FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2027711412> ?p ?o ?g. }

• W2027711412 endingPage "24452" @default.
• W2027711412 startingPage "24442" @default.
• W2027711412 abstract "Sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) pump Ca2+ into the endoplasmic reticulum. Recently, three human SERCA3 (h3a–c) proteins and a previously unknown rat SERCA3 (r3b/c) mRNA have been described. Here, we (i) document two novel human SERCA3 splice variants h3d and h3e, (ii) provide data for the expression and mechanisms regulating the expression of all known SERCA3 variants (r3a, r3b/c, and h3a–e), and (iii) show functional characteristics of the SERCA3 isoforms. h3d and h3e are issued from the insertion of an additional penultimate exon 22 resulting in different carboxyl termini for these variants. Distinct distribution patterns of the SERCA3 gene products were observed in a series of cell lines of hematopoietic, epithelial, embryonic origin, and several cancerous types, as well as in panels of rat and human tissues. Hypertension and protein kinase C, calcineurin, or retinoic acid receptor signaling pathways were found to differently control rat and human splice variant expression, respectively. Stable overexpression of each variant was performed in human embryonic kidney 293 cells, and the SERCA3 isoforms were fully characterized. All SERCA3 isoforms were found to pump Ca2+ with similar affinities. However, they modulated the cytosolic Ca2+ concentration ([Ca2+]c) and the endoplasmic reticulum Ca2+ content ([Ca2+]er) in different manners. A newly generated polyclonal antibody and a pan-SERCA3 antibody proved the endogenous expression of the three novel SERCA3 proteins, h3d, h3e, and r3b/c. All these data suggest that the SERCA3 gene products have a more widespread role in cellular Ca2+ signaling than previously appreciated. Sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) pump Ca2+ into the endoplasmic reticulum. Recently, three human SERCA3 (h3a–c) proteins and a previously unknown rat SERCA3 (r3b/c) mRNA have been described. Here, we (i) document two novel human SERCA3 splice variants h3d and h3e, (ii) provide data for the expression and mechanisms regulating the expression of all known SERCA3 variants (r3a, r3b/c, and h3a–e), and (iii) show functional characteristics of the SERCA3 isoforms. h3d and h3e are issued from the insertion of an additional penultimate exon 22 resulting in different carboxyl termini for these variants. Distinct distribution patterns of the SERCA3 gene products were observed in a series of cell lines of hematopoietic, epithelial, embryonic origin, and several cancerous types, as well as in panels of rat and human tissues. Hypertension and protein kinase C, calcineurin, or retinoic acid receptor signaling pathways were found to differently control rat and human splice variant expression, respectively. Stable overexpression of each variant was performed in human embryonic kidney 293 cells, and the SERCA3 isoforms were fully characterized. All SERCA3 isoforms were found to pump Ca2+ with similar affinities. However, they modulated the cytosolic Ca2+ concentration ([Ca2+]c) and the endoplasmic reticulum Ca2+ content ([Ca2+]er) in different manners. A newly generated polyclonal antibody and a pan-SERCA3 antibody proved the endogenous expression of the three novel SERCA3 proteins, h3d, h3e, and r3b/c. All these data suggest that the SERCA3 gene products have a more widespread role in cellular Ca2+ signaling than previously appreciated. endoplasmic reticulum sarco/endoplasmic reticulum Ca2+-ATPase plasma membrane Ca2+-ATPase reverse transcriptase inositol 1,4,5-trisphosphate receptor 4β-phorbol 12-myristate 13-acetate all-trans-retinoic acid Wistar Kyoto spontaneously hypertensive rat(s) protein kinase C human SERCA3 human SERCA3a, -3b, -3c, -3d, and -3e isoforms rat SERCA3a rat SERCA3b/c amino acid(s) human embryonic kidney glyceraldehyde-3-phosphate dehydrogenase The cytosolic free Ca2+ concentration is a dynamic signal regulating a variety of important cellular functions such as secretion, contraction, metabolism, neuronal plasticity, and gene transcription (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4493) Google Scholar). The free Ca2+ concentration is strictly controlled, in space, time, and amplitude. This very tight control is necessary to enable cells to extract relevant information from the Ca2+ signal, but also implies that disturbances in the intracellular Ca2+ signal can lead to a plethora of consequences. Ca2+ signaling is highly complex and comprises elementary and global oscillatory events involving endoplasmic reticulum (ER)1 membrane proteins. Among the better known proteins are Ca2+ channels, namely inositol 1,4,5-trisphosphate receptors (InsP3-Rs) (2Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (373) Google Scholar) and Ca2+ pumps or sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) (3Brandl C.J. Green N.M. Korczak B. MacLennan D.H. Cell. 1986; 44: 597-607Abstract Full Text PDF PubMed Scopus (593) Google Scholar, 4Sakuntabhai A. Ruiz-Perez V. Carter S. Jacobsen N. Burge S. Monk S. Smith M. Munro C.S. O'Donovan M. Craddock N. Kucherlapati R. Rees J.L. Owen M. Lathrop G.M. Monaco A.P. Strachan T. Hovnanian A. Nat. Genet. 1999; 21: 271-277Crossref PubMed Scopus (624) Google Scholar, 5Burk S.E. Lytton J. MacLennan D.H. Shull G.E. J. Biol. Chem. 1989; 264: 18561-18568Abstract Full Text PDF PubMed Google Scholar), which coordinate opposite Ca2+ ion fluxes. InsP3-Rs induce peaks in cytosolic Ca2+ concentration as a result of Ca2+ release from ER, whereas SERCAs are involved in the falling phase of Ca2+ transients because of Ca2+ resequestration into the ER Ca2+stores. Although many data are available on the role of InsP3-Rs in Ca2+ signaling, our knowledge of the role of SERCAs is more restricted. For 35 years research on these enzymes has focused on their biochemical and molecular characterization. The SERCA family includes three gene products, named SERCA1 (ATP2A1), -2 (ATP2A2), and -3 (ATP2A3), which give rise to alternatively spliced mRNA and protein isoforms. The SERCA1 and SERCA2 genes have been known for a while, and have two 3′ end splice variants encoding different carboxyl termini isoforms, mainly expressed in adult (SERCA1a) and neonatal (SERCA1b) skeletal muscles, in cardiac muscle (SERCA2a), and in all cell types (SERCA2b). Intriguingly as regards the variety of non-muscle cell functions, compared with muscle cells, until 1998, a unique so-called non-muscle SERCA3a isoform was described (5Burk S.E. Lytton J. MacLennan D.H. Shull G.E. J. Biol. Chem. 1989; 264: 18561-18568Abstract Full Text PDF PubMed Google Scholar, 6Bobe R. Bredoux R. Wuytack F. Quarck R. Kovacs T. Papp B. Corvazier E. Magnier C. Enouf J. J. Biol. Chem. 1994; 269: 1417-1424Abstract Full Text PDF PubMed Google Scholar, 7Wuytack F. Papp B. Verboomen H. Raeymaekers L. Dode L. Bobe R. Enouf J. Bokkala S. Authi K.S. Casteels R. J. Biol. Chem. 1994; 269: 1410-1416Abstract Full Text PDF PubMed Google Scholar). In recent years, there have been enormous advances in our understanding of SERCAs, including new insight into their structure (8Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1619) Google Scholar) and their relevant physiological functions in human diseases (4Sakuntabhai A. Ruiz-Perez V. Carter S. Jacobsen N. Burge S. Monk S. Smith M. Munro C.S. O'Donovan M. Craddock N. Kucherlapati R. Rees J.L. Owen M. Lathrop G.M. Monaco A.P. Strachan T. Hovnanian A. Nat. Genet. 1999; 21: 271-277Crossref PubMed Scopus (624) Google Scholar, 9Odermatt A. Kurzydlowski K. Maclennan D.H. J. Biol. Chem. 1996; 271: 14206-14213Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 10Varadi A. Lebel L. Hashim Y. Mehta Z. Ashcroft S.J. Turner R. Diabetologia. 1999; 42: 1240-1243Crossref PubMed Scopus (74) Google Scholar). SERCA1 gene mutations have been reported in some patients with Brody disease, a muscle disease. Various SERCA2 gene mutations have been reported in Darier-White disease, a rare dermatosis characterized by focal areas of separation between keratinocytes (4Sakuntabhai A. Ruiz-Perez V. Carter S. Jacobsen N. Burge S. Monk S. Smith M. Munro C.S. O'Donovan M. Craddock N. Kucherlapati R. Rees J.L. Owen M. Lathrop G.M. Monaco A.P. Strachan T. Hovnanian A. Nat. Genet. 1999; 21: 271-277Crossref PubMed Scopus (624) Google Scholar). Missense mutations in the SERCA3 gene are described in type II diabetic patients (10Varadi A. Lebel L. Hashim Y. Mehta Z. Ashcroft S.J. Turner R. Diabetologia. 1999; 42: 1240-1243Crossref PubMed Scopus (74) Google Scholar). In contrast, knockout of SERCA2 and SERCA3 genes leads to impaired cardiac performance (11Periasamy M. Reed T.D. Liu L.H. Loukianov E. Paul R.J. Nieman M.L. Riddle T. Duffy J.J. Doetschman T. Lorenz J.N. Shull G.E. J. Biol. Chem. 1999; 274: 2556-2562Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), followed by squamous cell tumors (12Liu L.H. Boivin G.P. Prasad V. Periasamy M. Shull G.E. J. Biol. Chem. 2001; 276: 26737-26740Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and defects in endothelium- and epithelium-dependent relaxation of vascular (13Liu L.H. Paul R.J. Sutliff R.L. Miller M.L. Lorenz J.N. Pun R.Y. Duffy J.J. Doetschman T. Kimura Y. MacLennan D.H. Hoying J.B. Shull G.E. J. Biol. Chem. 1997; 272: 30538-30545Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and tracheal (14Kao J. Fortner C.N. Liu L.H. Shull G.E. Paul R.J. Am. J. Physiol. 1999; 277: L264-L270PubMed Google Scholar) smooth muscles, respectively. These molecular genetic studies of causal genes in humans, coupled with the observation that distinct phenotypes were found in either SERCA2 or SERCA3 knock-out models, point to compensatory phenomena or to many more diseases than those known today caused by an altered function of one of the known or unknown Ca2+-transporting proteins (15Missiaen L. Robberecht W. Bosch L.V. Callewaert G. Parys J.B. Wuytack F. Raeymaekers L. Nilius B. Eggermont J. De Smedt H. Cell Calcium. 2000; 28: 1-21Crossref PubMed Scopus (182) Google Scholar). In the meanwhile, SERCA3 genes were suggested to possess a high degree of complexity. We and others showed that the human SERCA3 gene gives rise to two additional 3′ end transcripts, SERCA3b and -3c (16Bobe R. Lacabaratz-Porret C. Bredoux R. Martin V. Ozog A. Launay S. Corvazier E. Kovacs T. Papp B. Enouf J. FEBS Lett. 1998; 423: 259-264Crossref PubMed Scopus (21) Google Scholar, 17Dode L., De Greef C. Mountian I. Attard M. Town M.M. Casteels R. Wuytack F. J. Biol. Chem. 1998; 273: 13982-13994Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 18Poch E. Leach S. Snape S. Cacic T. MacLennan D.H. Lytton J. Am. J. Physiol. 1998; 275: C1449-C1458Crossref PubMed Google Scholar), that we recently found to encode the predicted proteins in platelets and lymphoid Jurkat T cells (19Kovacs T. Felfoldi F. Papp B. Paszty K. Bredoux R. Enyedi A. Enouf J. Biochem. J. 2001; 358: 559-568Crossref PubMed Scopus (31) Google Scholar). Moreover, these splice products would present an uncommonly high degree of species specificity, as mice express slightly different SERCA3b and -3c mRNAs (20Ozog A. Pouzet B. Bobe R. Lompré A.M. FEBS Lett. 1998; 427: 349-352Crossref PubMed Scopus (19) Google Scholar) from those of human origin, whereas rats are devoid of similar splice variants, but express a so-called SERCA3b/c mRNA (21Martin V. Bredoux R. Corvazier E. Papp B. Enouf J. Hypertension. 2000; 35: 91-102Crossref PubMed Scopus (43) Google Scholar). Evidence for the existence of these mouse and rat SERCA3 proteins, however, is still lacking. Here, we report (i) a growing family of SERCA3 isoforms, including the rat 3b/c and two novel human SERCA3d and SERCA3e proteins; (ii) their plural, diverse, and species-specific distribution patterns; (iii) factors involved in the regulation of their expressions; and (iv) their distinct functional properties determining both cytosolic and endoplasmic reticulum Ca2+ concentrations. Taken together, these results should help the understanding of pathophysiological cell Ca2+ signaling in a broad variety of cells. Human blood was obtained from different healthy volunteers, and the investigation was performed according to the requirements of the Declaration of Helsinki. The isolation of human platelets was performed as previously described (22Lacabaratz-Porret C. Launay S. Corvazier E. Bredoux R. Papp B. Enouf J. Biochem. J. 2000; 350: 723-734Crossref PubMed Scopus (39) Google Scholar). Sixteen-week-old WKY rats and SHR were supplied by the Centre d'Élevage R. Janvier (Le Genest, France). Approximately 10 WKY rats and SHR were used. Animals were anesthetized with ether. Rat blood was diluted in 0.9% NaCl, and the isolation of rat platelets was performed as detailed in Ref. 21Martin V. Bredoux R. Corvazier E. Papp B. Enouf J. Hypertension. 2000; 35: 91-102Crossref PubMed Scopus (43) Google Scholar. The isolation of rat tissues was as detailed in Ref. 21Martin V. Bredoux R. Corvazier E. Papp B. Enouf J. Hypertension. 2000; 35: 91-102Crossref PubMed Scopus (43) Google Scholar. The megakaryocytic CHRF-288 11, MEG 01, and Dami as well as adrenal pheochromocytoma PC12 cells were generously given by Dr. Lieberman, Pr. J. Peries, and Pr. Treiman, respectively. The KATO III (gastric cancer), lymphoblastoid Jurkat-T (JurE6–1 clone), promyelocytic HL-60, monocytic U937, HeLa (epithelial-like), NCI-H69 (lung cancer), and human embryonic kidney (HEK)-293 cell lines were obtained from the American Type Culture Collection (Rockville Pike, MD). All cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and 2 mm l-glutamine. The PC12 cells were cultured as described in Ref. 23Caspersen C. Pedersen P.S. Treiman M. J. Biol. Chem. 2000; 275: 22363-22372Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar. Lymphocyte activation and induction of HL-60 cell differentiation were performed as previously described (24Launay S. Bobe R. Lacabaratz-Porret C. Bredoux R. Kovàcs T. Enouf J. Papp B. J. Biol. Chem. 1997; 272: 10746-10750Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar,25Launay S. Gianni M. Kovàcs T. Bredoux R. Bruel A. Gélébart P. Zassadowski F. Chomienne C. Enouf J. Papp B. Blood. 1999; 93: 4395-4405Crossref PubMed Google Scholar). Preparation of whole cell lysates from transfected HEK-293 cells (human recombinants) was as in Ref. 24Launay S. Bobe R. Lacabaratz-Porret C. Bredoux R. Kovàcs T. Enouf J. Papp B. J. Biol. Chem. 1997; 272: 10746-10750Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar. Isolation of rat and human platelet and recombinant HEK-293 cell enriched intracellular membranes was as described in Refs.21Martin V. Bredoux R. Corvazier E. Papp B. Enouf J. Hypertension. 2000; 35: 91-102Crossref PubMed Scopus (43) Google Scholar and 22Lacabaratz-Porret C. Launay S. Corvazier E. Bredoux R. Papp B. Enouf J. Biochem. J. 2000; 350: 723-734Crossref PubMed Scopus (39) Google Scholar. For rat cerebellum membrane isolation, 20 mg of tissue was lysed by adding 1 ml of buffer containing 10 mm KCl, 20 mm Hepes (pH 7.4), 200 mm sucrose, 50 μm EGTA, 50 μm EDTA, 0.5 mmdithiothreitol, 0.2 unit/ml aprotinin, 50 μmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 50 μg/ml soybean trypsin inhibitor, 40 μg/ml Bowman-Birk trypsin-chymotrypsin inhibitor, and 5 μg/ml pepstatin A. The lysate was centrifuged at 1200 × g for 15 min at 4 °C. The supernatant was centrifuged at 100,000 × g for 1 h at 4 °C. The pellet was resuspended in a buffer containing 160 mmKCl, 17 mm Hepes (pH 7.4), and 0.2 mmdithiothreitol; aliquoted; frozen; and kept at −80 °C. The protein concentration of the different lysates and membrane fractions was determined using bovine serum albumin as a standard. For endogenous rat SERCA2b (r2b), and human SERCA2b (h2b), the pan-r and -h2b polyclonal antibody, anti-2b (21Martin V. Bredoux R. Corvazier E. Papp B. Enouf J. Hypertension. 2000; 35: 91-102Crossref PubMed Scopus (43) Google Scholar), and pan-h2 monoclonal antibody, IID8 (22Lacabaratz-Porret C. Launay S. Corvazier E. Bredoux R. Papp B. Enouf J. Biochem. J. 2000; 350: 723-734Crossref PubMed Scopus (39) Google Scholar), were used (BIOMOL, Plymouth Meeting, PA). For r3a and r3b/c transfectants, the pan-r and -h3 polyclonal antibody, the N89 (PA1–910, Affinity Bioreagents, Neshanic Station, NJ), directed against the extreme NH2-terminal part of the r3a protein (6Bobe R. Bredoux R. Wuytack F. Quarck R. Kovacs T. Papp B. Corvazier E. Magnier C. Enouf J. J. Biol. Chem. 1994; 269: 1417-1424Abstract Full Text PDF PubMed Google Scholar, 7Wuytack F. Papp B. Verboomen H. Raeymaekers L. Dode L. Bobe R. Enouf J. Bokkala S. Authi K.S. Casteels R. J. Biol. Chem. 1994; 269: 1410-1416Abstract Full Text PDF PubMed Google Scholar) and pan-SERCA polyclonal antibody, SERBIO (26Kovacs T. Corvazier E. Papp B. Magnier C. Bredoux R. Enyedi A. Sarkadi B. Enouf J. J. Biol. Chem. 1994; 269: 6177-6184Abstract Full Text PDF PubMed Google Scholar), were used. For h3a–e transfectants, the N89 and a pan-h3, monoclonal antibody, PL/IM430, directed against the NH2-terminal part of SERCA3 proteins, as well as our recently developed isoform-specific (h3a, h3b, and h3c) polyclonal antibodies (19Kovacs T. Felfoldi F. Papp B. Paszty K. Bredoux R. Enyedi A. Enouf J. Biochem. J. 2001; 358: 559-568Crossref PubMed Scopus (31) Google Scholar), were used. For PMCAs and InsP3-Rs, the pan-PMCA monoclonal 5F10, the polyclonal anti-InsP3-RI (Affinity BioReagents), the polyclonal anti-InsP3-RII (Santa Cruz Biotechnology, Santa Cruz, CA), and the monoclonal anti-InsP3-RIII (Transduction Laboratories, Lexington, KY) antibodies (22Lacabaratz-Porret C. Launay S. Corvazier E. Bredoux R. Papp B. Enouf J. Biochem. J. 2000; 350: 723-734Crossref PubMed Scopus (39) Google Scholar) were used. Secondary anti-rabbit, anti-guinea pig, and anti-mouse horseradish peroxidase-conjugated antibodies were from Jackson Immunoresearch (West Grove, PA). Antibody binding was detected using enhanced chemiluminescence Western blotting reagents according to the manufacturer's instructions (Amersham Biosciences, Little Chalfont, Bucks, UK). Luminograms were scanned using Adobe Photoshop and, where indicated, quantified by Molecular Analyst, version NIH Image 1. 62b7. The antibody was generated by immunizing New Zealand rabbits with the bovine serum albumin-conjugated synthetic peptide (see Fig. 6 A) as described for isoform-specific anti-h3a and anti-h3c antibodies (19Kovacs T. Felfoldi F. Papp B. Paszty K. Bredoux R. Enyedi A. Enouf J. Biochem. J. 2001; 358: 559-568Crossref PubMed Scopus (31) Google Scholar). A portion of the antibody was purified from the final serum on peptide-Sepharose 4B affinity column. The sensitivity and the cross-reactivity of the antibody were analyzed by the Crosslink Laboratory (Budapest, Hungary) by performing both enzyme-linked immunosorbent assay and dot-blot experiments using the corresponding peptide-bovine serum albumin conjugate (data not shown). Electrophoresis was performed on 8% SDS-PAGE, and Western blots were treated as in Refs. 19Kovacs T. Felfoldi F. Papp B. Paszty K. Bredoux R. Enyedi A. Enouf J. Biochem. J. 2001; 358: 559-568Crossref PubMed Scopus (31) Google Scholar, 21Martin V. Bredoux R. Corvazier E. Papp B. Enouf J. Hypertension. 2000; 35: 91-102Crossref PubMed Scopus (43) Google Scholar, and 22Lacabaratz-Porret C. Launay S. Corvazier E. Bredoux R. Papp B. Enouf J. Biochem. J. 2000; 350: 723-734Crossref PubMed Scopus (39) Google Scholar, for the N89, PL/IM430, 5F10, anti-InsP3-Rs, and anti-h3a–c antibodies. For the r3b/c protein, the nitrocellulose membranes were incubated with a 1:250 dilution of the anti-r3b/c antibody in Tris-buffered saline (pH 7.4), 0.1% Tween 20 for 4 h. After washing, the blots were treated with a 1:10,000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG, for 1 h. Total RNA extraction from human and rat platelets, human cell lines, and rat tissues was as described (21Martin V. Bredoux R. Corvazier E. Papp B. Enouf J. Hypertension. 2000; 35: 91-102Crossref PubMed Scopus (43) Google Scholar, 22Lacabaratz-Porret C. Launay S. Corvazier E. Bredoux R. Papp B. Enouf J. Biochem. J. 2000; 350: 723-734Crossref PubMed Scopus (39) Google Scholar, 25Launay S. Gianni M. Kovàcs T. Bredoux R. Bruel A. Gélébart P. Zassadowski F. Chomienne C. Enouf J. Papp B. Blood. 1999; 93: 4395-4405Crossref PubMed Google Scholar). For RT-PCR experiments, protocols essentially identical to those described in Refs. 21Martin V. Bredoux R. Corvazier E. Papp B. Enouf J. Hypertension. 2000; 35: 91-102Crossref PubMed Scopus (43) Google Scholar and 22Lacabaratz-Porret C. Launay S. Corvazier E. Bredoux R. Papp B. Enouf J. Biochem. J. 2000; 350: 723-734Crossref PubMed Scopus (39) Google Scholar were used. The primers used to amplify r2b and h2b, r3a, r3b/c, h3a, human SERCA3c (h3c), hPMCA1b, hPMCA4b, hInsP3-R types I, II, or III are detailed in Refs.21Martin V. Bredoux R. Corvazier E. Papp B. Enouf J. Hypertension. 2000; 35: 91-102Crossref PubMed Scopus (43) Google Scholar and 22Lacabaratz-Porret C. Launay S. Corvazier E. Bredoux R. Papp B. Enouf J. Biochem. J. 2000; 350: 723-734Crossref PubMed Scopus (39) Google Scholar. The primers used (Genosys Sigma-Aldrich, St. Louis, MO) to amplify human SERCA3b (h3b), SERCA3d (h3d) and SERCA3e (h3e) are in Table I. PCR was initiated by adding 1.25 units of Gold AmpliTaq DNA polymerase (Thermus aquaticus, PerkinElmer, Branchburg, NJ) and Touch Down-PCR was performed for 10 cycles with annealing temperature decrement from 65 to 55 °C. PCR was conducted for different cycles, each consisting of successive periods of denaturation at 95 °C for 1 min, annealing at 55 °C (r2b and h2b), and 58 or 68 °C (Fig. 4b) for 1 min, and extension at 72 °C for 1 min. GAPDH and r2b or h2b amplifications were used as internal RNA controls. PCR products were visualized on ethidium bromide-stained 1.5 and 2% agarose gels or by Southern blotting as in Ref. 22Lacabaratz-Porret C. Launay S. Corvazier E. Bredoux R. Papp B. Enouf J. Biochem. J. 2000; 350: 723-734Crossref PubMed Scopus (39) Google Scholar. They were scanned using Adobe Photoshop and, where indicated, quantified by Molecular Analyst, version NIH Image 1.62b7.Table IOligonucleotide sequences for the various human SERCA3 isoformsOligonucleotides1-af, forward; r, reverse.Sequences and localizationsReferencesf P1 (exon 18)2674GAG TCA CGC TTC CCC ACC ACC17Dode L., De Greef C. Mountian I. Attard M. Town M.M. Casteels R. Wuytack F. J. Biol. Chem. 1998; 273: 13982-13994Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholarf P2 (exon 20/21)2971CAC ATG CAC GCC TGT CTT TAT CC17Dode L., De Greef C. Mountian I. Attard M. Town M.M. Casteels R. Wuytack F. J. Biol. Chem. 1998; 273: 13982-13994Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholarf P3 (exon 21)2997CCT TCT CAG GAC AGT CTC17Dode L., De Greef C. Mountian I. Attard M. Town M.M. Casteels R. Wuytack F. J. Biol. Chem. 1998; 273: 13982-13994Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholarr P4 (exon 21)2997TGC GAG ACT GTC CTG AGA AGG17Dode L., De Greef C. Mountian I. Attard M. Town M.M. Casteels R. Wuytack F. J. Biol. Chem. 1998; 273: 13982-13994Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholarr P5 (intron 21/22)CCT CTC TGA GCA GCT CTGEMBL Y3021426r P6 (exon 22)3113GCC TGT CAT TTA TCC GGC G17Dode L., De Greef C. Mountian I. Attard M. Town M.M. Casteels R. Wuytack F. J. Biol. Chem. 1998; 273: 13982-13994Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholarr P6 (exon 23)3272GCC TGT CAT TTA TCC GGC GPresent workf P7 (exon 21/22)3058GAC CAC ACC GGG GCC AGG GAC ACAPresent workf P8 (exon 21/22)3069GTT GGC CTC TTT GGG CCA GGG ACA CAPresent workr P9 (exon 21/22)3058TGT GTC CCT GGC CCC GGT GTG GTCPresent workr P10 (exon 21/22)3069TGT GTC CCT GGC CCA AAG AGG CCA ACPresent workr P11 (exon 21/23)3058GGC TCA TTT CTT CCG GTG TGG TC17Dode L., De Greef C. Mountian I. Attard M. Town M.M. Casteels R. Wuytack F. J. Biol. Chem. 1998; 273: 13982-13994Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar1-a f, forward; r, reverse. Open table in a new tab For expression constructs, the cDNAs encoding the h2b in pcDNA3.1 (Pr. D. H. MacLennan), r3a in pBR322 (Dr. G. Shull), and the inactive h3a in pMT2 (Dr. J. Lytton) were used. The r3a cDNA wasNotI/NheI-excised and subcloned inEcoRI-digested and dephosphorylated pcDNA3 mammalian expression vector (Invitrogen, Cergy Pontoise, France) (Dr. A. M. Lompré). 3′ end r3b/c DNA was generated by PCR encompassing exons 17–21 (nucleotides 2605–3263). Products wereSalI/BspMI-excised after oriented subcloning in pCR2.1 vector. The r3b/c complete cDNA was created by switching aSalI/ApaI fragment of r3a by the specific 3′ end r3b/c product. The cDNA of the inactive h3a wasEcoRI-excised and subcloned in pUC18. The active h3a was obtained by substituting a PCR-amplified 1149-bpBclI/EcoRV fragment where Ile 817 was replaced by Met. This cDNA was subcloned into EcoRI site of pcDNA3. 3′ end variant-specific cDNAs were generated by overlap extension of two variant-specific PCR products covering the region encompassing exon 18–23 (nucleotides 2674–3290). Specific products were subcloned in pCR2.1, and thenEcoRV/XbaI-excised after oriented subcloning in pUC18 vector. The h3a–e cDNAs were created by switching theEcoRV/XbaI fragment of the active h3a construct, by the 3′ end variant-specific products. Each construct was verified by automated dye terminator sequencing (Applied Biosystems AB1 100 model 377, Genome Express, Grenoble, France) across the junctions and through the modified regions (data not shown). cDNA for transfection was purified using the EndoFree plasmid kit (Qiagen, Hilden, Germany). Cells were plated at a density of ∼1 × 104 cells/cm2 in 100-mm dishes. The following day, cells at ∼60% confluence were transfected with 10 μg of the different r3s and h3s and h2b cDNAs using the transfection agent ExGen 500 (Euromedex, Souffelweyersheim, France) according to the instructions of the manufacturer. In short, cell monolayers were exposed to DNA-polymer complexes in a small volume of serum-free RPMI 1640 in an incubator. After 1 h, serum-containing medium was carefully added to monolayers. To establish cell lines that stably express the different SERCA3 isoforms, the neomycin-resistant colonies were selected with 600 μg/ml G418 (Invitrogen) in RPMI 1640. Approximately 2 weeks later, colonies were picked and grown as individual cell lines in the presence of 200 μg/ml G418 for 2 weeks. A mean of 10–12 neomycin-resistant clones were screened for each SERCA transfection efficiencies by Western blotting (data not shown). A mean of three of the highest responsive clones was selected for characterization and Ca2+ response studies. Measurements of Ca2+ pumping activity was essentially performed as described (27Grynkiewics G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Google Scholar). A cell sample of 0.4 ml was diluted into 1.6 ml of calcium-free, Hepes/KCl (pH 7.4) composed of 100 mm KCl, 100 mm sucrose, 20 mmHepes, 1.4 mm MgCl2, 1.25 mmNaN3, 1 μm Fluo-3, 5 μg/ml oligomycin, and 37.5 μg/ml saponin. After 5 min at 37 °C, it was transferred to a constant temperature cuvette holder of a Shimadzu RF-1501 spectrofluorimeter (Shimadzu Europe, Duisburg, Germany). The free Ca2+ level was adjusted by stepwise additions from a concentrated CaCl2 solution, after which 2 mmATP was added. Fluorescence intensities (F) were continuously recorded at 488-nm excitation and 526-nm emission wavelengths (slits of 10 nm). Calibrations were performed by addition of CaCl2 or EGTA to obtain F max andF min values, respectively. Levels of [Ca2+] were calculated from the binding equation [Ca2+] = K d β (F −F min)/(F max −F). Confluent HEK-293 cells were loaded with 1.75 μm Fura-2-AM in culture medium for 45 min at 37 °C, and then incubated with fresh medium for another 15 min. Cells were rinsed with phosphate-buffered saline, detached, collected by centrifugation, and washed twice in 5 ml of Hepes buffer (pH 7.45) containing 136 mm NaCl, 10 mm Hepes, 2.7 mm KCl, 2 mm MgCl2, 1 mg/mld-glucose, and 1 mm CaCl2. Fluorescence measurements were performed with cuvets containing 2 ml of cell suspensions in Hepes buffer, using the same fluorimeter. For calculation of the [Ca2+]c, the ratio of fluorescence at excitation wavelengths of 340 and 380 nm (emission at 510 nm) was calibrated according to Ref. 28Keularts I.M. van Gorp R.M. Feijge M.A. Vuist W.M. Heemskerk J.W. J. Biol. Chem. 2000; 275: 1763-1772Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar. The human and rat SERCA3 genes were recently found to give rise to distinct and species-specific 3′ end splice variants, in addition to the h- and rSERCA3a mRNAs (h3a and r3a) (Fig.1 A). The hSERCA3b (h3b) and hSERCA3c (h3c) mRNAs result from the partial or complete insertion of a penultimate exon 21. In rat platelets a rSERCA3b/3c (r3b/c) variant (GenBank™ accession no. AF458230) is present, in which the 5′-part of exon 21 lacks an ACLYP sequence (black box) and its 3′-part is extended (red box) and terminated using a novel stop codon Sb/c (21Martin V. Bredoux R. Corvazier E. Papp B. Enouf J. Hypertension. 2000; 35: 91-102Crossref PubMed Scopus (43) Google Scholar). A search for similar 3b/c splice variant in human platelets (Fig.1 B), performing RT-PCR, with sets of primers located in exons 18–22, was unsuccessful. However, additional longer PCR products were detected either on agarose gels (Fig. 1 B, I,7) or by Southern blotting (Fig. 1 B,II, 7) when we used primers P3 and P6 amplifying exons 21 and 22. These data pointed to the insertion of a novel exon" @default.
• W2027711412 created "2016-06-24" @default.
• W2027711412 creator A5010112438 @default.
• W2027711412 creator A5039316771 @default.
• W2027711412 creator A5049363078 @default.
• W2027711412 creator A5066608441 @default.
• W2027711412 creator A5068603411 @default.
• W2027711412 creator A5083294960 @default.
• W2027711412 creator A5083308753 @default.
• W2027711412 date "2002-07-01" @default.
• W2027711412 modified "2023-10-01" @default.
• W2027711412 title "Three Novel Sarco/endoplasmic Reticulum Ca2+-ATPase (SERCA) 3 Isoforms" @default.
• W2027711412 cites W1486831897 @default.
• W2027711412 cites W1499927580 @default.
• W2027711412 cites W1544518972 @default.
• W2027711412 cites W1569777697 @default.
• W2027711412 cites W1599277155 @default.
• W2027711412 cites W1613210651 @default.
• W2027711412 cites W1915985314 @default.
• W2027711412 cites W1970182526 @default.
• W2027711412 cites W1976405409 @default.
• W2027711412 cites W1980830385 @default.
• W2027711412 cites W1982199817 @default.
• W2027711412 cites W1988406918 @default.
• W2027711412 cites W1992029300 @default.
• W2027711412 cites W2000786270 @default.
• W2027711412 cites W2004346548 @default.
• W2027711412 cites W2004487783 @default.
• W2027711412 cites W2005389122 @default.
• W2027711412 cites W2009860993 @default.
• W2027711412 cites W2016444884 @default.
• W2027711412 cites W2017763545 @default.
• W2027711412 cites W2022908241 @default.
• W2027711412 cites W2037568202 @default.
• W2027711412 cites W2045399399 @default.
• W2027711412 cites W2055836254 @default.
• W2027711412 cites W2060120692 @default.
• W2027711412 cites W2063083122 @default.
• W2027711412 cites W2069590168 @default.
• W2027711412 cites W2072682674 @default.
• W2027711412 cites W2089063033 @default.
• W2027711412 cites W2092197701 @default.
• W2027711412 cites W2095245627 @default.
• W2027711412 cites W2103450164 @default.
• W2027711412 cites W2111416247 @default.
• W2027711412 cites W2116747657 @default.
• W2027711412 cites W2119949003 @default.
• W2027711412 cites W2127019250 @default.
• W2027711412 cites W2130653887 @default.
• W2027711412 cites W2133624980 @default.
• W2027711412 cites W2141260882 @default.
• W2027711412 cites W2153331570 @default.
• W2027711412 cites W2157581901 @default.
• W2027711412 cites W2161549748 @default.
• W2027711412 cites W2167047446 @default.
• W2027711412 cites W2396712276 @default.
• W2027711412 cites W4229860806 @default.
• W2027711412 cites W4231008067 @default.
• W2027711412 cites W4249759827 @default.
• W2027711412 cites W4376453153 @default.
• W2027711412 cites W7245226 @default.
• W2027711412 doi "https://doi.org/10.1074/jbc.m202011200" @default.
• W2027711412 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11956212" @default.
• W2027711412 hasPublicationYear "2002" @default.
• W2027711412 type Work @default.
• W2027711412 sameAs 2027711412 @default.
• W2027711412 citedByCount "91" @default.
• W2027711412 countsByYear W20277114122012 @default.
• W2027711412 countsByYear W20277114122013 @default.
• W2027711412 countsByYear W20277114122014 @default.
• W2027711412 countsByYear W20277114122015 @default.
• W2027711412 countsByYear W20277114122016 @default.
• W2027711412 countsByYear W20277114122017 @default.
• W2027711412 countsByYear W20277114122018 @default.
• W2027711412 countsByYear W20277114122019 @default.
• W2027711412 countsByYear W20277114122020 @default.
• W2027711412 countsByYear W20277114122021 @default.
• W2027711412 countsByYear W20277114122022 @default.
• W2027711412 countsByYear W20277114122023 @default.
• W2027711412 crossrefType "journal-article" @default.
• W2027711412 hasAuthorship W2027711412A5010112438 @default.
• W2027711412 hasAuthorship W2027711412A5039316771 @default.
• W2027711412 hasAuthorship W2027711412A5049363078 @default.
• W2027711412 hasAuthorship W2027711412A5066608441 @default.
• W2027711412 hasAuthorship W2027711412A5068603411 @default.
• W2027711412 hasAuthorship W2027711412A5083294960 @default.
• W2027711412 hasAuthorship W2027711412A5083308753 @default.
• W2027711412 hasBestOaLocation W20277114121 @default.
• W2027711412 hasConcept C104317684 @default.
• W2027711412 hasConcept C142579037 @default.
• W2027711412 hasConcept C158617107 @default.
• W2027711412 hasConcept C181199279 @default.
• W2027711412 hasConcept C185592680 @default.
• W2027711412 hasConcept C23265538 @default.
• W2027711412 hasConcept C2781414619 @default.
• W2027711412 hasConcept C53345823 @default.
• W2027711412 hasConcept C55493867 @default.
• W2027711412 hasConcept C86803240 @default.

• next