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PubMed2001

The novel long PDE4A10 cyclic AMP phosphodiesterase shows a pattern of expression within brain that is distinct from the long PDE4A5 and short PDE4A1 isoforms.

In situ hybridisation methods were used to map the distribution of the novel long PDE4A10 isoform in the brain. PDE4A10 distribution was compared to that of the long PDE4A5 isoform and the short PDE4A1 isoform using probes specific for unique sequences within each of these isoforms. Coronal sections of the brain, taken at the level of the olfactory bulb, prefrontal cortex, striatum, thalamus, hippocampus and cerebellum, were analysed. Strongest expression of PDE4A isoforms was found in the olfactory bulb granular layer with high signals also in the piriform cortex, the dentate gyrus and the CA1 and CA2 pyramidal cells. For the two long forms, level general staining was noted throughout the striatum, thalamus and hippocampus but no signal was evident in the cerebellum. The long PDE4A10 and PDE4A5 isoforms localised to essentially the same regions throughout the brain, although PDE4A10 was uniquely expressed in the major island of Calleja. A signal for the short PDE4A1 isoform was found in regions in which the two long isoforms were both expressed, with the exception of the medial nucleus of the amygdala where weak signals for PDE4A5 and PDE4A10 were detected but PDE4A1 was absent. Uniquely, strong signals for PDE4A1 were detected in the glomerular layer of the olfactory bulb, the CA3 pyramidal cell region and the cerebellum; areas where signals for the two long forms were not evident. PDE4A transcripts for both PDE4A5 and PDE4A10 were not apparent in the brain stem and those for PDE4A1 were low. PDE4A isoforms are present in several key areas of the brain and therefore present valid targets for therapeutic interventions. Whilst the two long PDE4A isoforms show a remarkably similar distribution, in at least three regions there is clear segregation between their pattern of expression and that of the PDE4A1 short form. This identifies differential regulation of the expression of PDE4A long and short isoforms. We suggest that specific PDE4A isoforms may have distinct functional roles in the brain, indicating that PDE4A isoform-selective inhibitors may have specific therapeutic and pharmacologic properties.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Isoforms of rat liver fatty acid binding protein differ in structure and affinity for fatty acids and fatty acyl CoAs.

Although native rat liver fatty acid binding protein (L-FABP) is composed of isoforms differing in isoelectric point, their comparative structure and function are unknown. These properties of apo- and holo-L-FABP isoforms were resolved by circular dichroism, time-resolved fluorescence spectroscopy, and binding/displacement of fluorescent ligands. Both apo-isoforms had similar hydrodynamic radii of 18.5 A, but apo-isoform I had a greater alpha-helical content and exhibited a longer Tyr lifetime, indicative of secondary and tertiary structural differences from isoform II. Isoforms I and II both had two fatty acid or fatty acyl CoA binding sites. Ligand binding decreased the isoform hydrodynamic radii by 3-4 A and increased Tyr rotational motions in a more restricted range. Fatty acyl CoAs were more effective than fatty acids in altering the isoform structures. Scatchard analysis showed that both isoforms bound cis- parinaric acid with high affinity (Kd values 41 and 60 nM, respectively) and bound trans-parinaric acid with 2- and 7-fold, respectively, higher affinity than for cis-parinaric acid. In contrast, isoform I had higher affinity for cis- and trans-parinaroyl CoAs (Kd values of 33 and 14 nM) than did isoform II (Kd values of 110 and 97 nM), thereby resulting in biphasic plots of parinaroyl-CoA binding to native L-FABP. Finally, displacement studies indicated that each isoform displayed distinct specificities for fatty acid/fatty acyl CoA chain length and unsaturation. Thus, rat L-FABP isoforms differ markedly in both structure and ligand binding function.

Acyl Coenzyme A↗

PubMed1995

Beta 2 microglobulin isoforms in healthy individuals and in amyloid deposits.

beta 2 microglobulin (beta 2m) is classically known to have isoforms with isoelectric points (pI) 5.7 and 5.3. New isoforms of beta 2m with lower pI, probably due to modifications with advanced glycation end products, were found in the amyloid deposits of dialysis related amyloidosis (DRA), and they were proposed as the amyloidogenic forms of beta 2m. The other modifications in beta 2m from amyloid deposits are partial proteolysis and single amino acid replacement (Asn by ASp at position 17). However, there are no data on the sequence of the different isoforms of beta 2 m from amyloid deposits. Amyloid deposits surgically obtained from the carpal tunnel from 13 dialysis treated patients and urine from 10 healthy volunteers and 5 living-related kidney donors were analyzed for beta 2m content. Two-dimensional gel electrophoresis (2D-PAGE) of beta 2m from amyloid deposits showed the presence of four or more isoforms with pIs < 5.7. All the spots migrating at 12 kDa Mr region and between 4 and 6 pH reacted with rabbit anti-human beta 2m antibody by Western blotting, confirming that they were beta 2m isoforms. beta 2m isoforms from the amyloid deposits were then separately purified with an IEF column (PB94, Pharmacia) for analysis. Enough quantities of three pure beta 2m isoforms could be obtained in two cases. The sequence analysis showed an intact N-terminus in all the isoforms. There was Asn in the 17th residue in all the isoforms sequenced. 2D-PAGE of urine from 8 out of the 10 healthy volunteers showed the presence of beta 2m. In two of them beta 2m also displayed four different isoforms. At least four isoforms were observed in urine of all the kidney donors. The present study shows that the elution peaks of three different beta 2m isoforms in gel isoelectrofocusing contain beta 2m with intact N-terminus. None of them have deamidated their 17th residue. More importantly, the beta 2m isoforms with lower pI are not specific for amyloidosis as they were found in urine from kidney donors and in normal volunteers. These results bring into question the hypothesis that dialysis related amyloidosis is due to the known modifications on beta 2m. They suggest that the precipitation of beta 2m into amyloid fibrils should result from the interaction of beta 2m with other factors with amyloid enhancing activity.

Adult↗

PubMed1998

A GR-motif functions in nuclear accumulation of the large FGF-2 isoforms and interferes with mitogenic signalling.

Nuclear translocation has been documented for members of the fibroblast growth factor (FGF) family in addition to their roles as extra-cellular signalling molecules. Fusing different parts of the chicken FGF-2 open reading frame to pyruvate kinase shows that direct nuclear accumulation is mediated by the amino-termini of the two leucine initiated FGF-2 isoforms (Leu-isoforms; 21.5 and 20.0 kDa). An evolutionarily conserved glycine-arginine (GR)-motif is present in the 21.5 kDa Leu-isoform and a shorter GR-repeat in the 20.0 kDa Leu-isoform, whereas no such repeats are present in the 18.5 kDa FGF-2 isoform (Met-isoform). Expression in NIH3T3 fibroblasts shows that the 21.5 kDa Leu-isoform is predominantly nuclear, whereas the Met-isoform is predominantly cytoplasmic. Most importantly, insertion of the GR-motif into the Met-isoform results in a protein with characteristics similar to the Leu-isoforms, as shown by nuclear accumulation of the chimeric MGR-protein. Furthermore, only NIH3T3 fibroblasts expressing the Met-isoform proliferate under serum starvation conditions, whereas cells expressing either the MGR- or Leu-isoforms stay growth arrested. These studies show that the GR-signal mediates nuclear translocation of endogenous Leu-isoforms and blocks their mitogenic activity.

3T3 Cells↗

PubMed1997

Expression, purification, and regulation of two isoforms of the inositol 1,4,5-trisphosphate 3-kinase.

The level of inositol 1,4,5-trisphosphate in the cytoplasm is tightly regulated by two enzymes, the inositol 1,4,5,5-phosphatase and the inositol 1,4,5-trisphosphate 3-kinase. Two isoforms of the inositol 1,4,5-trisphosphate 3-kinase have been identified, the A form and the B form. The regulatory properties of the two isoforms were compared following overexpression and purification of the proteins from a v-src transformed mammalian cell line. The highly purified, recombinant inositol 1,4,5-trisphosphate 3-kinases were differentially regulated by calcium/calmodulin and via phosphorylation by protein kinase C or the cyclic AMP-dependent protein kinase. Both enzymes had similar affinities for inositol 1,4, 5-trisphosphate (Km 2-5 mu M). Calcium/calmodulin stimulated the activity of isoform A about 2.5-fold, whereas the activity of isoform B was increased 20-fold. The cyclic AMP-dependent protein kinase phosphorylated the inositol 1,4,5-trisphosphate 3-kinase A to the extent of 0.9 mol/mol and isoform B to 1 mol/mol. Protein kinase C phosphorylated isoform A to the extent of 2 mol/mol and isoform B to 2.7 mol/mol. Phosphorylation of isoform A by the cyclic AMP-dependent protein kinase caused a 2.5-fold increase in its activity when assayed in the absence of calcium/calmodulin, whereas phosphorylation by protein kinase C decreased activity by 72%. The activity of isoform B in the absence of calcium/calmodulin was not affected by phosphorylation using either kinase. When assayed in the presence of calcium/calmodulin, phosphorylation of isoform A by the cyclic AMP-dependent protein kinase increased activity 1.5-fold, whereas phosphorylation of isoform B decreased activity by 45%. Phosphorylation of either isoform A or B by protein kinase C resulted in a 70% reduction of calcium/calmodulin-stimulated activity. Differential expression and regulation of the two inositol 1,4,5-trisphosphate 3-kinase isoforms provides multiple mechanisms for regulating the cytosolic level of inositol 1,4,5-trisphosphate in cells.

Animals↗

PubMed2000

Plasma membrane Ca(2+) pump isoform 3f is weakly stimulated by calmodulin.

Isoform 3f of the plasma membrane Ca(2+) pump is a major isoform of this pump in rat skeletal muscle. It has an unusual structure, with a short carboxyl-terminal regulatory region of only 33 residues when compared with the 77 to 124 residues found in the other isoforms. Also, whereas the regulatory regions of the other isoforms, downstream of the alternative splice, consist of two homologous groups, the sequence of 3f is not related to either group. A synthetic peptide representing the calmodulin binding domain of isoform 3f had a much lower calmodulin affinity (with a K(d) of 15 nM) than the corresponding peptide of isoform 2b (K(d) value was 0.2 nM). The characteristics of this domain were further studied by making chimeras of the 3f regulatory region with the catalytic core of isoform 4 and by making the full-length isoform 3f. Both constructs bound to calmodulin-Sepharose. The chimera was fully active without calmodulin, showing no stimulation of activity when calmodulin was added. The full-length isoform 3f was slightly activated by calmodulin. These data show that the regulatory region of isoform 3f is only a weak autoinhibitor of the enzyme, in contrast to the properties of all the other isoforms studied so far. Rather, this isoform is a special-purpose, constitutively active form of the enzyme, expressed primarily in skeletal muscle and as a minor isoform in brain.

Amino Acid Sequence↗

PubMed2004

The alpha 1 isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart.

The primary objective of this study was to examine the functional role of the Na,K-ATPase alpha 1 isoform in the regulation of cardiac contractility. Previous studies using knock-out mice showed that the hearts of animals lacking one copy of the alpha 1 or alpha 2 isoform gene exhibit opposite phenotypes. Hearts from alpha 2(+/-) animals are hypercontractile, whereas those of the alpha 1(+/-) animals are hypocontractile. The cardiac phenotype of the alpha 1(+/-) animals was unexpected as other studies suggest that inhibition of either isoform increases contraction. To help resolve this difference, we have used genetically engineered knock-in mice expressing a ouabain-sensitive alpha 1 isoform and a ouabain-resistant alpha 2 isoform of the Na,K-ATPase, and we analyzed cardiac contractility following selective inhibition of the alpha1 isoform by ouabain. Administration of ouabain to these animals and to isolated heart preparations selectively inhibits only the activity of the alpha 1 isoform without affecting the activity of the alpha 2 isoform. Low concentrations of ouabain resulted in positive cardiac inotropy in both isolated hearts and intact animals expressing the modified alpha 1 and alpha 2 isoforms. Pretreatment with 10 microm KB-R7943, which inhibits the reverse mode of the Na/Ca exchanger, abolished the cardiotonic effects of ouabain in isolated wild type and knock-in hearts. Immunoprecipitation analysis demonstrated co-localization of the alpha1 isoform and the Na/Ca exchanger in cardiac sarcolemma. The alpha 1 isoform co-immunoprecipitated with the Na/Ca exchanger and vice versa. These results demonstrate that the alpha 1 isoform regulates cardiac contractility, and that both the alpha 1 and alpha 2 isoforms are functionally and physically coupled with the Na/Ca exchanger in heart.

Animals↗

PubMed1994

Characterization of isoform diversity in smooth muscle myosin heavy chains.

In this paper we review some of our recent work on the structural and biochemical characterization of isoforms of the heavy chain of vertebrate smooth muscle myosin II. There exist both amino-terminal and carboxyl-terminal alternatively spliced isoforms of the smooth muscle myosin heavy chain (MHC). mRNA splicing at the 3' end generates two MHCs, which differ in length and amino acid sequence in the carboxyl terminus. We will refer to the longer, 204-kDa isoform as MHC204 and the shorter, 200-kDa isoform as MHC200. We found that MHC204, but not MHC200, can be phosphorylated by casein kinase II on a serine near the carboxyl terminus, suggesting that these isoforms may be differentially regulated. The physiological significance of this phosphorylation is not known. However, as demonstrated in this paper, phosphorylation does not appear to affect filament formation, velocity of movement of actin filaments by myosin in an in vitro motility assay, actin-activated Mg2+ ATPase activity, or myosin conformation. Our results also show that MHC204 and MHC200 form homodimers, but not heterodimers. Purified MHC204 and MHC200 homodimers are not enzymatically different, at least as measured using an in vitro motility assay. The amino-terminal spliced MHC204 and MHC200 isoforms are the result of the specific insertion or deletion of seven amino acids near the ATP-binding region in the myosin head. We refer to these isoforms as inserted (MHC204-I; MHC200-I) or noninserted (MHC204; MHC200), respectively. In contrast to the carboxyl-terminal spliced isoforms, the amino-terminal spliced inserted and noninserted myosin heavy chain isoforms are enzymatically different. The inserted isoform, which is expressed in intestinal, phasic-type smooth muscle, has a higher actin-activated Mg ATPase activity and moves actin filaments at a greater velocity in an in vitro motility assay than the noninserted MHC isoform, which is expressed in tonic-type vascular smooth muscle. The results presented in this review suggest that the alternative splicing of smooth muscle mRNA results in at least four different isoforms of the myosin heavy chain molecule. The potential relevance of these molecular isoforms to smooth muscle function is discussed.

Amino Acid Sequence↗

PubMed1986

Differential isoform distribution between stored and secreted prolactin.

PRL exists within the mammotroph population in a number of different molecular forms. Three of these forms are best described as isoforms, as they have the same mol wt (24K) but differ in their net molecular charges. In this study we have examined the relative proportions of newly synthesized isoforms found in stored (intracellular) vs. secreted (extracellular) PRL. Dissociated cells from female rat anterior pituitaries were cultured for 48 h and then incubated in [35S]methionine (6 h; 37 C). Intracellular and medium proteins were then extracted and resolved by one- and two-dimensional polyacrylamide gel electrophoresis, followed by silver staining or autoradiography. Control experiments, in which biosynthetically labeled PRL was re-extracted, ensured that the isolation conditions did not in themselves promote isoform interconversion. The relative proportions of the PRL isoforms were determined by densitometric scanning of developed autoradiograms. In the cell extracts, the relative proportions were 13.6 +/- 2.1% isoform 1 (least negatively charged), 71.5 +/- 3.26% isoform 2, and 14.7 +/- 1.9% isoform 3 (most negatively charged). In the medium, the relative proportions were 60 +/- 2.89% isoform 1, 20 +/- 1.75% isoform 2, and 11 +/- 1.14% isoform 3. When the labeling was performed in the presence of 0.5 mM cysteamine (an agent we show to distinguish between newly synthesized and older stored hormone and, hence, between the previously described functional subpopulations of mammotrophs), the same ratios of newly synthesized isoforms were secreted from the cells. We conclude that secretion of the different isoforms is more complex than simple proportional release of each form, and based on the cysteamine results, this nonproportional release cannot be attributed to release of one isoform per functional subpopulation of mammotrophs.

Animals↗

PubMed1991

4 S oestrogen receptor isoforms and their distribution in breast cancer samples.

The variability in the profile of oestrogen receptor (ER) isoforms in breast tumours has been studied. Using low-resolution isoelectric focussing (IEF), two major ER isoforms with isoelectric point (pI) values of 6.1 and 6.6 could be identified, with corresponding sedimentation coefficients in sucrose density gradients of 8 S and 4 S respectively. Using high-resolution IEF or immunoblotting, the pI 6.6 form (4 S) was shown to be composed of three different species, with pI values of 6.3, 6.6 and 6.8, while the oligomeric pI 6.1 protein (8 S) did not show charge heterogeneity. Data were obtained on the soluble receptors from supernatants of 42 ER-positive primary breast tumour homogenates using high-resolution IEF to obtain ER isoform profiles. It was found that 54.7% of tumours contained the isoforms at pI 6.6 and 6.1, while only 11.9% contained the full complement of isoforms (pI 6.1, 6.3, 6.6 and 6.8). Of the tumours studied, 11.9% contained isoforms of pI 6.1, 6.6 and 6.8, with 14.3% containing isoforms with pI 6.1, 6.6 and 6.3. Very few tumours contained only one isoform, with 4.8% of tumours containing a single isoform at pI 6.1 and 2.4% of tumours containing only the isoform at pI 6.6. All four ER isoforms were also shown to be present in some tumours by immunoblotting using antibody H222 and, in addition, high-resolution IEF indicated that all isoforms bind oestradiol, diethylstilboestrol and tamoxifen. The variability in the ER isoform profile may have a bearing on the known variability of tumour response to endocrine therapy and prognosis.

Breast Neoplasms↗

PubMed1991

Metabolism of two Go alpha isoforms in neuronal cells during differentiation.

We have previously shown that undifferentiated N1E-115 neuroblastoma cells express only one isoform of Go alpha (pI = 5.8), whereas differentiated neuroblastoma cells expressed, in addition to this isoform, another Go alpha with a more acidic pI (5.55). Moreover, primary cultures of cerebellar granule cells, which are extremely well differentiated cells yielding a high density of synapses, expressed only a single Go alpha isoform with a pI of 5.55 (Brabet, P., Pantaloni, C., Rodriguez Martinez, J., Bockaert, J., and Homburger, V. (1990) J. Neurochem. 54, 1310-1320). In this report, using biosynthetic labeling with [35S]methionine and specific quantitative immunoprecipitation with a polyclonal antibody raised against the purified Go alpha protein, we have determined 1) the degradation rate of total Go alpha (sum of the two isoforms) in differentiated as well as in undifferentiated neuroblastoma cells and in cerebellar granule cells, 2) the degradation rates of each isoform in differentiated neuroblastoma cells. The t 1/2 for total Go alpha protein degradation was very different in the three neuronal cell populations and was 28 +/- 5 h (n = 5), 58 +/- 9 h (n = 5), and 154 +/- 22 h (n = 6) in undifferentiated, differentiated neuroblastoma, and granule cells, respectively. Using two-dimensional gel analysis of immunoprecipitates, we have also determined the individual t 1/2 for degradation of each Go alpha isoform in differentiated neuroblastoma cells, in which the two Go alpha isoforms were expressed. Results indicated that the two Go alpha isoforms exhibit similar t1/2 for degradation (49 +/- 5 h, n = 3). Thus, the t1/2 for degradation of the more basic Go alpha isoform is higher in differentiated neuroblastoma cells (49 +/- 5 h, n = 3) than in undifferentiated neuroblastoma cells (28 +/- 5 h, n = 5) which expressed only the more basic Go alpha isoform. It can be concluded that the degradation rate of the more basic Go alpha isoform is not a characteristic of the protein itself but depends on the state of the cell differentiation. The comparison between the t1/2 for degradation of the more acidic Go alpha isoform is differentiated neuroblastoma cells (51 +/- 6 h, n = 3) with that of cerebellar granule cells (154 +/- 22 h, n = 6) suggests that there is also a decrease in the degradation rate of the more acidic Go alpha isoform during differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

CD44 isoforms containing exons V6 and V7 are differentially expressed on mitogenically stimulated normal and Epstein-Barr virus-transformed human B cells.

CD44, a cell adhesion molecule, exists in multiple isoforms that are generated by RNA alternative splicing. CD44 isoforms containing exon V6 (CD44 V6) have been associated with tumorigenesis and metastasis. We investigated the association between human B-cell activation and CD44 V6 isoform expression by analysing its expression in resting and mitogenically stimulated B cells. Results showed that resting B cells expressed the CD44 H (no variable exon) isoform alone. Activation of B cells [phorbol myristate acetate (PMA), surface immunoglobulin cross-linking alone or in the presence of interleukin-2 (IL-2)] induced CD44E (variable exon V8-10), R2 (VIO) and CD44 isoforms containing exons V6 and/or V7 (CD44 V6/V7). Epstein-Barr virus (EBV) infection of B cells, an alternative method of B-cell activation, induced the expression of CD44 E and R2 but not CD44 V6/V7. These results indicate that CD44 V6/V7 expression depends on the mode of activation. CD44 isoform expression was also investigated in a panel of EBV-negative and EBV-positive Burkitt's lymphoma (BL) B-cell lines. EBV-negative BL cells did not express CD44. In contrast, EBV-positive BL cells expressed CD44 H, R2 and E but not CD44 V6/V7 isoforms, suggesting an association between EBV infection and CD44 isoform induction. To determine directly the role of EBV in CD44 isoform induction, an EBV-negative BL cell line, BL30 (negative for all isoforms of CD44), BL30 infected in vitro with the EBNA-2-defective P3HR1 (BL30/P3HR1), and the wild-type B95-8 strain of EBV (BL30/B95-8) were examined. The parental BL30 cells infected with the wild-type EBV strain, but not with the P3HR-1 strain, expressed CD44 H, R2 and E isoforms, as seen in EBV-immortalized B cells. These studies suggest that (1) alternative splicing of CD44 isoforms is differentially regulated depending on the mode and state of cell activation, and that (2) the CD44 V6/V7 isoforms may represent B-cell activation antigens that are induced by mitogenic stimulation but not following EBV infection.

B-Lymphocytes↗

PubMed1997

The role of a single formin isoform in the limb and renal phenotypes of limb deformity.

BACKGROUND: Mutations of the murine limb deformity (ld) locus are responsible for a pleiotropic phenotype of completely penetrant limb malformations and incompletely penetrant renal agenesis and/or dysgenesis. The ld locus encodes a complex family of mRNA and protein isoforms. MATERIALS AND METHODS: To examine the role of one of the more prominent of these isoforms, isoform IV, we specifically eliminated it by gene targeting. RESULTS: Unlike other mutant ld mice, homozygous mice bearing this isoform IV disruption display incompletely penetrant renal agenesis, but have perfectly normal limbs. Whole mount in situ hybridization demonstrated that this targeted disruption was specific for isoform IV and did not interfere with the expression of other ld isoforms. The isoform IV-disrupted allele of ld does not complement the renal agenesis phenotype of other ld alleles, in a manner consistent with its penetrance, and like the isoform IV-deficient mice, these compound heterozygotes have normal limbs. Sequence analysis of formin isoform IV in other ld mutant alleles did not detect any amino acid changes relative to the strain of origin of the mutant allele. CONCLUSIONS: Thus, the disruption of isoform IV is sufficient for the renal agenesis phenotype, but not the limb phenotype of ld mutant mice. Structural mutations in this isoform are only one of several genetic mechanisms leading to the renal phenotype, since amino acid changes in this isoform were not detected. These results demonstrate that this gene is limb deformity, and that variable isoform expression may play a role in generating the pleiotropic ld phenotype.

Abnormalities, Multiple↗

PubMed1998

Isoform-specific regulation of the Na+/Ca2+ exchanger in rat astrocytes and neurons by PKA.

The Na+/Ca2+ exchanger is a major transporter of Ca2+ in neurons and glial cells. The Na+/Ca2+ exchanger gene NCX1 expresses tissue-specific isoforms of the Na+/Ca2+ exchanger, and the isoforms have been examined here quantitatively using primary cultures of astrocytes and neurons. We present a PCR-based quantitative method, quantitative end-labeled reverse transcription-PCR (QERT-PCR), to determine the relative amounts of the NCX1 isoforms present in these cells. Six exons (A, B, C, D, E, and F) are alternatively spliced to produce the known NCX1 isoforms. Three exon B-containing isoforms (BDEF, BDF, and BD) are the predominant transcripts in primary rat cortical astrocytes and in C6 glioma cells. In contrast, exon A-containing isoforms (ADF and AD) are the predominant transcripts in primary rat hippocampal neurons. Functional differences between full-length constructs of NCX1 containing either the astrocyte isoform BD or the neuron isoform AD were examined in a Xenopus oocyte expression system. Although both isoforms function normally, the activity of the AD isoform can be increased 39% by activation of protein kinase A (PKA), whereas that of the BD isoform is not affected. We conclude that specific NCX1 isoforms are expressed in distinct patterns in astrocytes and neurons. Furthermore, the activity of a neuronal (but not glial) isoform of the Na+/Ca2+ exchanger can be altered by the activation of the PKA pathway.

Animals↗

PubMed2025

Single-Cell Splicing Isoform Atlas of the Adult Human Heart and Heart Failure.

BACKGROUND: Alternative splicing plays crucial roles in normal heart development and cardiac disease by influencing protein-coding sequences, functional domains, and molecular networks. However, a detailed characterization of the human heart isoform landscape remains incomplete. METHODS: Leveraging long-read single-nucleus RNA sequencing and computational analysis, we dissected full-length isoform heterogeneities, expression patterns, and usage shifts across cell types, cell states, and cardiac conditions of the adult left ventricle. We applied in silico approaches to assess the functional relevance of identified isoforms; validated isoform compositions of representative cardiac genes using reverse transcription quantitative polymerase chain reaction and targeted amplicon sequencing; and developed a web server for interactive navigation of our results. RESULTS: The data revealed that isoform heterogeneity is widespread in the cardiac cellular system, serving as a posttranscriptional buffer mechanism that calibrates the molecule reservoirs in human hearts. In healthy left ventricles, ≈30% of cell type-specific genes were polyform, using multiple isoforms tailored to cell type-specific programs. Among ubiquitously expressed genes, >300 showed differential isoform usage with cell type specificity in normal hearts. Comparisons of cardiomyocytes across conditions uncovered 379 genes with marked isoform usage shifts, most of which are predicted to change protein coding outcomes through direct changes in protein coding sequences and switches between intron retention and non-protein-coding biotypes. In contrast, cell state-specific programs tend to operate on monoform genes associated with changes among cell states. In addition, our data revealed heart failure-associated differential isoform usage events in stromal and immune cell types in the cardiac microenvironment. CONCLUSIONS: We present a comprehensive atlas of splicing isoforms in the normal adult heart and heart failure through long-read single-nucleus RNA sequencing and computational analyses. The results suggest crucial roles of isoforms in buffering core cellular programs and contributing to disease-associated cell states. The full-length details of these cell-specific isoforms serve as an important reference for downstream translational and mechanistic studies and are available on our online data portal at https://github.com/gaolabtools/heart-isoform-atlas.

Humans↗

PubMed2003

Effect of overexpressing fibroblast growth factor 2 protein isoforms in osteoblastic ROS 17/2.8 cells.

Fibroblast growth factor-2 (FGF-2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF-2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF-2 protein that is exported from cells. Since FGF-2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF-2 as a novel means of assessing different FGF-2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb alpha1(I)-collagen 5' regulatory elements (Col 3.6). A set of expression vectors, called Col3.6-Fgf2 isoforms-IRES-GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF-2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6-IRES-GFPsaph) versus each of the Col3.6-Fgf2-IRES-GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF-2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF-2 isoforms. A neutralizing FGF-2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF-2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF-2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF-2 proteins isoforms is important in modulating OB function.

Animals↗

PubMed2006

Fiber type-specific immunostaining of the Na+,K+-ATPase subunit isoforms in skeletal muscle: age-associated differential changes.

The expression of the Na(+),K(+)-ATPase alpha and beta subunit isoforms in rat skeletal muscle and its age-associated changes have been shown to be muscle-type dependent. The cellular basis underlying these findings is not completely understood. In this study, we examined the expression of Na(+),K(+)-ATPase isoforms in individual fiber types and tested the hypothesis that, with age, the changes in the expression of the isoforms differ among individual fibers. We utilized immunohistochemical techniques to examine the expression of the subunit isoforms at the individual fiber levels. Immunofluorescence staining of the subunit isoforms in both white gastrocnemius (GW) and red gastrocnemius (GR) revealed a predominance of staining on the sarcolemmal membrane. Compared to the skeletal muscle of 6-month-old rats, there were substantial increases in the levels of alpha1, beta1, and beta3 subunit isoforms, and decreases in the levels of alpha2 and beta2 in 30-month-old rats. In addition, we found distinct patterns of staining for the alpha1, alpha2, beta1, and beta2 isoforms in tissue sections from young and aged rats. Muscle fiber-typing was performed to correlate the pattern of staining with specific fiber types. Staining for alpha1 and alpha2 isoforms in the skeletal muscle of young rats was generally evenly distributed among the fibers of GW and GR, with the exception of higher alpha1 levels in slow-twitch oxidative Type I fibers of GR. By contrast, staining for the beta1 and beta2 isoforms in the mostly oxidative fibers and the mostly glycolytic fibers, respectively, was almost mutually exclusive. With age, there was a fiber-type selective qualitative decrease of alpha2 and beta2 in Type IIB fibers, and increase of beta1 in Type IIB fibers and beta2 in Type IID fibers of white gastrocnemius. These results provide, at the individual fiber level, a cellular basis for the differential expression of the Na(+),K(+)-ATPase subunit isoforms in the muscle groups. The data further indicate that the aged-associated changes in expression of the subunit isoforms occur in both a fiber-type specific as well as an across fiber-type manner. Because of the differing biochemical properties of the subunit isoforms, these changes add another layer of complexity in our understanding of the adaptation of the Na-pump in skeletal muscle with advancing age.

Age Factors↗

PubMed2006

Characterization of protein kinase C isoforms in primary cultured cerebellar granule cells.

Protein kinase C (PKC) is a family of serine/threonine kinases comprised of 10 isoforms. Although commercial antibodies are available for all 10 isoforms, the specificity of these antibodies has been questioned. We have identified immunoblot conditions in which commercially purchased PKC antibodies are specific for their respective isoform. We then used these conditions to determine that PKC isoforms alpha, betaI, betaII, delta, epsilon, gamma, lambda, theta, and zeta are present in rat primary cultured cerebellar granule cells (CGCs) 6-14 days in vitro (DIV). This PKC profile is identical to that observed in cerebellar homogenates taken from 6-, 14- and 21-day-old rats. Western blot analysis indicated that the classical and the atypical PKC isoforms were more prevalent in the cytosolic subcellular fraction compared to the particulate fraction under basal conditions. Immunoreactivity for the novel isoforms tended to be higher in the particulate fraction under basal conditions. Phorbol 12-myristate 13-acetate (PMA) treatment resulted in translocated immunoreactivity from the cytosolic to the particulate fraction for all of the classical and novel PKC isoforms, but not for the atypical isoforms. However, the degree of translocation as well as the speed of translocation varied among the isoforms. The stability of the individual isoforms after PMA-induced activation also varied among the isoforms. Differences in these parameters were dependent upon culture batches and PKC isoform groups. We have identified experimental conditions in which reproducible results can be obtained with primary cultured CGCs in the study of PKC. We discuss possible solutions for problems encountered when utilizing primary cultured neurons to study PKC-mediated signal transduction.

Animals↗