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PubMed1999

Neurofibrillary degeneration in progressive supranuclear palsy and corticobasal degeneration: tau pathologies with exclusively "exon 10" isoforms.

Pathological tau proteins that constitute the basic matrix of neuronal inclusions observed in numerous neurodegenerative disorders are disease specific. This is mainly the consequence of the aggregation of specific sets of tau isoforms according to the diseases, i.e., six isoforms in Alzheimer's disease (AD) and exclusively the three tau isoforms lacking the corresponding sequence of exon 10 (E10-) in Pick's disease (PiD). By using antibodies specific to the different tau isoforms and one- and two-dimensional gel electrophoresis followed by western blots, we demonstrate herein a third group of neurodegenerative disorders characterized by intraneuronal inclusions exclusively constituted of tau isoforms containing the sequence corresponding to exon 10, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Together, tau isoforms with exon 10 clearly differentiate three groups of neurodegenerative diseases: AD, PiD, and PSP/CBD. For each group, the neuropathological and clinical phenotypes are most likely related to specific sets of tau isoforms expressed by the vulnerable neuronal populations. The recently described mutations of the tau gene responsible for familial frontotemporal dementias also support this hypothesis.

Alzheimer Disease↗
PubMed1999

Selective decrease of mRNAs encoding plasma membrane calcium pump isoforms 2 and 3 in rat kidney.

BACKGROUND: Although the existence of multiple isoforms of plasma membrane calcium ATPase (PMCA) is now well documented, their biological functions are not yet known. In this study, we set out to investigate the potential role of PMCA isoforms, previously identified in renal cortical tissue, in tubular reabsorption of calcium (Ca2+). METHODS: With use of reverse transcription-polymerase chain reaction analysis, we determined levels of mRNAs encoding isoforms of PMCA1 through PMCA4 in renal cortex, liver, and brain of rats with hypercalciuria induced by feeding with a low-phosphate diet (LPD) as compared with Ca2+-retaining rats that were fed a high-phosphate diet (HPD). RESULTS: We observed that in hypercalciuric LPD-fed rats, the mRNAs encoding isoforms PMCA2b and PMCA3(a + c) are significantly lower (Delta approximately-50%) than in HPD-fed hypocalciuric rats, whereas no changes in mRNAs encoding isoforms PMCA1b and PMCA4 were observed, and mRNA encoding calbindin 28 kDa was increased. On the other hand, the content of mRNAs encoding PMCA2b and PMCA3(a + c) in liver and brain, respectively, was not changed. CONCLUSION: These findings are evidence that expression of PMCA isoforms in the kidney can be selectively modulated in response to pathophysiologic stimuli. The association of a decrease in mRNA encoding PMCA2b and PMCA3(a + c) with hypercalciuria suggests that the two PMCA isoforms may be operant in tubular reabsorption of Ca2+ and its regulation.

Animals↗
PubMed2001

Effect of chronic renal failure on bone turnover and bone alkaline phosphatase isoforms.

BACKGROUND: Biochemical markers of bone turnover are used to monitor metabolic bone disease associated with renal failure. We have applied a comprehensive panel of markers to patients with chronic renal failure (CRF), with particular focus on the isoforms of bone alkaline phosphatase (BALP). METHODS: Twenty CRF patients undergoing hemodialysis (N = 9) and peritoneal dialysis (N = 11) were measured for serum parathyroid hormone (PTH), osteocalcin, total ALP, and four BALP isoforms (B/I, B1x, B1, and B2) by high-performance liquid chromatography. These BALP isoforms were also compared with BALP measured by three commercial immunoassays (Alkphase-B, Tandem-R Ostase, and Tandem-MP Ostase). Type I collagen turnover was assessed by serum samples using the type I procollagen intact amino- and carboxy-terminal propeptides (PINP and PICP) and two fragments (ICTP and CrossLaps) derived from the carboxy-terminal telopeptide of mature matrix collagen by different degradative pathways. RESULTS: Mean levels of bone turnover markers were elevated in CRF, with marked increases in those markers, osteocalcin, ICTP, and CrossLaps, cleared by the kidney. Total ALP activities were increased corresponding to elevated B/I and B2 isoform levels. The B1 isoform level was not significantly different from healthy controls. B1x was detected in 60% of the patients but was not resolved in healthy individuals. Kendall's tau rank correlation showed that B1x correlated significantly (P < 0.05) with B1 (0.53) and PINP (0.55), and was the only marker to correlate with PTH (0.49). B1x was not significantly correlated with any of the commercial BALP immunoassays. Interestingly, the immunoassay calibrators contained high activities of the B/I peak (39 to 80%) compared with human serum (4%). CONCLUSION: There are selective differences between the BALP isoforms in CRF compared with healthy adults. The commercial BALP immunoassays are comparable with each other but are unable to distinguish the BALP isoform-specific differences in CRF patients.

Adult↗
PubMed2002

Isolation and sequencing of a novel tropomyosin isoform preferentially associated with colon cancer.

BACKGROUND & AIMS: Nonmuscle human tropomyosin (hTM) isoforms have distinct functions and may play important roles in various disease processes. METHODS: In an attempt to identify colon epithelial tropomyosin isoform, a complementary DNA library prepared from a human colon cancer cell line T84 was screened by an oligonucleotide probe complementary to messages of all known hTM isoforms. A novel clone called TC22 was obtained. The amino acid sequence of TC22 isoform is identical to isoform 5 (hTM5) apart from the C terminal domain, amino acids 222-247 coding the exon 9. RESULTS: Northern blot analysis showed that TC22 message is expressed in transformed epithelial cell lines and tumor tissues but not in normal epithelial cells. We developed a monoclonal antibody specific to TC22 isoform (TC22-4). By Western blot and immunoperoxidase assays, we analyzed 105 colonic specimens (fresh frozen and formalin fixed) from 96 patients with colon polyps (hyperplastic) or adenomas with or without dysplasia and cancer. Twenty-one of 22 (95%) of colon cancer specimens showed the presence of TC22, compared with only 1 of the 17 normal colon specimens and none of the 13 hyperplastic polyps (P < 0.0001). As assayed by immunoperoxidase staining, TC22 expression progressively increased in benign adenomatous polyps (35%) and polyps with mild and severe dysplasia (57% and 100%, respectively). CONCLUSIONS: We cloned and sequenced a novel hTM isoform, TC22, which is strongly associated with colonic neoplasia and carcinoma. TC22 may provide a useful biomarker for surveillance of colon cancer.

Adenoma↗
PubMed2002

Characterization of protein kinase C beta isoform's action on retinoblastoma protein phosphorylation, vascular endothelial growth factor-induced endothelial cell proliferation, and retinal neovascularization.

Retinal neovascularization is a major cause of blindness and requires the activities of several signaling pathways and multiple cytokines. Activation of protein kinase C (PKC) enhances the angiogenic process and is involved in the signaling of vascular endothelial growth factor (VEGF). We have demonstrated a dramatic increase in the angiogenic response to oxygen-induced retinal ischemia in transgenic mice overexpressing PKC beta 2 isoform and a significant decrease in retinal neovascularization in PKC beta isoform null mice. The mitogenic action of VEGF, a potent hypoxia-induced angiogenic factor, was increased by 2-fold in retinal endothelial cells by the overexpression of PKC beta 1 or beta 2 isoforms and inhibited significantly by the overexpression of a dominant-negative PKC beta 2 isoform but not by the expression of PKC alpha, delta, and zeta isoforms. Association of PKC beta 2 isoform with retinoblastoma protein was discovered in retinal endothelial cells, and PKC beta 2 isoform increased retinoblastoma phosphorylation under basal and VEGF-stimulated conditions. The potential functional consequences of PKC beta-induced retinoblastoma phosphorylation could include enhanced E2 promoter binding factor transcriptional activity and increased VEGF-induced endothelial cell proliferation.

Animals↗
PubMed1997

Na,K-ATPase subunit isoforms in human reticulocytes: evidence from reverse transcription-PCR for the presence of alpha1, alpha3, beta2, beta3, and gamma.

The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium efflux mode between the human red blood cell Na,K-ATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoform-specific primers were used. The alpha1 and alpha3 isoforms of the alpha subunit, and the beta2 and beta3 isoforms of the beta subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found beta2 but not beta1 in reticulocyte single-stranded cDNA, and beta1 but not beta2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that alpha1, alpha2, and alpha3 as well as all three beta isoforms were present. The extent to which the kinetic properties of uncoupled sodium efflux might depend on different isoform combinations is not yet known.

Base Sequence↗
PubMed1997

Na+ pump low and high ouabain affinity alpha subunit isoforms are differently distributed in cells.

Three isoforms (alpha1, alpha2, and alpha3) of the catalytic (alpha) subunit of the plasma membrane (PM) Na+ pump have been identified in the tissues of birds and mammals. These isoforms differ in their affinities for ions and for the Na+ pump inhibitor, ouabain. In the rat, alpha1 has an unusually low affinity for ouabain. The PM of most rat cells contains both low (alpha1) and high (alpha2 or alpha3) ouabain affinity isoforms, but precise localization of specific isoforms, and their functional significance, are unknown. We employed high resolution immunocytochemical techniques to localize alpha subunit isoforms in primary cultured rat astrocytes, neurons, and arterial myocytes. Isoform alpha1 was ubiquitously distributed over the surfaces of these cells. In contrast, high ouabain affinity isoforms (alpha2 in astrocytes, alpha3 in neurons and myocytes) were confined to a reticular distribution within the PM that paralleled underlying endoplasmic or sarcoplasmic reticulum. This distribution is identical to that of the PM Na/Ca exchanger. This raises the possibility that alpha1 may regulate bulk cytosolic Na+, whereas alpha2 and alpha3 may regulate Na+ and, indirectly, Ca2+ in a restricted cytosolic space between the PM and reticulum. The high ouabain affinity Na+ pumps may thereby modulate reticulum Ca2+ content and Ca2+ signaling.

Animals↗
PubMed1998

Prominence of the dopamine D2 short isoform in dopaminergic pathways.

As a result of alternative splicing, the D2 gene of the dopamine receptor family exists in two isoforms. The D2 long is characterized by the insertion of 29 amino acids in the third cytoplasmic loop, which is absent in the short isoform. We have produced subtype-specific antibodies against both the D2 short and D2 long isoforms and found a unique compartmentalization between these two isoforms in the primate brain. The D2 short predominates in the cell bodies and projection axons of the dopaminergic cell groups of the mesencephalon and hypothalamus, whereas the D2 long is more strongly expressed by neurons in the striatum and nucleus accumbens, structures targeted by dopaminergic fibers. These results show that the splice variants of the dopamine D2 receptor are differentially distributed and possess distinct functions. The strategic localization of the D2 short isoform in dopaminergic cell bodies and axons strongly suggests that this isoform is the likely dopamine autoreceptor, whereas the D2 long isoform is primarily a postsynaptic receptor.

Alternative Splicing↗
PubMed1995

Casein kinase I gamma subfamily. Molecular cloning, expression, and characterization of three mammalian isoforms and complementation of defects in the Saccharomyces cerevisiae YCK genes.

Casein kinase I, one of the first protein kinases identified biochemically, is known to exist in multiple isoforms in mammals. Using a partial cDNA fragment corresponding to an isoform termed CK1 gamma, three full-length rat testis cDNAs were cloned that defined three separate members of this subfamily. The isoforms, designated CK1 gamma 1, CK1 gamma 2, and CK1 gamma 3, have predicted molecular masses of 43,000, 45,500, and 49,700. CK1 gamma 3 may also exist in an alternatively spliced form. The proteins are more than 90% identical to each other within the protein kinase domain but only 51-59% identical to other casein kinase I isoforms within this region. Messages for CK1 gamma 1 (2 kilobases (kb)), CK1 gamma 2 (1.5 and 2.4 kb), and CK1 gamma 3 (2.8 kb) were detected by Northern hybridization of testis RNA. Message for CK1 gamma 3 was also observed in brain, heart, kidney, lung, liver, and muscle whereas CK1 gamma 1 and CK1 gamma 2 messages were restricted to testis. All three CK1 gamma isoforms were expressed as active enzymes in Escherichia coli and partially purified. The enzymes phosphorylated typical in vitro casein kinase I substrates such as casein, phosvitin, and a synthetic peptide, D4. Phosphorylation of the D4 peptide was activated by heparin whereas phosphorylation of the protein substrates was inhibited. The known casein kinase I inhibitor CK1-7 also inhibited the CK1 gamma s although less effectively than the CK1 alpha or CK1 delta isoforms. All three CK1 gamma s underwent autophosphorylation when incubated with ATP and Mg2+. The YCK1 and YCK2 genes in Saccharomyces cerevisiae encode casein kinase I homologs, defects in which lead to aberrant morphology and growth arrest. Expression of mammalian CK1 gamma 1 or CK1 gamma 3 restored growth and normal morphology to a yeast mutant carrying a disruption of YCK1 and a temperature-sensitive allele of YCK2, suggesting overlap of function between the yeast Yck proteins and these CK1 isoforms.

Amino Acid Sequence↗
PubMed1996

Tissue-specific versus isoform-specific differences in cation activation kinetics of the Na,K-ATPase.

The experiments described in this report reconcile some of the apparent differences in isoform-specific kinetics of the Na,K-ATPase reported in earlier studies. Thus, tissue-specific differences in Na+ and K+ activation kinetics of Na,K-ATPase activity of the same species (rat) were observed when the same isoform was assayed in different tissues or cells. In the case of alpha1, alpha1-transfected HeLa cell, rat kidney, and axolemma membranes were compared. For alpha3, the ouabain-insensitive alpha3*-transfected HeLa cell (cf. Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930), pineal gland, and axolemma (mainly alpha3) membranes were compared. The order of apparent affinities for Na+ of alpha1 pumps was axolemma approximately rat alpha1-transfected HeLa > kidney, and for K+, kidney approximately alpha1-transfected HeLa > axolemma. For alpha3, the order of apparent affinities for Na+ was pineal gland approximately axolemma > alpha3*-transfected HeLa, and for K+, alpha3*-transfected HeLa > axolemma approximately pineal gland. In addition, the differences in apparent affinities for Na+ of either kidney alpha1 or HeLa alpha3* as compared to the same isoform in other tissues were even greater when the K+ concentration was increased. A kinetic analysis of the apparent affinities for Na+ as a function of K+ concentration indicates that isoform-specific as well as tissue-specific differences are related to the apparent affinities for both Na+ and K+, the latter acting as a competitive inhibitor at cytoplasmic Na+ activation sites. Although the nature of the tissue-specific modulation of K+/Na+ antagonism remains unknown, an analysis of the nature of the beta isoform associated with alpha1 or alpha3 using isoform-specific immunoprecipitation indicates that the presence of distinct beta subunits does not account for differences of alpha1 of kidney, axolemma, and HeLa, and of alpha3 of axolemma and HeLa; in both instances beta1 is the predominant beta isoform present or associated with either alpha1 or alpha3. However, a kinetic difference in K+/Na+ antagonism due to distinct betas may apply to alpha3 of axolemma (alpha3beta1) and pineal gland ( alpha3beta2).

Animals↗
PubMed1997

Rapid, agonist-dependent phosphorylation in vivo of human thromboxane receptor isoforms. Minimal involvement of protein kinase C.

Thromboxane A2 (TxA2) is a potent vasoconstrictor and platelet agonist. Its biological function is tightly regulated. G protein-coupled membrane receptors transduce the effects of TxA2. However, although a single thromboxane receptor (TP) gene has been identified, two splice variants have been cloned from human placenta and megakaryocytic lines (TPalpha) and from human endothelial cells (TPbeta). These differ in the length of their carboxyl-terminal extensions (15 versus 79 residues), which contain multiple potential sites for receptor phosphorylation. Given that TP agonists activate protein kinase C (PKC), it would seem possible that PKC-dependent phosphorylation of TPs might play a central role in homologous desensitization of these receptors. To determine if the TP isoforms were differentially phosphorylated in response to agonist in vivo, human embryonic kidney (HEK) 293 cells were stably transfected with TPalpha and TPbeta. Isoform-specific anti-peptide antibodies were developed and used to immunoprecipitate the phosphorylated receptors. U46619, a PGH2/TxA2 mimetic, induced specific phosphorylation of both isoforms. Phosphorylation of the two isoforms was similar in dose and time dependence, reaching a plateau at around 100 nM U46619. Inhibition of PKC with either GF 109203X (5 microM) or RO 31-8220 (5 microM) or of protein kinase A with H-89 (50 microM) marginally influenced agonist-dependent phosphorylation of either isoform and failed to modulate homologous desensitization of agonist-induced stimulation of inositol phosphate formation. Similar results were obtained when PKC was down-regulated by long term incubation with the phorbol ester, phorbol myristate acetate. Although short term stimulation with phorbol myristate acetate caused PKC-dependent phosphorylation of TPs in vivo, thrombin stimulation of the TP-transfected HEK cells in vivo failed to phosphorylate either of the TP isoforms. Thus, despite the capacity of PKC to phosphorylate TPs in HEK 293 cells and the likely activation of PKC by TP stimulation, this enzyme, like protein kinase A, contributes marginally to rapid, agonist-induced phosphorylation of either TP isoform.

Cell Line↗
PubMed1997

Plasma membrane Ca2+ pump in rat brain. Patterns of alternative splices seen by isoform-specific antibodies.

The expression at the protein level of plasma membrane calcium pump (PMCA) isoforms in rat brain was detected by new antibodies that distinguished the four gene products and their alternatively spliced variants. All four gene products were distributed throughout hippocampus, cortex, and cerebellum, but the alternate splices showed more distinct distribution patterns. The b splice of isoform 1 was not detectable in any of the brain regions, which makes it unlikely that this isoform performs an essential housekeeping role as is frequently proposed. The b splices of isoforms 3 and 4, although expressed in all three regions, showed evidence of proteolysis, which removed a portion of the carboxyl terminus. In contrast, isoform 2b retained its full length, indicating that PMCA2b is more resistant to proteolysis than the other b forms. Whereas substantial amounts of isoforms 1a, 2a, and 3a were expressed in all regions, 4a was found only in frontal cortex. The distinct patterns of expression of the PMCA isoforms in brain suggest that some of them play a special role in intracellular Ca regulation.

Alternative Splicing↗
PubMed1997

Protein kinase C phosphorylates the "a" forms of plasma membrane Ca2+ pump isoforms 2 and 3 and prevents binding of calmodulin.

Phosphorylation by protein kinase C of the "a" and "b" variants of plasma membrane Ca2+ pump isoforms 2 and 3 was studied. Full-length versions of these isoforms were assembled and expressed in COS cells. Whereas the "a" forms were phosphorylated easily with PKC, isoform 2b was phosphorylated only a little, and isoform 3b was not phosphorylated at all. Phosphorylation of isoforms 2a and 3a did not affect their basal activity, but prevented the stimulation of their activity by calmodulin and their binding to calmodulin-Sepharose. This indicated that phosphorylation prevented activation of these isoforms by preventing calmodulin binding. Based on these results, phosphorylation of the pump with PKC would be expected to increase free intracellular Ca2+ levels in those cells where isoforms 2a and 3a are expressed.

Amino Acid Sequence↗
PubMed1999

Human pancreatic islets express mRNA species encoding two distinct catalytically active isoforms of group VI phospholipase A2 (iPLA2) that arise from an exon-skipping mechanism of alternative splicing of the transcript from the iPLA2 gene on chromosome 22q13.1.

An 85-kDa Group VI phospholipase A2 enzyme (iPLA2) that does not require Ca2+ for catalysis has recently been cloned from three rodent species. A homologous 88-kDa enzyme has been cloned from human B-lymphocyte lines that contains a 54-amino acid insert not present in the rodent enzymes, but human cells have not previously been observed to express catalytically active iPLA2 isoforms other than the 88-kDa protein. We have cloned cDNA species that encode two distinct iPLA2 isoforms from human pancreatic islet RNA and a human insulinoma cDNA library. One isoform is an 85-kDa protein (short isoform of human iPLA2 (SH-iPLA2)) and the other an 88-kDa protein (long isoform of human iPLA2 (LH-iPLA2)). Transcripts encoding both isoforms are also observed in human promonocytic U937 cells. Recombinant SH-iPLA2 and LH-iPLA2 are both catalytically active in the absence of Ca2+ and inhibited by a bromoenol lactone suicide substrate, but LH-iPLA2 is activated by ATP, whereas SH-iPLA2 is not. The human iPLA2 gene has been found to reside on chromosome 22 in region q13.1 and to contain 16 exons represented in the LH-iPLA2 transcript. Exon 8 is not represented in the SH-iPLA2 transcript, indicating that it arises by an exon-skipping mechanism of alternative splicing. The amino acid sequence encoded by exon 8 of the human iPLA2 gene is proline-rich and shares a consensus motif of PX5PX8HHPX12NX4Q with the proline-rich middle linker domains of the Smad proteins DAF-3 and Smad4. Expression of mRNA species encoding two active iPLA2 isoforms with distinguishable catalytic properties in two different types of human cells demonstrated here may have regulatory or functional implications about the roles of products of the iPLA2 gene in cell biologic processes.

Alternative Splicing↗
PubMed1999

The expression of plasma membrane Ca2+ pump isoforms in cerebellar granule neurons is modulated by Ca2+.

Plasma membrane Ca2+ ATPase (PMCA) pump isoforms 2, 3, and 1CII are expressed in large amounts in the cerebellum of adult rats but only minimally in neonatal cerebellum. These isoforms were almost undetectable in rat neonatal cerebellar granule cells 1-3 days after plating, but they became highly expressed after 7-9 days of culturing under membrane depolarizing conditions (25 mM KCl). The behavior of isoform 4 was different: it was clearly detectable in adult cerebellum but was down-regulated by the depolarizing conditions in cultured cells. 25 mM KCl-activated L-type Ca2+ channels, significantly increasing cytosolic Ca2+. Changes in the concentration of Ca2+ in the culturing medium affected the expression of the pumps. L-type Ca2+ channel blockers abolished both the up-regulation of the PMCA1CII, 2, and 3 isoforms and the down-regulation of PMCA4 isoform. When granule cells were cultured in high concentrations of N-methyl-D-aspartic acid, a condition that increased cytosolic Ca2+ through the activation of glutamate-operated Ca2+ channels, up-regulation of PMCA1CII, 2, and 3 and down-regulation of PMCA4 was also observed. The activity of the isoforms was estimated by measuring the phosphoenzyme intermediate of their reaction cycle: the up-regulated isoforms, the activity of which was barely detectable at plating time, accounted for a large portion of the total PMCA activity of the cells. No up-regulation of the sarcoplasmic/endoplasmic reticulum calcium pump was induced by the depolarizing conditions.

Animals↗
PubMed1999

Identification by chimera formation and site-selected mutagenesis of a key amino acid residue involved in determining stereospecificity of guinea pig 3-hydroxysteroid sulfotransferase isoforms.

The 3-hydroxysteroid sulfotransferases that have been isolated and cloned from humans and rodents appear to have broad substrate specificities. In the guinea pig, however, two 3-hydroxysteroid sulfotransferases have been isolated that function according to an innate stereospecificity: the alpha-isoform acts on steroids with a 3-hydroxyl group oriented in the alpha position, whereas the beta-isoform acts on steroids where the 3-hydroxyl group is in a beta orientation. To examine the structural basis for this remarkable stereoselectivity, chimeras of the two enzymes, which are 87% identical, were constructed. A chimera consisting of the NH(2)-terminal 91 amino acids of the alpha-isoform and the COOH-terminal 196 amino acids of the beta-isoform displayed activity similar to that of the alpha-isoform. Site-selected mutagenesis of this 3alpha/beta-hydroxysteroid sulfotransferase chimera involving the 12 amino acid differences that exist between the two isoforms within the 91 amino acid NH(2)-terminal region revealed that the amino acid residue at position 51 plays a fundamental role in determining the stereospecificity exhibited by the alpha- and beta-isoforms, i.e. if residue 51 is an asparagine, alpha activity predominates, whereas if an isoleucine is in that position, beta activity prevails.

Amino Acid Sequence↗
PubMed2000

The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine.

We previously cloned heparan sulfate 6-O-sulfotransferase (HS6ST) (Habuchi, H., Kobayashi, M., and Kimata, K. (1998) J. Biol. Chem. 273, 9208-9213). In this study, we report the cloning and characterization of three mouse isoforms of HS6ST, a mouse homologue to the original human HS6ST (HS6ST-1) and two novel HS6STs (HS6ST-2 and HS6ST-3). The cDNAs have been obtained from mouse brain cDNA library by cross-hybridization with human HS6ST cDNA. The three cDNAs contained single open reading frames that predicted type II transmembrane proteins composed of 401, 506, and 470 amino acid residues, respectively. Amino acid sequence of HS6ST-1 was 51 and 57% identical to those of HS6ST-2 and HS6ST-3, respectively. HS6ST-2 and HS6ST-3 had the 50% identity. Overexpression of each isoform in COS-7 cells resulted in about 10-fold increase of HS6ST activity. The three isoforms purified with anti-FLAG antibody affinity column transferred sulfate to heparan sulfate and heparin but not to other glycosaminoglycans. Each isoform showed different specificity toward the isomeric hexuronic acid adjacent to the targeted N-sulfoglucosamine; HS6ST-1 appeared to prefer the iduronosyl N-sulfoglucosamine while HS6ST-2 had a different preference, depending upon the substrate concentrations, and HS6ST-3 acted on either substrate. Northern analysis showed that the expression of each message in various tissues was characteristic to the respective isoform. HS6ST-1 was expressed strongly in liver, and HS6ST-2 was expressed mainly in brain and spleen. In contrast, HS6ST-3 was expressed rather ubiquitously. These results suggest that the expression of these isoforms may be regulated in tissue-specific manners and that each isoform may be involved in the synthesis of heparan sulfates with tissue-specific structures and functions.

Amino Acid Sequence↗
PubMed2001

Role of myosin phosphatase isoforms in cGMP-mediated smooth muscle relaxation.

In vitro experiments showing the activation of the myosin phosphatase via heterophilic leucine zipper interactions between its targeting subunit (MYPT1) and cGMP-dependent protein kinase I suggested a pathway for smooth muscle relaxation (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). The relationship between MYPT1 isoform expression and smooth muscle responses to cGMP signaling in vivo has not been explored. MYPT1 isoforms that contain or lack a C-terminal leucine zipper are generated in birds and mammals by cassette-type alternative splicing of a 31-nucleotide exon. The avian and mammalian C-terminal isoforms are highly conserved and expressed in a tissue-specific fashion. In the mature chicken the tonic contracting aorta and phasic contracting gizzard exclusively express the leucine zipper positive and negative MYPT1 isoforms, respectively. Expression of the MYPT1 isoforms is also developmentally regulated in the gizzard, which switches from leucine zipper positive to negative isoforms around the time of hatching. This switch coincides with the development in the gizzard of a cGMP-resistant phenotype, i.e. inability to dephosphorylate myosin and relax in response to 8-bromo-cGMP after calcium activation. Furthermore, association of cGMP-dependent protein kinase I with MYPT1 is detected by immunoprecipitation only in the tissue that expresses the leucine zipper positive isoform of MYPT1. These results suggest that the regulated splicing of MYPT1 is an important determinant of smooth muscle phenotypic diversity and the variability in the response of smooth muscles to the calcium desensitizing effect of cGMP signaling.

Amino Acid Sequence↗