as well in which both serines were replaced with alanine. Phosphorylation of the single mutants was greatly reduced and was nearly abolished in the double mutants in both in vivo and in vitro labeling assays. Analysis by MALDI-MS of (32)P-labeled proteolytic peptides derived from wild type 1c and the 1c mutants supported both Ser(18) and Ser(20) as phosphorylated residues. These data demonstrate the first finding that hECE-1 is constitutively phosphorylated within its cytoplasmic domain in an isoform-specific manner.

Amino Acid Sequence↗
PubMed2002

Heterodimerization of endothelin-converting enzyme-1 isoforms regulates the subcellular distribution of this metalloprotease.

Endothelin-converting enzyme (ECE) is a membrane metalloprotease that generates endothelin from its direct precursor big endothelin. Four isoforms of ECE-1 are produced from a single gene through the use of alternate promoters. These isoforms share the same extracellular catalytic domain and contain unique cytosolic tails, which results in their specific subcellular targeting. We investigated the distribution of ECE-1 isoforms in transfected AtT-20 neuroendocrine cells. Whereas ECE-1a and 1c were present at the plasma membrane, ECE-1b and ECE-1d were retained inside the cells. We found that both intracellular isoforms were concentrated in the endosomal system: ECE-1d in recycling endosomes, and ECE-1b in late endosomes/multivesicular bodies. Leucine-based motifs were involved in the intracellular retention of these isoforms, and the targeting of ECE-1b to the degradation pathway required an additional signal in the N terminus. The concentration of ECE-1 isoforms in the endosomal system suggested new functions for these enzymes. Potential novel functions include redistribution of other isoforms through direct interaction. We have showed that ECE-1 isoforms could heterodimerize, and that in such heterodimers the ECE-1b targeting signal was dominant. Interaction of a plasma membrane isoform with ECE-1b resulted in its intracellular localization and decreased its extracellular activity. These data demonstrated that the targeting signals specific for ECE-1b constitute a regulatory domain per se that could modulate the localization and the activity of other isoforms.

Acetylcysteine↗
PubMed1993

Differential effects of over-expressed neural cell adhesion molecule isoforms on myoblast fusion.

We have used a transfection based approach to analyze the role of neural cell adhesion molecule (NCAM) in myogenesis at the stage of myoblast fusion to form multinucleate myotubes. Stable cell lines of myogenic C2 cells were isolated that express the transmembrane 140- or 180-kD NCAM isoforms or the glycosylphosphatidylinositol (GPI) linked isoforms of 120 or 125 kD. We found that expression of the 140-kD transmembrane isoform led to a potent enhancement of myoblast fusion. The 125-kD GPI-linked NCAM also enhanced the rate of fusion but less so when a direct comparison of cell surface levels of the 140-kD transmembrane form was carried out. While the 180-kD transmembrane NCAM isoform was effective in promoting C2 cell fusion similar to the 140-kD isoform, the 120-kD isoform did not have an effect on fusion parameters. It is possible that these alterations in cell fusion are associated with cis NCAM interactions in the plane of the membrane. While all of the transfected human NCAMs (the transmembrane 140- and 180-kD isoforms and the 125- and 120-kD GPI isoforms) could be clustered in the plane of the plasma membrane by species-specific antibodies there was a concomitant clustering of the endogenous mouse NCAM protein in all cases except with the 120-kD human isoform. These studies show that different isoforms of NCAM can undergo specific interactions in the plasma membrane which are likely to be important in fusion. While the transmembrane and the 125-kD GPI-anchored NCAMs are capable of enhancing fusion the 120-kD GPI NCAM is not. Thus it is likely that interactions associated with NCAM intracellular domains and also the muscle specific domain (MSD) region in the extracellular domain of the GPI-linked 125-kD NCAM are important. In particular this is the first role ascribed to the O-linked carbohydrate containing MSD region which is specifically expressed in skeletal muscle.

Animals↗
PubMed1996

Studies on the relative in-vitro biological potency of the naturally-occurring isoforms of intrapituitary follicle stimulating hormone.

In the present study, we analysed and compared the relative in-vitro biological activity of the various intrapituitary human follicle stimulating hormone (FSH) isoforms employing two different bioassay systems. FSH was fractionated by chromatofocusing (pH range 7.10 to < 3.80) and the several isoforms isolated were quantified at multiple dose levels by three highly specific immunoassay systems: radioimmunoassay (RIA), enzyme-immunoassay (EIA) and immunoradiometric assay (IRMA), as well as by two in-vitro bioassays, one that measures the amount of oestrogen produced by rat granulosa cells in culture and the other that determines the amount of cAMP produced by a human fetal cell line (293) expressing the recombinant human FSH receptor. The relative in-vitro biological activity of each FSH isoform, expressed as the bioassay/ immunoassay (B/I) activity ratio (B/RIA, B/EIA and B/IRMA ratios) varied with its elution pH value. Regardless of the immunoassay or bioassay method employed, less acidic FSH isoforms exhibited higher B/I ratios than their more acidic counterparts [B/RIA, B/EIA and B/IRMA ratios for isoforms with elution pH values > 4.5 = 1.05 +/- 0.13, 0.99 +/- 0.10 and 1.15 +/- 0.08 (rat oestrogen bioassay), and 2.75 +/- 0.34, 2.20 +/- 0.25 and 2.96 +/- 0.35 (human cAMP production bioassay) respectively. Ratios for isoforms with pH values < 4.5 = 0.71 +/- 0.06, 0.47 +/- 0.05 and 0.63 +/- 0.06 (rat oestrogen assay), and 1.80 +/- 0.26, 1.10 +/- 0.09 and 1.44 +/- 0.13 (cAMP assay) respectively (P < 0.05 for isoforms with pH < 4.5 compared with those isoforms with pH > 4.5)]. Furthermore, statistically significant direct relationships between the B/RIA, B/EIA and B/IRMA ratios and elution pH value of each isoform was identified by regression analysis [rat assay: r = 0.844, 0.800 and 0.780 (P < 0.01); human assay: r = 0.730, 0.845 and 0.821 (P < 0.01), for their corresponding B/RIA, B/EIA and B/IRMA ratios respectively]. The finding of significant differences in relative in-vitro biological potency among the various intrapituitary FSH isoforms strongly suggests that the shifts towards the production and secretion of more basic or acidic FSH molecules occurring in certain specific physiological conditions (e.g. puberty and menstrual cycle), may represent an important mechanism through which the anterior pituitary regulates gonadal function.

Adult↗
PubMed1997

Characterization of tyrosine-phosphorylated delta isoform of protein kinase C isolated from Chinese hamster ovary cells.

Phorbol ester treatment of Chinese hamster ovary cells stably overexpressing the delta isoform of protein kinase C induced the association of the isoform with the particulate fraction and the tyrosine phosphorylation of a small portion of the delta isoform. The delta isoform without tyrosine phosphorylation was recovered as an enzyme dependent on phospholipid and diacylglycerol, whereas the tyrosine-phosphorylated delta isoform was recovered in two fractions, one dependent on, and the other independent of, phospholipid and diacylglycerol. The tyrosine-phosphorylated delta isoform independent of lipid activators might be associated with phorbol ester and phospholipids. Immunoblot analysis revealed that the delta isoform is a doublet protein of 76 and 78 kDa, and that the delta isoform fraction without tyrosine phosphorylation contained 76- and 78-kDa proteins, whereas the tyrosine-phosphorylated delta isoform contained the 78-kDa protein but not the 76-kDa protein. In vitro analysis showed that the 78-kDa protein of the delta isoform without tyrosine phosphorylation is an efficient substrate of tyrosine kinase only when phosphatidylserine and either diacylglycerol or phorbol ester are present; however, the 76-kDa protein can not be tyrosine-phosphorylated even in the presence of these lipid activators. The phospholipid and diacylglycerol-dependent form of the tyrosine-phosphorylated enzyme isolated from the cell line required lower concentrations of phosphatidylserine and phorbol ester for its activity in vitro as compared with the enzyme without tyrosine phosphorylation. These results suggest that the tyrosine-phosphorylated enzyme generated upon stimulation of the cells may associate with membranes and exert its full activity even with the lower concentrations of the lipid activators.

Animals↗
PubMed1992

Retinoic acid-specific induction of a protein kinase C isoform during differentiation of HL-60 cells.

Human promyelocytic leukemia cells (HL-60) were treated with several differentiation inducers, then the changes in the activity of cytosolic protein kinase C (PKC) isoforms were examined by hydroxylapatite chromatography and the species of the isoforms were determined immunologically. In three undifferentiated HL-60 cell lines examined, PKC alpha and beta isoforms were present, but PKC gamma isoform was not detected. When the cells were induced by dimethylsulfoxide, dibutyryl cAMP, or nicotinamide to differentiate into granulocytes, these two PKC isoforms each increased to about 2- to 3-fold. When retinoic acid was used as the inducer, in addition to PKC alpha and beta, a third PKC isoform appeared. This isoform was clearly distinct from rat PKC alpha, beta, and gamma, immunologically. This isoform showed a distinctly lower Ca(2+)-requirement (3 microM) than that of PKC alpha or beta (100 microM) and was more dependent on cardiolipin and phosphatidylethanolamine, compared with PKC alpha, beta, and gamma. These results suggest that while the increases in the activities of PKC alpha and beta isoforms are common in the differentiation program initiated by several inducers, including retinoic acid, the emergence of an unclassified PKC isoform is a retinoic acid-specific process.

Animals↗
PubMed1995

Genomic organization and isoforms of the mouse ELP gene.

Analysis was made on the genomic structure, functions, and expression of the mouse ELP gene, which codes for the embryonal long terminal repeat binding protein. Extensive screening of the cDNA library of embryonal carcinoma cells (EC cells) identified four isoforms of ELP: ELP1 (the original ELP isolate), ELP2, ELP3, and Ad4BP/SF1. Analysis of the genomic sequences revealed that these ELP isoforms were generated by alternative promoter usage and differential splicing. The mRNAs of isoforms initiated at four transcription start sites distributed on three exons. Sequence analysis of the four isoforms identified three polypeptides. The N-terminal portion of ELP1 and ELP2 was longer than ELP3, and Ad4BP/SF1 by 77 aa. The DNA-binding domain and region II were shared by all four isoforms. The C-terminal portion shared by ELP2, ELP3, and Ad4BP/SF1 was 131 aa in length, and that specific to ELP1 was 57 aa in length. The ELP3 and Ad4BP/SF1 isoforms were identical for the coding sequence, but the two differed at the 5' noncoding region. Region II and III domains of nuclear receptors were thought to be involved in ligand-binding and transcriptional activation. ELP1, which lacked region III, functioned as a repressor. The isoforms carrying intact region II and region III functioned as transactivators. Expression of the four isoforms was studied in mouse tissues and in tissue culture cells by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Complex patterns of expression of these isoforms were observed in various tissues. All four ELP isoforms were expressed only in EC cells.

3T3 Cells↗
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↗