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PubMed2000

Three subunit a isoforms of mouse vacuolar H(+)-ATPase. Preferential expression of the a3 isoform during osteoclast differentiation.

Vacuolar H(+)-ATPase (V-ATPase) is a multi-subunit enzyme with a membrane peripheral catalytic (V(1)) and an intrinsic (V(o)) sector. We have identified three cDNA clones coding for isoforms of mouse V(o) subunit a (a1, a2, and a3). They exhibit 48-52% identity with each other and high similarity to subunit a of other species. The a1 isoform was mainly expressed in brain and liver. The a2 isoform was observed in heart and kidney in addition to brain and liver. Transcripts for the a3 isoform were strongly expressed in heart and liver. The a3 isoform was induced during osteoclast differentiation, and localized in the plasma membrane and cytoplasmic filamentous structures. In contrast to a3, the a1 isoform was constitutively expressed and localized in the cytoplasmic endomembrane compartments of the same cells. These findings suggest that the a3 isoform is a component of the plasma membrane V-ATPase essential for bone resorption.

Amino Acid Sequence↗
PubMed1993

Human fibroblast tropomyosin isoforms: characterization of cDNA clones and analysis of tropomyosin isoform expression in human tissues and in normal and transformed cells.

A tropomyosin-specific oligonucleotide probe (REN29) designed to hybridize to all known human tropomyosin isoforms was used to study tropomyosin mRNA levels in normal and transformed human cells. At least four different sizes of RNAs were detected in normal human fibroblast KD cells by Northern blot analysis. The major bands of 1.1 kb RNA for hTM1 and 3.0 kb RNA for hTM4 were decreased substantially in various transformed cell lines. One of the minor RNA bands (2.0 kb for hTM2 and hTM3) appeared to be absent in a human pancreatic carcinoma cell line. The level of the other minor RNA band (2.5 kb for hTM5) was found to be unchanged or slightly decreased in transformed cells. This differential expression of tropomyosin isoforms at the RNA level was not totally in agreement with the difference in the protein amounts found in normal and transformed cells, suggesting that translational control may also play an important role in the expression of some tropomyosin isoforms. The REN29 probe was further used to screen lambda gt10 and lambda gt11 cDNA libraries, which were constructed from poly(A)+ RNAs of human fibroblast cell lines HuT-14 and WI-38, respectively. In addition to cDNA clones encoding known isoforms, we obtained three classes of new cDNA clones that encode two low M(r) isoforms (hTM5a and hTM5b), and a high M(r) isoform (hTMsm alpha). Sequence comparison revealed that hTM5a and hTM5b are alternatively spliced products derived from the same gene that encodes hTM2 and hTM3. Northern blot analysis and amino acid sequence comparison suggested that the hTMsm alpha represents a smooth muscle tropomyosin which is also expressed in human fibroblasts. The exon specific for, and common to, hTM5a and hTM5b was found to be highly expressed in small intestine. However, there was no detectable expression of this exon in stomach and skeletal muscle. The difference in tissue-specific expression suggests that different isoforms may perform distinct functions in different tissues.

Amino Acid Sequence↗
PubMed1991

Organ-specific occurrence and expression of the isoforms of nonspecific lipid transfer protein in castor bean seedlings, and molecular cloning of a full-length cDNA for a cotyledon-specific isoform.

Four kinds of nonspecific lipid transfer proteins (nsLTP) were purified from different organs of castor bean (Ricinus communis L.) seedlings. Amino acid compositions and amino-terminal sequences of the four nsLTPs were determined and compared with those of castor bean isoforms, nsLTP-A, -B, and -C, previously reported [Takishima et al. (1986) Biochim. Biophys. Acta 870, 248-255; Takishima et al. (1988) Eur. J. Biochem. 177, 241-249]. Two isoforms from the cotyledons were identified as nsLTP-A and -C, one isoform from the endosperms as nsLTP-B, and the other was a new isoform from the axes. This new isoform was named nsLTP-D and its amino acid sequence was determined. These results demonstrated organ-specific occurrence of the nsLTP isoforms in castor bean seedlings. The isoforms nsLTP-A, -B, -C, and -D showed similar transfer activity not only for phosphatidylcholine and phosphatidylethanolamine but also for monogalactosyldiacylglycerol, although the homology among their amino acid sequences ranged from 70 to 30%. Two cDNA clones (pnsLTP-C and pnsLTP-D) for nsLTPs of castor bean seedlings were isolated and sequenced. pnsLTP-C was the cDNA clone for nsLTP-C expressed in the cotyledons, and pnsLTP-D was that for nsLTP-D in the axis. A coupled in vitro transcription-translation analysis of both cDNA clones revealed that pnsLTP-C encodes the full-length of nsLTP-C precursor (pro-nsLTP-C), while pnsLTP-D encodes a part of nsLTP-D precursor. PronsLTP-C contained a 24-amino acid pre-sequence preceding the mature nsLTP-C (92 amino acids).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in mouse tissues: male-specific isoforms are expressed in the gonads and liver.

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta HSD) are expressed in various mouse tissues in a tissue-specific, sex-specific, and developmental manner. Three distinct immunoreactive species [molecular masses, 47, 44, and 42 kilodaltons (kDa)] are detectable by Western immunoblot analysis using a 3 beta HSD antiserum. Different immunoreactive isoforms are expressed in steroidogenic (44 and 47 kDa in gonads) and nonsteroidogenic (42 and 47 kDa in liver and kidney) tissues. Two of these isoforms are sex-specific in the gonads (47 kDa) and liver (42 kDa), because they are detectable only in male mice. Sex-specific expression in the liver is developmentally regulated. Low levels of this male-specific hepatic isoform are first detectable at 23-25 days of age, but its level of expression increases progressively during sexual maturation to adult levels. NAD(+)-dependent 3 beta HSD activity is detectable in homogenates of all tissues examined, but the kinetic characteristics of this activity differ among tissues and are sexually dimorphic in the liver. Apparent Michaelis constants for dehydroepiandrosterone are much lower in steroidogenic (0.24 +/- 0.07 microM for testis) than in nonsteroidogenic (range, 10-100 microM for liver and kidney) tissues and are lower in male mouse liver (16 +/- 1 microM) than in female mouse liver (82 +/- 20 microM). Oligonucleotides with unique sequences but encoding homologous regions of the mouse type I, II, and III 3 beta HSD cDNAs were used for Northern blot analyses. A type I oligomer hybridizes with RNA from steroidogenic (adrenal, ovary, and testis) tissues, and a type III oligomer hybridizes with RNA from nonsteroidogenic (liver and kidney) tissues. A type II oligomer, however, hybridizes specifically with RNA from testis and liver of male mice, tissues that express a male-specific 3 beta HSD. These results suggest that type II-like transcripts may encode a 47-kDa sex-specific 3 beta HSD in testis and a 42-kDa sex-specific 3 beta HSD in liver of male mice. It is unclear how many members of subfamilies of the 3 beta HSD gene family will be discovered. The mouse may prove to be a valuable experimental model, as this is the first species in which multiple immunoreactive isoforms can be identified in a single tissue. This multiplicity makes it difficult to correlate the size and number of immunoreactive isoforms with the diverse kinetic characteristics of NAD(+)-dependent 3 beta HSD activities in tissue homogenates and to develop isoform-specific probes.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed2005

Tissue expression of alternatively spliced GFRalpha1, NCAM and RET isoforms and the distinct functional consequence of ligand-induced activation of GFRalpha1 isoforms.

Glial-cell-line-derived neurotrophic factor (GDNF) exerts its effect through a multi-component receptor system consisting of GFRalpha1, RET and NCAM. Two highly homologous alternatively spliced GFRalpha1 isoforms (GFRalpha1a and GFRalpha1b) have previously been identified. In this study, isoform specific real-time PCR assays were used to quantify the expression levels of GFRalpha1, RET and NCAM isoforms in murine embryonic and adult tissues. The expression levels of GFRalpha1b were found to be comparable to that of GFRalpha1a in peripheral tissues. However, GFRalpha1a was the predominant isoform expressed in the whole brain. The co-expressions of GFRalpha1 and the co-receptors were developmentally regulated and differentially expressed in some tissues. Microarray analyses of GFRalpha1 isoforms transfected cells stimulated with NTN showed distinct and non-overlapping gene profiles. These observations are consistent with the emerging view that the combinatorial interactions of the spliced isoforms of GFRalpha, RET and NCAM may contribute to the pleiotropic biological responses.

Alternative Splicing↗
PubMed2006

Autoimmunity against human tropomyosin isoforms in ulcerative colitis: localization of specific human tropomyosin isoforms in the intestine and extraintestinal organs.

BACKGROUND: Tropomyosins (TMs) are microfilament cytoskeletal proteins, and 5 major human TM isoforms (hTM1-5) are described. hTMs, particularly isoform 5 (hTM5), is capable of inducing autoantibodies and T-cell response in ulcerative colitis (UC). However, cellular localization of hTM isoforms in the colon and in extraintestinal organs commonly involved in UC is unknown. METHODS: Using isoform-specific monoclonal antibodies, we localized hTMs through immunoperoxidase assay in normal colon (n = 12), small intestine (n = 14), esophagus (n = 10), skin (n = 19), eye (n = 12), gallbladder (n = 16), liver, including bile duct at the porta hepatis (n = 4), lungs (n = 4), and pancreas (n = 4). RESULTS: There is intense expression of hTM5, but not other isoforms, in the epithelium of the colon, gallbladder, and skin. In the eye, hTM5 is expressed only in the nonpigmented ciliary epithelium. Although extrahepatic and interlobar large ductal biliary epithelium was positive, bile canaliculi at the portal tract are negative. The immunoreactivity in epithelial cells from these organs is diffuse cytoplasmic and along the periphery. In colon epithelium, there is intense expression along basolateral areas and luminal (apical) surface. In the small intestinal epithelium, however, hTM5 expression is weak and distinctly different than in the colon. hTM5 was not detected in the squamous epithelium of the esophagus, although it was strongly positive in the skin. hTM1, hTM2, and hTM3 are localized predominantly in smooth muscle of the intestine and blood vessel wall but not the epithelium. HTM4 is localized in the endothelial cells and basement membrane of the colonic epithelium. CONCLUSIONS: hTM5 is the predominant isoform in the epithelium of colon and extraintestinal organs commonly involved in UC. The unique expression of hTM5 may allow its interaction with effector immune cells involved in the immunopathogenesis of UC and its extraintestinal manifestations.

Antibodies, Monoclonal↗
PubMed2004

Genomic organization of mouse ZAKI-4 gene that encodes ZAKI-4 alpha and beta isoforms, endogenous calcineurin inhibitors, and changes in the expression of these isoforms by thyroid hormone in adult mouse brain and heart.

OBJECTIVE: ZAKI-4 was identified as a thyroid hormone-responsive gene in cultured human fibroblasts. A single ZAKI-4 gene encodes two isoforms, ZAKI-4 alpha and beta, both inhibiting calcineurin activity. ZAKI-4 alpha and beta differ at their N termini, and show distinct distribution profiles in human tissues. The aim of this study was to elucidate the organization of the mouse ZAKI-4 gene and to determine the effect of thyroid hormone on the expression of ZAKI-4 isoforms in vivo. DESIGN: We cloned mouse homologues of human ZAKI-4 alpha and beta cDNA. Fluorescence in situ hybridization and bioinformatics analysis were employed to determine the gene organization. The effect of thyroid hormone on the expression of ZAKI-4 isoforms in mouse brain and heart was also studied. METHODS: Total RNA extracted from mouse cerebellum was used to clone ZAKI-4 alpha and beta cDNAs by RT-PCR followed by rapid amplification of cDNA ends. Mice were rendered hypothyroid by feeding a low iodine diet supplemented with propylthiouracil for 2 weeks. In one group (hyperthyroid) L-T(3) was injected i.p. for the last 4 days whereas another group (hypothyroid) received vehicle only. Non-treated mice were controls. RESULTS AND CONCLUSION: Mouse ZAKI-4 alpha and beta cDNAs were highly homologous to the human isoforms. The gene was mapped on chromosome 17qC, syntenic to human chromosome 6 where the human ZAKI-4 gene is located. As observed in human, ZAKI-4 alpha mRNA was expressed only in brain whereas beta mRNA was distributed in other tissues as well, such as heart and skeletal muscle. ZAKI-4 alpha mRNA was lower in the cerebral cortex of hypothyroid mice. Injection of L-T(3) caused an increase in ZAKI-4 beta mRNA in heart; however, expression of neither ZAKI-4 alpha nor beta mRNA was influenced by thyroid status in other tissues. These results indicate that expression of ZAKI-4 alpha and beta isoforms is regulated by thyroid hormone in vivo, and the regulation is isoform- and tissue-specific.

Amino Acid Sequence↗
PubMed1996

Protein kinase C isoform expression during the differentiation of 3T3-L1 preadipocytes: loss of protein kinase C-alpha isoform correlates with loss of phorbol 12-myristate 13-acetate activation of nuclear factor kappaB and acquisition of the adipocyte phenotype.

The regulated expression of protein kinase C (PKC) isoforms was examined during the differentiation program of 3T3-L1 preadipocytes. In a parallel analysis, differentiation was blocked by treatment of the cells with tumor necrosis factor-alpha (TNF) to determine differentiation-specific changes in isoform expression from growth or treatment-induced effects. This analysis revealed that the expression of the conventional PKC-alpha isoform was reduced by 85% as cells attained the adipocyte phenotype. PKC-beta expression was measurable only during the early stages of the differentiation process and was not detectable in fully differentiated cells. An upregulation of PKC-theta, a novel PKC isoform, occurred during the latter stage of differentiation. Expression of PKC-zeta an atypical PKC isoform suggested to participate in TNF signal transduction, occurred throughout the time course with similar levels of expression in both preadipocytes and adipocytes. Nuclear run-on analysis demonstrated an approximately 85% reduction in the transcription of the PKC-alpha gene during differentiation. The reduced expression of this isoform corresponded with the decreased ability to activate nuclear factor kapppaB (NF-kappaB) in response to phorbol 12-myristate 13-acetate (PMA) treatment in the adipocytes. These data suggest that PMA responsiveness in 3T3-L1 adipocytes is markedly diminished.

Adipocytes↗
PubMed1994

Cloning and expression of fibroblast growth factor receptor-1 isoforms in the mouse heart: evidence for isoform switching during heart development.

Basic (b) fibroblast growth factor (FGF) mediates various biological responses including mitogenesis and angiogenesis by binding to specific cell surface receptors of the tyrosine kinase family. The bFGF receptor-1 FGFR1) exists in short and long isoforms due to alternate RNA splicing. Minor alterations in the amino acid sequence have also led to reports of different FGFR1 isoforms in different tissues even in the same species. In the absence of any sequence for heart FGFR1 and accumulating evidence for a role of bFGF in heart growth and differentiation, we cloned FGFR1 from embryonic mouse hearts. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to generate full-length short (2259 base pairs) and long (2526 base pairs) forms of FGFR1 cDNAs which generated 86 and 102 kDa proteins, respectively, following in vitro translation. Embryonic mouse heart FGFR1 differed by seven amino acids from the reported sequence for mouse neuroepithelial FGFR1 and appeared more similar to human placental FGFR1. A single FGFR1 transcript of approximately 4.3 kb was seen in RNA isolated from embryonic as well as adult mouse hearts. There was a decrease (approximately 8.5-fold) in FGFR1 RNA levels in the adult. The majority of FGFR1 transcripts in the adult as well as embryonic heart contained exon IIIc (FGFR1-IIIc) which is associated with isoforms that display the highest affinity for bFGF. However, the relative ratio of short versus long FGFR1 RNA expression was 0.5 in the embryonic heart compared to 5.9 in the adult heart. These results indicate that: (i) structurally distinct short and long FGFR1 isoform RNAs are expressed in the embryonic and adult heart; (ii) FGFR1-IIIc is the major form of receptor expressed in the embryonic as well as adult heart; (iii) the transition from the embryo to the adult stage is associated with a decrease but not absence of FGFR1 RNA expression; and (iv) long FGFR1-isoforms are more abundant in the embryo while short FGFR1 isoforms predominate in the adult.

Amino Acid Sequence↗
PubMed1998

LAR tyrosine phosphatase receptor: a developmental isoform is present in neurites and growth cones and its expression is regional- and cell-specific.

Transgenic mice and Drosophila mutant studies demonstrate that the leukocyte common antigen-related (LAR) protein tyrosine phosphatase (PTPase) receptor is required for formation of neural networks. We assessed the hypothesis that alternative splicing of the LAR extracellular region contributes to this function by establishing temporospatial expression patterns of LAR isoforms containing an alternatively spliced extracellular nine amino acid segment (LAR alternatively spliced element-c; LASE-c). LASE-c was present in multiple alternatively spliced and truncated LAR transcripts. In contrast to LAR isoforms without LASE-c, levels of LAR transcripts and protein isoforms containing LASE-c were primarily present during development, suggesting a mechanism for developmental regulation of LAR function. In situ analysis demonstrated increasingly region- and cell-specific expression of LASE-c during maturation. Immunostaining revealed LASE-c-containing LAR protein along neurites and in growth cones. The discovery of highly regulated, temporospatial extracellular domain alternative splicing of LAR-type PTPase receptors points to a novel mechanism by which these receptors might influence network formation.

Animals↗
PubMed1989

Chromosomal assignment of two myosin alkali light-chain genes encoding the ventricular/slow skeletal muscle isoform and the atrial/fetal muscle isoform (MYL3, MYL4).

In all eukaryotes, myosin plays a major role in the maintenance of cell shape and in cellular movement; in association with actin and other contractile proteins it is also a major structural component of the muscle sarcomere. Several isoforms of myosin alkali light chain have been identified, associated with different muscle types. We have recently localized the gene encoding the fast skeletal muscle alkali light-chain isoforms MLC1F and MLC3F (HGM symbol, MYL1) to human chromosome 2q32.1-qter (Cohen-Haguenauer 1988). We present here the chromosomal assignment of two loci encoding the ventricular muscle isoform MLC1V (equivalent to the slow skeletal muscle isoform MLC1Sb) and the atrial muscle isoform MLC1A (equivalent to the fetal isoform MLC1emb) using a panel of 25 independent man-rodent somatic cell hybrids. The MLC1V gene (HGM symbol, MYL3) was mapped to human chromosome 3 using a human full-length cDNA probe that hybridizes to a single major human TaqI2.8-kb fragment. The MLC1A probe (HGM symbol, MYL4) was a 360-bp mouse cDNA fragment that gave a distinct signal with human DNA using low stringency conditions of hybridization and washings and after presaturation of the Southern blots with rodent DNA. A single PstI 7.8-kb fragment gives an intense signal, and its presence correlates with the presence of chromosome 17 among the hybrids. These data are in keeping with the localizations of the MLC1V gene to mouse chromosome 9, and of the MLC1A gene to mouse chromosome 11, which share some markers in common with human chromosomes 3 and 17 respectively.

Animals↗
PubMed1995

Regulation of the inducible soybean nitrate reductase isoform in mutants lacking constitutive isoform(s).

In soybean, three nitrate reductase isoforms have been identified based on metabolic regulation, substrate specificity, and kinetic parameters. Two isoforms have been termed constitutive, as their activities are present without the addition exogenous nitrate to soybean seedlings. The third activity is termed inducible, as its activity is present only when soybean plants have been supplied with nitrate. The purpose of this study was to examine the regulation of the inducible nitrate reductase isoform in soybean mutants lacking one or both of the constitutive isoforms. Based on evidence obtained through measurements of enzyme activity, Western blotting, and RNA determinations, the absence of one or both of the constitutive nitrate reductase isoforms has no effect on the metabolite regulation of the inducible nitrate reductase isoform.

Enzyme Induction↗
PubMed2004

Up-regulation of Na(+),K(+)-ATPase alpha 3-isoform and down-regulation of the alpha1-isoform in human colorectal cancer.

We investigated expression levels of Na(+),K(+)-ATPase alpha-isoforms and their ATPase activities in human colorectal cancer tissue and the accompanying normal mucosa. A decrease in expression of the alpha1-isoform protein was observed in all sampled cancer tissues compared with the normal mucosae. The level of ouabain (5 microM)-sensitive Na(+),K(+)-ATPase activity in carcinomas was 81+/-5% that of in the normal mucosae. The mRNA expression of alpha2- and alpha 4-isoforms was decreased in almost all the carcinoma samples. Interestingly, the expression level of the alpha 3-isoform protein in the cancer tissue was higher than that of the normal mucosa. These results indicate that a decrease in the alpha1-isoform expression and an increase in the alpha 3-isoform expression may be associated with colorectal cancer.

Asian People↗
PubMed1997

Distribution of inositol 1,4,5-trisphosphate receptor isoforms, SERCA isoforms and Ca2+ binding proteins in RBL-2H3 rat basophilic leukemia cells.

RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) isoforms (2b and 3) as well as four Ca2+ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP3 binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca2+ binding proteins in fraction F3 and the sucrose density gradient fractions. InsP3R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP3R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP3R-1 and InsP3R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP3R-1 and InsP3R-2 and the SERCAs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP3R isoforms, the two SERCA isoforms and the four Ca2+ binding proteins investigated. This heterogeneity may underlie specialization of the Ca2+ stores and the subsequent initiation of intracellular Ca2+ signals.

Animals↗
PubMedDate not supplied

Displaced starburst amacrine cells of the rabbit retina contain the 67-kDa isoform, but not the 65-kDa isoform, of glutamate decarboxylase.

The cholinergic identity of retinal starburst amacrine neurons is well established, but recent evidence suggests that these cells are GABAergic as well. Confirmation of this dual transmitter function requires the demonstration of glutamate decarboxylase (GAD), the biosynthetic enzyme for GABA, within starburst cells. The current work was undertaken to determine whether rabbit retinal starburst amacrine neurons contain either of the two known isoforms of GAD. To do this, we have examined the localization of the following: (1) the 65-kDa isoform of GAD; (2) the 67-kDa isoform of GAD; (3) choline acetyltransferase; and (4) the fluorescent dye DAPI, a marker for cholinergic amacrine cells. In addition, we labeled displaced starburst neurons directly, by injecting them with Lucifer Yellow in vitro. Four strata within the inner plexiform layer contained immunoreactive GAD65. A non-GAD65-immunoreactive zone separated the two innermost strata (G3 and G4); this zone contained (1) the dendrites of individual Lucifer Yellow-injected, displaced starburst amacrine cells; (2) dendrites immunoreactive for choline acetyltransferase; and (3) processes of DAPI-labeled amacrine cells. Immunoreactive GAD67 appeared in the same strata that contained GAD65, and in at least two additional strata, one of which lay at precisely the same depth as the proximal cholinergic stratum. In addition, the somas of displaced starburst cells were strongly immunoreactive for GAD67, but not for GAD65. These results demonstrate (1) that displaced starburst amacrine cells contain the 67-kDa isoform of GAD, but not the 65-kDa isoform; and (2) that the dendrites of starburst (67-kDa GAD) amacrines, and the dendrites of 65-kDa-GAD-containing amacrines, occupy different strata within the inner plexiform layer. Thus, displaced starburst cells do contain GAD, and can, presumably, manufacture GABA. The reasons for their preferential use of the 67-kDa GAD isoform remain to be elucidated.

Animals↗
PubMed1996

Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies.

The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51-75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156-1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719-738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, we could identify both hPMCA4a and hPMCA4b. Heart tissue also showed isoform 4b, and probably 4a. In contrast, kidney and lung showed primarily hPMCA4b. In brain, overlapping bands that did not correspond to either variant of hPMCA4 were detected, and in kidney a band migrating in the same position as hPMCA1b was observed. The distribution of the a and b forms of hPMCA4 at the protein level, as analysed by these antibodies, is consistent with the available data about the abundance of mRNAs for the hPMCA isoforms. The presence of hPMCA4b in all the samples supports the proposed role of this isoenzyme as a constitutive form of the pump.

Alternative Splicing↗
PubMed2000

Properties of three isoforms of the 116-kDa subunit of vacuolar H+-ATPase from a single vertebrate species. Cloning, gene expression and protein characterization of functionally distinct isoforms in Gallus gallus.

Vacuolar H+-ATPases (V-ATPases) are involved in a wide variety of essential cellular processes. An unresolved question is how the cell regulates the activity of these proton pumps and their targeting to distinct cellular compartments. There is growing evidence for the presence of subunit diversity amongst V-pumps, particularly regarding the 116-kDa subunit (called the a subunit). We have cloned and characterized three isoforms (a1, a2 and a3) of this subunit from chicken. The amino-acid sequences of these homologues are approximately 50% similar and their nucleotide differences indicate that they are products of distinct genes. The levels of mRNA expression of these isoforms was quantified by ribonuclease protection analysis. The a1 and a2 isoforms have a similar tissue distribution, with the highest level of mRNA expression in brain, an intermediate level in kidney and relatively low levels in liver and bone. In contrast, the highest level of expression of the a3 isoform is in bone and liver, with a moderate level in kidney, and the lowest level in brain. An antibody against the a1 isoform reacted with a 116 kDa protein in a brain V-ATPase preparation that was not detected in bone or liver V-ATPase preparations, whereas an antibody against the a3 isoform reacted with a 116-kDa peptide in bone and liver, but not brain V-ATPases preparations. The bone and brain V-ATPases showed differential sensitivity to the inhibitors bafilomycin and (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-[4-(2, 2,6,6-tetramethyl)piperidinyl]-2,4-pentadienamide. Thus, this work demonstrates the presence of structurally and functionally distinct V-ATPases in a single vertebrate species.

Amino Acid Sequence↗
PubMed1995

The B isoform of the insulin receptor signals more efficiently than the A isoform in HepG2 cells.

We have demonstrated previously that dexamethasone treatment of HepG2 cells caused an enhancement of insulin's metabolic effects (Kosaki, A., and Webster, N. J. (1993) J. Biol. Chem. 268, 21990-21996). This correlated with increased expression of the mRNA encoding the B isoform of the insulin receptor (IR). In the present study, we have demonstrated that dexamethasone treatment caused in addition an enhancement in insulin-stimulated immediate-early gene expression (c-fos and egr-1). Dexamethasone treatment caused an increase in in vivo IR autophosphorylation and insulin receptor substrate-1 (IRS-1) phosphorylation both early events in the insulin signaling pathway. Furthermore, the IRS-1 phosphorylation was distinctly left shifted, although the level of IRS-1 protein was unchanged. Total cellular tyrosine phosphatase activity was unaltered when assayed with 32P-labeled IR and IRS-1. Studies in vitro on wheat-germ agglutinin-purified receptors showed that the B isoform of the IR had increased kinase activity both toward itself and the exogenous substrates poly.glu4:tyr1 and recombinant IRS-1 protein. In addition, two-dimensional tryptic phosphopeptide maps indicated that the B isoform has an additional phosphopeptide that is not seen for the A isoform. In conclusion, it appears that the B isoform of the IR signals more efficiently than the A isoform in HepG2 cells.

Base Sequence↗