G2Cdb::Gene report

Gene id
G00001320
Gene symbol
ATP2B1 (HGNC)
Species
Homo sapiens
Description
ATPase, Ca++ transporting, plasma membrane 1
Orthologue
G00000071 (Mus musculus)

Databases (8)

Gene
ENSG00000070961 (Ensembl human gene)
490 (Entrez Gene)
410 (G2Cdb plasticity & disease)
ATP2B1 (GeneCards)
Literature
108731 (OMIM)
Marker Symbol
HGNC:814 (HGNC)
Protein Expression
5605 (human protein atlas)
Protein Sequence
P20020 (UniProt)

Synonyms (1)

  • PMCA1

Literature (40)

Pubmed - other

  • Genetic variations in ATP2B1, CSK, ARSG and CSMD1 loci are related to blood pressure and/or hypertension in two Korean cohorts.

    Hong KW, Go MJ, Jin HS, Lim JE, Lee JY, Han BG, Hwang SY, Lee SH, Park HK, Cho YS and Oh B

    Department of Biomedical Engineering, School of Medicine, Kyung Hee University, Seoul, Republic of Korea.

    Blood pressure, one of the important vital signs, is affected by multiple genetic and environmental factors. Recently, several genome-wide association (GWA) studies have successfully identified genetic factors that influence blood pressure and hypertension risk. In this study, we report results of the Korean Association REsource (KARE, 8842 subjects) GWA study on blood pressure and hypertension risk. In all, 10 single-nucleotide polymorphisms (SNPs) that showed significant association with hypertension were further analysed for replication associations in the Health2 project (7861 subjects). Among these 10 SNPs, 3 were replicated in the Health2 cohort for an association with systolic or diastolic blood pressure. The most significant SNP (rs17249754 located in ATPase, Ca(++) transporting, plasma membrane 1 (ATP2B1)) has been previously reported, and the other two SNPs are rs1378942 in the c-src tyrosine kinase (CSK) gene and rs12945290 in the arylsulphatase G (ARSG) gene. An additional hypertension case-control study confirmed that rs17249754 (in ATP2B1) increases hypertension risk in both the KARE and Health2 (meta-analysis, P-value=4.25 x 10(-9)) cohorts. One more SNP, rs995322, located in the CUB and Sushi multiple domains 1 (CSMD1), is also associated with increased risk of hypertension (meta-analysis, P-value=1.00 x 10(-4)). Despite the difficulty of obtaining replication results for a complex trait genetic association between blood pressure and hypertension, we were able to identify consistent genetic factors in both the Korean cohorts in ATP2B1, CSK, ARSG and CSMD1 genes.

    Journal of human hypertension 2010;24;6;367-72

  • Determination of the dissociation constants for Ca2+ and calmodulin from the plasma membrane Ca2+ pump by a lipid probe that senses membrane domain changes.

    Mangialavori I, Ferreira-Gomes M, Pignataro MF, Strehler EE and Rossi JP

    Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Junín 956, 1113 Buenos Aires, Argentina.

    The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca(2+)-pump (PMCA) in the membrane region upon interaction with Ca(2+), calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16, measuring the shift of conformation E(2) to the auto-inhibited conformation E(1)I and to the activated E(1)A state, titrating the effect of Ca(2+) under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca(2+) regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca(2+) transport but does not modify the affinity for Ca(2+) at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E(1)I to the activated E(1)A conformation and thus modulating the transport of Ca(2+). This is reflected in the different apparent constants for Ca(2+) in the absence of CaM (calculated by Ca(2+)-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca(2+) site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca(2+) and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca(2+) binding and activation.

    Funded by: FIC NIH HHS: R03 TW006837, R03 TW006837-03, R03TW006837

    The Journal of biological chemistry 2010;285;1;123-30

  • A locally-induced increase in intracellular Ca2+ propagates cell-to-cell in the presence of plasma membrane Ca2+ ATPase inhibitors in non-excitable cells.

    Nakano T, Koujin T, Suda T, Hiraoka Y and Haraguchi T

    Frontier Research Base for Global Young Researchers, Frontier Research Center, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan. tnakano@wakate.frc.eng.osaka-u.ac.jp

    Intercellular Ca(2+) waves are commonly observed in many cell types. In non-excitable cells, intercellular Ca(2+) waves are mediated by gap junctional diffusion of a Ca(2+) mobilizing messenger such as IP(3). Since Ca(2+) is heavily buffered in the cytosolic environment, it has been hypothesized that the contribution of the diffusion of Ca(2+) to intercellular Ca(2+) waves is limited. Here, we report that in the presence of plasma membrane Ca(2+) ATPase inhibitors, locally-released Ca(2+) from the flash-photolysis of caged-Ca(2+) appeared to induce further Ca(2+) release and were propagated from one cell to another, indicating that Ca(2+) was self-amplified to mediate intercellular Ca(2+) waves. Our findings support the notion that non-excitable cells can establish a highly excitable medium to communicate local responses with distant cells.

    FEBS letters 2009;583;22;3593-9

  • Altered Ca2+ dependence of synaptosomal plasma membrane Ca2+-ATPase in human brain affected by Alzheimer's disease.

    Berrocal M, Marcos D, Sepúlveda MR, Pérez M, Avila J and Mata AM

    Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, Avda de Elvas s/n, 06071 Badajoz, Spain.

    High-affinity Ca(2+) transport ATPases play a crucial role in controlling cytosolic Ca(2+). The amyloid beta-peptide (Abeta) is a neurotoxic agent found in affected neurons in Alzheimer's disease (AD) that has been implicated in dysregulation of Ca(2+) homeostasis. Using kinetic assays, we have shown that the Ca(2+) dependencies of intracellular Ca(2+)-ATPase (SERCA and SPCA) activity are the same in human AD and normal brain but that of plasma membrane Ca(2+)-ATPase (PMCA) is different. The addition of Abeta to normal brain decreases the PMCA activity measured at pCa 5.5, resulting in the same Ca(2+)dependency as that seen in AD brain, whereas the addition of Abeta to AD brain has no effect on PMCA activity. Abeta also decreases the activity of PMCA purified from pig cerebrum, the effect being isoform specific. The level of inhibition of purified PMCA caused by Abeta is reduced by cholesterol, and the level of inhibition of PMCA activity by Abeta in the raft fraction of pig synaptosomal membranes is lower than for the nonraft fraction. We conclude that the effect of Abeta on PMCA activity could be important in amyloid toxicity, resulting in cytoplasmic Ca(2+) dysregulation and could explain the different Ca(2+) dependencies of PMCA activity observed in normal and AD brain.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2009;23;6;1826-34

  • Genome-wide association study of blood pressure and hypertension.

    Levy D, Ehret GB, Rice K, Verwoert GC, Launer LJ, Dehghan A, Glazer NL, Morrison AC, Johnson AD, Aspelund T, Aulchenko Y, Lumley T, Köttgen A, Vasan RS, Rivadeneira F, Eiriksdottir G, Guo X, Arking DE, Mitchell GF, Mattace-Raso FU, Smith AV, Taylor K, Scharpf RB, Hwang SJ, Sijbrands EJ, Bis J, Harris TB, Ganesh SK, O'Donnell CJ, Hofman A, Rotter JI, Coresh J, Benjamin EJ, Uitterlinden AG, Heiss G, Fox CS, Witteman JC, Boerwinkle E, Wang TJ, Gudnason V, Larson MG, Chakravarti A, Psaty BM and van Duijn CM

    National Heart, Lung, and Blood Institute's Framingham Heart Study, Framingham, Massachusetts, USA. levyd@nhlbi.nih.gov

    Blood pressure is a major cardiovascular disease risk factor. To date, few variants associated with interindividual blood pressure variation have been identified and replicated. Here we report results of a genome-wide association study of systolic (SBP) and diastolic (DBP) blood pressure and hypertension in the CHARGE Consortium (n = 29,136), identifying 13 SNPs for SBP, 20 for DBP and 10 for hypertension at P < 4 × 10(-7). The top ten loci for SBP and DBP were incorporated into a risk score; mean BP and prevalence of hypertension increased in relation to the number of risk alleles carried. When ten CHARGE SNPs for each trait were included in a joint meta-analysis with the Global BPgen Consortium (n = 34,433), four CHARGE loci attained genome-wide significance (P < 5 × 10(-8)) for SBP (ATP2B1, CYP17A1, PLEKHA7, SH2B3), six for DBP (ATP2B1, CACNB2, CSK-ULK3, SH2B3, TBX3-TBX5, ULK4) and one for hypertension (ATP2B1). Identifying genes associated with blood pressure advances our understanding of blood pressure regulation and highlights potential drug targets for the prevention or treatment of hypertension.

    Funded by: Intramural NIH HHS; NCRR NIH HHS: UL1 RR025005, UL1 RR025005-01, UL1RR025005; NHGRI NIH HHS: U01 HG004402, U01 HG004402-01, U01HG004402; NHLBI f1d NIH HHS: N01 HC085079; NHLBI NIH HHS: N01 HC-55222, N01 HC015103, N01 HC025195, N01 HC035129, N01 HC045133, N01 HC055015, N01 HC055016, N01 HC055018, N01 HC055019, N01 HC055020, N01 HC055021, N01 HC055022, N01 HC055222, N01 HC075150, N01 HC085086, N01-HC-25195, N01-HC-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-55021, N01-HC-55022, N01-HC-75150, N01-HC-85079, N01-HC-85080, N01-HC-85081, N01-HC-85082, N01-HC-85083, N01-HC-85084, N01-HC-85085, N01-HC-85086, N01HC25195, N01HC55015, N01HC55016, N01HC55018, N01HC55019, N01HC55020, N01HC55021, N01HC55022, N01HC55222, N01HC75150, N01HC85079, N01HC85086, N02 HL64278, N02-HL-6-4278, R01 HL059367, R01 HL059367-02, R01 HL086694, R01 HL086694-01A1, R01 HL087641, R01 HL087641-01, R01 HL087652, R01 HL087652-01, R01HL086694, R01HL087641, R01HL59367, R37 HL051021, R37 HL051021-06, R37 HL051021-10, R37 HL051021-11, R37 HL051021-12, R37 HL051021-13, R37 HL051021-14, R37 HL051021-15, R37HL051021, U01 HL080295, U01 HL080295-01, U10 HL054512, U10 HL054512-05, U10HL054512; NIA NIH HHS: N01 AG012100, N01AG12100; NIDDK NIH HHS: P30 DK063491, P30 DK063491-019004, P30 DK063491-029004, P30 DK063491-049004; PHS HHS: HHSN268200625226C

    Nature genetics 2009;41;6;677-87

  • A large-scale genome-wide association study of Asian populations uncovers genetic factors influencing eight quantitative traits.

    Cho YS, Go MJ, Kim YJ, Heo JY, Oh JH, Ban HJ, Yoon D, Lee MH, Kim DJ, Park M, Cha SH, Kim JW, Han BG, Min H, Ahn Y, Park MS, Han HR, Jang HY, Cho EY, Lee JE, Cho NH, Shin C, Park T, Park JW, Lee JK, Cardon L, Clarke G, McCarthy MI, Lee JY, Lee JK, Oh B and Kim HL

    Center for Genome Science, National Institute of Health, Seoul, Korea.

    To identify genetic factors influencing quantitative traits of biomedical importance, we conducted a genome-wide association study in 8,842 samples from population-based cohorts recruited in Korea. For height and body mass index, most variants detected overlapped those reported in European samples. For the other traits examined, replication of promising GWAS signals in 7,861 independent Korean samples identified six previously unknown loci. For pulse rate, signals reaching genome-wide significance mapped to chromosomes 1q32 (rs12731740, P = 2.9 x 10(-9)) and 6q22 (rs12110693, P = 1.6 x 10(-9)), with the latter approximately 400 kb from the coding sequence of GJA1. For systolic blood pressure, the most compelling association involved chromosome 12q21 and variants near the ATP2B1 gene (rs17249754, P = 1.3 x 10(-7)). For waist-hip ratio, variants on chromosome 12q24 (rs2074356, P = 7.8 x 10(-12)) showed convincing associations, although no regional transcript has strong biological candidacy. Finally, we identified two loci influencing bone mineral density at multiple sites. On chromosome 7q31, rs7776725 (within the FAM3C gene) was associated with bone density at the radius (P = 1.0 x 10(-11)), tibia (P = 1.6 x 10(-6)) and heel (P = 1.9 x 10(-10)). On chromosome 7p14, rs1721400 (mapping close to SFRP4, a frizzled protein gene) showed consistent associations at the same three sites (P = 2.2 x 10(-3), P = 1.4 x 10(-7) and P = 6.0 x 10(-4), respectively). This large-scale GWA analysis of well-characterized Korean population-based samples highlights previously unknown biological pathways.

    Nature genetics 2009;41;5;527-34

  • Preserved function of the plasma membrane calcium pump of red blood cells from diabetic subjects with high levels of glycated haemoglobin.

    Bookchin RM, Etzion Z, Lew VL and Tiffert T

    Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

    The activity of the plasma membrane Ca(2+)-pump decreases steeply throughout the 120 days lifespan of normal human red blood cells. Experiments with isolated membrane preparations showed that glycation of a lysine residue near the catalytic site of the pump ATPase had a powerful inhibitory effect. This prompted the question of whether glycation is the mechanism of age-related decline in pump activity in vivo. It is important to investigate this mechanism because the Ca(2+) pump is a major regulator of Ca(2+) homeostasis in all cells. Its impaired activity in diabetic patients, continuously exposed to high glycation rates, may thus contribute to varied tissue pathology in this disease. We measured Ca(2+)-pump activity as a function of red cell age in red cells from diabetics continuously exposed to high glucose concentrations, as documented by their high mean levels of glycated haemoglobin. The distribution of Ca(2+)-pump activities was indistinguishable from that in non-diabetics, and the pattern of activity decline with cell age in the diabetics' red cells was identical to that observed in red cells from non-diabetics. These results indicate that in intact cells the Ca(2+) pump is protected from glycation-induced inactivation.

    Funded by: NCRR NIH HHS: RR12248; NHLBI NIH HHS: HL585121; NIDDK NIH HHS: DK070996; Wellcome Trust

    Cell calcium 2009;45;3;260-3

  • Phosphatidylserine externalization in caveolae inhibits Ca2+ efflux through plasma membrane Ca2+-ATPase in ECV304.

    Zhang J, Xiao P and Zhang X

    National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.

    It has been evidenced that plasma membrane Ca(2+)-ATPase (PMCA) is localized at caveolae. However, the caveolar function of PMCA in living cells has never been demonstrated. In the present study, PMCA is exclusively localized at caveolae from ECV 304 cells demonstrated by sucrose gradient fractionation and the co-localization of PMCA with caveolin-1 was visualized by confocal microscopy. We found that PMCA is the main mechanism involved in Ca(2+) efflux in ECV 304 cells. Treatment of cells with MbetaCD to disrupt caveolae significantly reduced the Ca(2+) efflux, and the rate of decay is 4.45+/-0.14 min(-1) in the absence of MbetaCD and 1.99+/-0.038 min(-1) in the presence of MbetaCD. Moreover, the replenishment of cholesterol restored the reduction of the PMCA-mediated Ca(2+) efflux in the presence of MbetaCD. Consistent with Ca(2+) efflux in living cells, the activity of the reconstituted PMCA in membranes extracted from cells in vitro was decreased in the presence of MbetaCD. It was found that phosphatidylserine, which is normally in the inner leaflet of plasma membranes and is able to stimulate PMCA was relatively enriched in caveolae. Importantly, the treatment of cells with MbetaCD concomitantly increased the phosphatidylserine externalization. Taken together, our results suggest that activation of PMCA in caveolae is modulated by phosphatidylserine, and phosphatidylserine externalization induced by MbetaCD reduced the interaction of phosphatidylserine with PMCA, subsequently PMCA-mediated Ca(2+) efflux in ECV 304 cells.

    Cell calcium 2009;45;2;177-84

  • Muscarinic-induced recruitment of plasma membrane Ca2+-ATPase involves PSD-95/Dlg/Zo-1-mediated interactions.

    Kruger WA, Yun CC, Monteith GR and Poronnik P

    School of Biomedical Sciences and School of Pharmacy, The University of Queensland, Brisbane QLD 4072, Australia.

    Efflux of cytosolic Ca2+ mediated by plasma membrane Ca2+-ATPases (PMCA) plays a key role in fine tuning the magnitude and duration of Ca2+ signaling following activation of G-protein-coupled receptors. However, the molecular mechanisms that underpin the trafficking of PMCA to the membrane during Ca2+ signaling remain largely unexplored in native cell models. One potential mechanism for the recruitment of proteins to the plasma membrane involves PDZ interactions. In this context, we investigated the role of PMCA interactions with the Na+/H+ exchanger regulatory factor 2 (NHERF-2) during muscarinic-induced Ca2+ mobilization in the HT-29 epithelial cell line. GST pull-downs in HT-29 cell lysates showed that the PDZ2 module of NHERF-2 bound to the PDZ binding motif on the C terminus of PMCA. Co-immunoprecipitations confirmed that PMCA1b and NHERF-2 associated under normal conditions in HT-29 cells. Cell surface biotinylations revealed significant increases in membrane-associated NHERF-2 and PMCA within 60 s following muscarinic activation, accompanied by increased association of the two proteins as seen by confocal microscopy. The recruitment of NHERF-2 to the membrane preceded that of PMCA, suggesting that NHERF-2 was involved in nucleating an efflux complex at the membrane. The muscarinic-mediated translocation of PMCA was abolished when NHERF-2 was silenced, and the rate of relative Ca2+ efflux was also reduced. These experiments also uncovered a NHERF-2-independent PMCA retrieval mechanism. Our findings describe rapid agonist-induced translocation of PMCA in a native cell model and suggest that NHERF-2 plays a key role in scaffolding and maintaining PMCA at the cell membrane.

    Funded by: NIDDK NIH HHS: DK061418, R01 DK061418, R01 DK061418-05, R01 DK061418-06A1

    The Journal of biological chemistry 2009;284;3;1820-30

  • Functional and structural demonstration of the presence of Ca-ATPase (PMCA) in both microvillous and basal plasma membranes from syncytiotrophoblast of human term placenta.

    Marín R, Riquelme G, Godoy V, Díaz P, Abad C, Caires R, Proverbio T, Piñero S and Proverbio F

    Laboratorio de Bioenergética Celular, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), A.P. 21827, Caracas 1020A, Venezuela. rmarin@ivic.ve

    It is known that human syncytiotrophoblast (hSCT) actively transports more than 80% of the Ca2+ that goes from maternal to fetal circulation. Transepithelial transport of Ca2+ is carried out through channels, transporters and exchangers located in both microvillous (MVM) and basal (BM) plasma membranes. The plasma membrane Ca-ATPase (PMCA) is the most important mechanism of Ca2+ homeostasis control in the human placenta. In this work, we reexamined the distribution of PMCA in isolated hSCT of term placenta. The PMCA activity was determined in isolated hSCT plasma membranes. A partial characterization of the PMCA activity was performed, including an evaluation of the sensitivity of this enzyme to an in vitro induced lipid peroxidation. Expression of the PMCA in hSCT plasma membranes and tissue sections was investigated using Western blots and immunohistochemistry, respectively. Our study demonstrates, for the first time, a correlation between the activity and structural distribution of PMCA in both MVM and BM of hSCT. It also demonstrates a higher PMCA activity and expression in MVM as compared to BM. Finally, PMCA4 seems to be preferentially distributed in both hSCT plasma membranes, while PMCA1 is shown to be present in the hSCT homogenate. However, the membrane fractions did not show any PMCA1 labeling. Our results must be taken into account in order to propose a new model for the transport of calcium across the hSCT.

    Placenta 2008;29;8;671-9

  • Sarco(endo)plasmic reticulum and plasmalemmal Ca(2+)-ATPase activities in cremaster muscles and sacs differ according to the associated inguinal pathology.

    Ulusu NN, Tandoğan B and Tanyel FC

    Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, Turkey. nnulusu@hacettepe.edu.tr

    Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and plasmalemmal Ca(2+)-ATPase (PMCA) activities in cremaster muscles and sacs, which have been subjected to different autonomic tonuses, were determined and compared. Samples of cremaster muscles and sacs associated with male or female inguinal hernia, hydrocele or undescended testis were obtained from children during operations and activities of SERCA and PMCA were determined. While highest SERCA and PMCA activities were encountered among cremaster muscles and sacs associated with undescended testis, least activities were encountered among structures associated with hydrocele. The alterations in SERCA and PMCA activities in cremaster muscles associated with undescended testis appear to reflect the attempts at maintaining the levels of cytosolic calcium. Despite similar total calcium contents, lower SERCA and PMCA activities were found in sacs associated with hydrocele compared to those associated with undescended testis suggest a difference among the levels of cytosolic calcium.

    Cell biochemistry and function 2007;25;5;515-9

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • Deletions in the A(L) region of the h4xb plasma membrane Ca(2+) pump. High apparent affinity for Ca(2+) of a deletion mutant resembling the alternative spliced form h4zb.

    de Tezanos Pinto F and Adamo HP

    IQUIFIB-Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires Junín 956, 1113 Buenos Aires, Argentina.

    Mutants of the plasma membrane Ca(2+) pump (human isoform 4xb) with deletions in the linker between domain A and transmembrane segment M3 (A(L) region) were constructed and expressed in Chinese hamster ovary cells. The total or partial removal of the amino acid segment 300-349 did not change the maximal Ca(2+) transport activity, but mutants with deletions involving residues 300-338 exhibited a higher apparent affinity for Ca(2+) than the wild type h4xb enzyme. Deletion of the putative acidic lipid interacting sequence (residues 339-349) had no observable functional consequences. The removal of either residues 300-314 or 313-338 resulted in a similar increase in the apparent Ca(2+) affinity of the pump although the increase was somewhat lower than that obtained by the deletion 300-349 suggesting that both deletions affected the same structural determinant. The results show that alterations in the region of the alternative splicing site A change the sensitivity to Ca(2+) of the human isoform 4 of the PMCA.

    FEBS letters 2006;580;6;1576-80

  • The influence of membrane lipid structure on plasma membrane Ca2+ -ATPase activity.

    Tang D, Dean WL, Borchman D and Paterson CA

    Department of Ophthalmology and Visual Science, University of Louisville School of Medicine, KY 40202, USA.

    Lipid composition and Ca(2+)-ATPase activity both change with age and disease in many tissues. We explored relationships between 1f40 lipid composition/structure and plasma membrane Ca(2+)-ATPase (PMCA) activity. PMCA was purified from human erythrocytes and was reconstituted into liposomes prepared from human ocular lens membrane lipids and synthetic lipids. Lens lipids were used in this study as a model for naturally ordered lipids, but the influence of lens lipids on PMCA function is especially relevant to the lens since calcium homeostasis is vital to lens clarity. Compared to fiber cell lipids, epithelial lipids exhibited an ordered to disordered phase transition temperature that was 12 degrees C lower. Reconstitution of PMCA into lipids was essential for maximal activity. PMCA activity was two to three times higher when the surrounding phosphatidylcholine molecules contained acyl chains that were ordered (stiff) compared to disordered (fluid) acyl chains. In a completely ordered lipid hydrocarbon chain environment, PMCA associates more strongly with the acidic lipid phosphatidylserine in comparison to phosphatidylcholine. PMCA associates much more strongly with phosphatidylcholine containing disordered hydrocarbon chains than ordered hydrocarbon chains. PMCA activity is influenced by membrane lipid composition and structure. The naturally high degree of lipid order in plasma membranes such as those found in the human lens may serve to support PMCA activity. The absence of PMCA activity in the cortical region of human lenses is apparently not due to a different lipid environment. Changes in lipid composition such as those observed with age or disease could potentially influence PMCA function.

    Funded by: PHS HHS: EYO6916

    Cell calcium 2006;39;3;209-16

  • Plasma membrane Ca2+ ATPase isoform 1 down-regulated in human oral cancer.

    Saito K, Uzawa K, Endo Y, Kato Y, Nakashima D, Ogawara K, Shiba M, Bukawa H, Yokoe H and Tanzawa H

    Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, Japan.

    The plasma membrane Ca(2+) ATPase (PMCA) is an essential regulator of free intracellular calcium. Recent studies have reported aberrant expression of the PMCA1 gene, a member of the PMCA family, in several cancer cell types. To elucidate the contribution of PMCA1 to oral carcinogenesis, we analyzed genetic and epigenetic changes and mRNA and protein expression in primary oral squamous cell carcinomas (OSCCs), oral premalignant lesions (OPLs), and OSCC-derived cell lines. The PMCA1 gene was epigenetically inactivated, but not mutated in the eight OSCC-derived cell lines tested. In clinical samples, frequent down-regulation of PMCA1 protein expression was found not only in primary OSCCs (43%), but also in OPLs (40%). Real-time quantitative reverse transcriptase-polymerase chain reaction data were consistent with the protein expression status. These results suggest that inactivation of the PMCA1 gene is a frequent and early event during oral carcinogenesis, and gene expression may be regulated by an epigenetic mechanism.

    Oncology reports 2006;15;1;49-55

  • Collaborative effect of SERCA and PMCA in cytosolic calcium homeostasis in human platelets.

    Redondo PC, Rosado JA, Pariente JA and Salido GM

    Department of Physiology, University of Extremadura, 10071 Cáceres, Spain. pcr@unex.es

    Intracellular free Ca2+ concentration ([Ca2+]c) is finely regulated by several mechanisms that either increase or reduce [Ca2+]c. Two different Ca2+ pumps have been described so far as the main mechanisms for Ca2+ removal from the cytosol, either by its sequestration into the stores, mediated by the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) or by Ca2+ extrusion to the extracellular medium, by the plasma membrane Ca2+-ATPase (PMCA). We have used inhibitors of these pumps to analyze their Ca2+ clearance efficacy in human platelets stimulated by the physiological agonist thrombin. Results demonstrate that, after platelet stimulation with thrombin, activation of SERCA precedes that of PMCA, although the ability of PMCA to remove Ca2+ from the cytosol last longer than that of SERCA. The efficacy of SERCA and PMCA removing Ca2+ from the cytosol is reduced when the concentration of thrombin increases. This phenomenon correlates with the greater increase in [Ca2+]c induced by higher concentrations of thrombin, which further confirms that SERCA and PMCA activities are regulated by [Ca2+]c.

    Journal of physiology and biochemistry 2005;61;4;507-16

  • Antisense-mediated Inhibition of the plasma membrane calcium-ATPase suppresses proliferation of MCF-7 cells.

    Lee WJ, Robinson JA, Holman NA, McCall MN, Roberts-Thomson SJ and Monteith GR

    School of Pharmacy, University of Queensland, Brisbane, Queensland 4072, Australia.

    Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anti-cancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.

    The Journal of biological chemistry 2005;280;29;27076-84

  • Calcium pumps of plasma membrane and cell interior.

    Strehler EE and Treiman M

    Department of Biochemistry and Molecular Biology, Mayo Clinic, College of Medicine, Rochester, Minnesota, USA.

    Calcium entering the cell from the outside or from intracellular organelles eventually must be returned to the extracellular milieu or to intracellular storage organelles. The two major systems capable of pumping Ca2+ against its large concentration gradient out of the cell or into the sarco/endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue- and cell type-specific patterns of expression. Different PMCA and SERCA isoforms are characterized by different regulatory and kinetic properties that likely are optimized for the distinct functional tasks fulfilled by each pump in setting resting cytosolic or intra-organellar Ca2+ levels, and in shaping intracellular Ca2+ signals with spatial and temporal resolution. The loss or malfunction of specific Ca2+ pump isoforms is associated with defects such as deafness, ataxia or heart failure. Understanding the involvement of different Ca2+ pump isoforms in the pathogenesis of disease allows their identification as therapeutic targets for the development of selective strategies to prevent or combat the progression of these disorders.

    Funded by: NIDCD NIH HHS: DC-04200; NIGMS NIH HHS: GM-58710

    Current molecular medicine 2004;4;3;323-35

  • Expression, purification, and characterization of isoform 1 of the plasma membrane Ca2+ pump: focus on calpain sensitivity.

    Guerini D, Pan B and Carafoli E

    Institute of Biochemistry, Swiss Federal Institute of Technology, CH-8092 Zürich, Switzerland.

    The plasma membrane Ca2+ ATPase isoform 1(PMCA1) is ubiquitously distributed in tissues and cells, but only scarce information is available on its properties. The isoform was overexpressed in Sf9 cells, purified on calmodulin columns, and characterized functionally. The level of expression was very low, but sufficient amounts of the protein could be isolated for biochemical characterization. The affinity of PMCA1 for calmodulin was similar to that of PMCA4, the other ubiquitous PMCA isoform. The affinity of PMCA1 for ATP, evaluated by the formation of the phosphorylated intermediate, was higher than that of the PMCA4 pump. The recombinant PMCA1 pump was a much better substrate for the cAMP-dependent protein kinase than the PMCA2 and PMCA4 isoforms. Pulse and chase experiments on Sf9 cells overexpressing the PMCA pumps showed that PMCA1 was much less stable than the PMCA4 and PMCA2 isoforms, i.e. PMCA1 had a much higher sensitivity to degradation by calpain. The effect of calpain was not the result of a general higher susceptibility of the PMCA1 to proteolytic degradation, because the pattern of degradation by trypsin was the same in the three isoforms.

    The Journal of biological chemistry 2003;278;40;38141-8

  • Expression and role of calcium-ATPase pump and sodium-calcium exchanger in differentiated trophoblasts from human term placenta.

    Moreau R, Daoud G, Masse A, Simoneau L and Lafond J

    Département des Sciences Biologiques, Université du Québec á Montréal, Québec, Canada.

    Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are not completely elucidated. Especially, mechanisms of syncytiotrophoblast Ca(2+) extrusion into fetal circulation remain to be established. In the current study we have investigated the characteristics of Ca(2+) efflux in syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblasts isolated from human term placenta. Time-courses of Ca(2+) uptake by differentiated human trophoblasts displayed rapid initial entry (initial velocity (V(i)) of 8.82 +/- 0.86 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid decline of cell-associated (45)Ca(2+) with a V(i) of efflux (V(ie)) of 8.90 +/- 0.96 nmol/mg protein/min. Expression of membrane systems responsible for intracellular Ca(2+) extrusion from differentiated human trophoblast were investigated by RT-PCR. Messenger RNAs of four known isoforms of PMCA (PMCA 1-4) were detected. Messenger RNAs of two cloned human NCX isoforms (NCX1 and NCX3) were also revealed. More specifically, both splice variants NCX1.3 and NCX1.4 were amplified by PCR with total RNA of differentiated human trophoblast cells. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. However, erythrosine B (inhibitor of PMCA) time- and dose-dependently increased cell associated (45)Ca(2+) suggesting a principal role of plasma membrane Ca(2+)-ATPase (PMCA) in the intracellular Ca(2+) extrusion of syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblast cells isolated from human term placenta.

    Molecular reproduction and development 2003;65;3;283-8

  • Plasma membrane Ca2+ ATPase isoform 2b interacts preferentially with Na+/H+ exchanger regulatory factor 2 in apical plasma membranes.

    DeMarco SJ, Chicka MC and Strehler EE

    Program in Molecular Neuroscience, Department of Biochemistry and Molecular Biology, Mayo Graduate School, Mayo Clinic, Rochester, Minnesota 55905, USA.

    Spatial and temporal regulation of Ca(2+) signaling require the assembly of multiprotein complexes linking molecules involved in Ca(2+) influx, sensing, buffering, and extrusion. Recent evidence indicates that plasma membrane Ca(2+) ATPases (PMCAs) participate in the control of local Ca(2+) fluxes, but the mechanism of multiprotein complex formation of specific PMCAs is poorly understood. Using the PMCA2b COOH-terminal tail as bait in a yeast two-hybrid screen, we identified the PSD-95, Dlg, ZO-1 (PDZ) domain-containing Na(+)/H(+) exchanger regulatory factor-2 (NHERF2) as an interacting partner. Protein pull-down and coimmunoprecipitation experiments using recombinant PMCA2b and PMCA4b as well as NHERF1 and NHERF2 showed that the interaction of PMCA2b with NHERF2 was specific and selective. PMCA4b did not interact with either of the NHERFs, and PMCA2b selectively preferred NHERF2 over NHERF1. Green fluorescent protein-tagged PMCA2b was expressed at the apical membrane in Madin-Darby canine kidney epithelial cells, where it colocalized with apically targeted NHERF2. Our study identifies NHERF2 as the first specific PDZ partner for PMCA2b not shared with PMCA4b, and demonstrates that PMCA splice forms differing only minimally in their COOH-terminal residues interact with unique PDZ proteins. NHERFs have been implicated in the targeting, retention and regulation of membrane proteins including the beta(2)-adrenergic receptor, cystic fibrosis transmembrane conductance regulator, and Trp4 Ca(2+) channel, and NHERF2 is now shown to also interact with PMCA2b. This interaction may allow the functional assembly of PMCA2b in a multiprotein Ca(2+) signaling complex, facilitating integrated cross-talk between local Ca(2+) influx and efflux.

    Funded by: NIGMS NIH HHS: GM58710

    The Journal of biological chemistry 2002;277;12;10506-11

  • Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps.

    Strehler EE and Zacharias DA

    Department of Biochemistry and Molecular Biology, Mayo Graduate School, Mayo Clinic/Foundation, Rochester, Minnesota, USA. strehler.emanuel@mayo.edu

    Calcium pumps of the plasma membrane (also known as plasma membrane Ca(2+)-ATPases or PMCAs) are responsible for the expulsion of Ca(2+) from the cytosol of all eukaryotic cells. Together with Na(+)/Ca(2+) exchangers, they are the major plasma membrane transport system responsible for the long-term regulation of the resting intracellular Ca(2+) concentration. Like the Ca(2+) pumps of the sarco/endoplasmic reticulum (SERCAs), which pump Ca(2+) from the cytosol into the endoplasmic reticulum, the PMCAs belong to the family of P-type primary ion transport ATPases characterized by the formation of an aspartyl phosphate intermediate during the reaction cycle. Mammalian PMCAs are encoded by four separate genes, and additional isoform variants are generated via alternative RNA splicing of the primary gene transcripts. The expression of different PMCA isoforms and splice variants is regulated in a developmental, tissue- and cell type-specific manner, suggesting that these pumps are functionally adapted to the physiological needs of particular cells and tissues. PMCAs 1 and 4 are found in virtually all tissues in the adult, whereas PMCAs 2 and 3 are primarily expressed in excitable cells of the nervous system and muscles. During mouse embryonic development, PMCA1 is ubiquitously detected from the earliest time points, and all isoforms show spatially overlapping but distinct expression patterns with dynamic temporal changes occurring during late fetal development. Alternative splicing affects two major locations in the plasma membrane Ca(2+) pump protein: the first intracellular loop and the COOH-terminal tail. These two regions correspond to major regulatory domains of the pumps. In the first cytosolic loop, the affected region is embedded between a putative G protein binding sequence and the site of phospholipid sensitivity, and in the COOH-terminal tail, splicing affects pump regulation by calmodulin, phosphorylation, and differential interaction with PDZ domain-containing anchoring and signaling proteins. Recent evidence demonstrating differential distribution, dynamic regulation of expression, and major functional differences between alternative splice variants suggests that these transporters play a more dynamic role than hitherto assumed in the spatial and temporal control of Ca(2+) signaling. The identification of mice carrying PMCA mutations that lead to diseases such as hearing loss and ataxia, as well as the corresponding phenotypes of genetically engineered PMCA "knockout" mice further support the concept of specific, nonredundant roles for each Ca(2+) pump isoform in cellular Ca(2+) regulation.

    Funded by: NIGMS NIH HHS: GM-58710

    Physiological reviews 2001;81;1;21-50

  • Regulation of plasma membrane Ca2+-ATPase by small GTPases and phosphoinositides in human platelets.

    Rosado JA and Sage SO

    Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom.

    We have investigated the restoration of [Ca(2+)](i) in human platelets following the discharge of the intracellular Ca(2+) stores. We found that the plasma membrane Ca(2+)-ATPase is the main mechanism involved in Ca(2+) extrusion in human platelets. Treatment of platelets with the farnesylcysteine analogs, farnesylthioacetic acid and N-acetyl-S-geranylgeranyl-l-cysteine, inhibitors of activation of Ras proteins, accelerated the rate of decay of [Ca(2+)](i) to basal levels after activation with thapsigargin combined with a low concentration of ionomycin, indicating that Ras proteins are involved in the negative regulation of Ca(2+) extrusion. Rho A, which is involved in actin polymerization, was not responsible for this effect. Consistent with this, the actin polymerization inhibitors, cytochalasin D and latrunculin A, did not alter the recovery of [Ca(2+)](i). Activation of human platelets with thapsigargin and ionomycin stimulated the tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase, a mechanism that was inhibited by farnesylcysteine analogs, suggesting that Ras proteins could regulate Ca(2+) extrusion by mediating tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase. Treatment of platelets with LY294002, a specific inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinase, resulted in a reduction in the rate of recovery of [Ca(2+)](i) to basal levels, suggesting that the products of these kinases are involved in stimulating Ca(2+) extrusion in human platelets.

    The Journal of biological chemistry 2000;275;26;19529-35

  • NMR solution structure of a complex of calmodulin with a binding peptide of the Ca2+ pump.

    Elshorst B, Hennig M, Försterling H, Diener A, Maurer M, Schulte P, Schwalbe H, Griesinger C, Krebs J, Schmid H, Vorherr T and Carafoli E

    Institute of Organic Chemistry, University of Frankfurt, Germany.

    The three-dimensional structure of the complex between calmodulin (CaM) and a peptide corresponding to the N-terminal portion of the CaM-binding domain of the plasma membrane calcium pump, the peptide C20W, has been solved by heteronuclear three-dimensional nuclear magnetic resonance (NMR) spectroscopy. The structure calculation is based on a total of 1808 intramolecular NOEs and 49 intermolecular NOEs between the peptide C20W and calmodulin from heteronuclear-filtered NOESY spectra and a half-filtered experiment, respectively. Chemical shift differences between free Ca(2+)-saturated CaM and its complex with C20W as well as the structure calculation reveal that C20W binds solely to the C-terminal half of CaM. In addition, comparison of the methyl resonances of the nine assigned methionine residues of free Ca(2+)-saturated CaM with those of the CaM/C20W complex revealed a significant difference between the N-terminal and the C-terminal domain; i.e., resonances in the N-terminal domain of the complex were much more similar to those reported for free CaM in contrast to those in the C-terminal half which were significantly different not only from the resonances of free CaM but also from those reported for the CaM/M13 complex. As a consequence, the global structure of the CaM/C20W complex is unusual, i.e., different from other peptide calmodulin complexes, since we find no indication for a collapsed structure. The fine modulation in the peptide protein interface shows a number of differences to the CaM/M13 complex studied by Ikura et al. [Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638]. The unusual binding mode to only the C-terminal half of CaM is in agreement with the biochemical observation that the calcium pump can be activated by the C-terminal half of CaM alone [Guerini, D., Krebs, J., and Carafoli, E. (1984) J. Biol. Chem. 259, 15172-15177].

    Biochemistry 1999;38;38;12320-32

  • Large-scale concatenation cDNA sequencing.

    Yu W, Andersson B, Worley KC, Muzny DM, Ding Y, Liu W, Ricafrente JY, Wentland MA, Lennon G and Gibbs RA

    A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

    Funded by: NHGRI NIH HHS: 1F32 HG00169-01, F32 HG000169, F33 HG000210, P30 HG00210-05, R01 HG00823, U54 HG003273

    Genome research 1997;7;4;353-8

  • Investigation of the Met-267 Arg exchange in isoform 1 of the human plasma membrane calcium pump in patients with essential hypertension by the amplification-created restriction site technique.

    Benkwitz C, Kubisch C, Kraft K and Neyses L

    Department of Medicine, University of Würzburg, Germany.

    Alterations in Ca2+ homeostasis have been proposed to be a primary factor in the pathogenesis of essential hypertension. In this disease increased intracellular Ca2+ levels have repeatedly been reported in various cell types. Because of its prominent role in cellular calcium homeostasis in vascular smooth muscle cells, modifications of the plasma membrane Ca2+-ATPase (PMCA) pump have been suggested to contribute to an increased contractile tone of small blood vessels. This pump is a calmodulin-dependent Ca2+-ATPase that ejects Ca2+ from the cytosol into the extracellular space. Recently a mutational thymidine (T)-->guanosine (G) transversion in isoform 1 of the PMCA has been identified resulting in the substitution of a methionine (Met) by an arginine (Arg) at amino acid position 267 in a highly conserved domain of the pump molecule. The aim of our study was to determine the prevalence of this polymorphism in the normal population and to investigate whether the Met-267 Arg occurs more frequently in patients with essential hypertension than in normotensives. To detect the mutational change we modified a method based on the technique of amplification-created restriction sites (ACRS) using three base exchanges in the diagnostic primer. Samples from 100 hypertensive and 60 normotensive subjects revealed a thymidine at nucleotide position 981. These data suggest that ACRS is feasible in spite of extensive primer modifications (e.g., three mismatched bases) in contrast to the previously used one or two and may therefore be conceptually suitable to detect almost any base changes in the genome. The described T-->G transversion is a rare polymorphism and is presumably not related to common forms of essential hypertension.

    Journal of molecular medicine (Berlin, Germany) 1997;75;1;62-6

  • Structural organization, ion transport, and energy transduction of P-type ATPases.

    Møller JV, Juul B and le Maire M

    Department of Biophysics, University of Aarhus, Denmark.

    Biochimica et biophysica acta 1996;1286;1;1-51

  • A "double adaptor" method for improved shotgun library construction.

    Andersson B, Wentland MA, Ricafrente JY, Liu W and Gibbs RA

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.

    The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

    Funded by: NHGRI NIH HHS: R01 HG00823

    Analytical biochemistry 1996;236;1;107-13

  • Quantitative analysis of alternative splicing options of human plasma membrane calcium pump genes.

    Stauffer TP, Hilfiker H, Carafoli E and Strehler EE

    The Journal of biological chemistry 1994;269;50;32022

  • Quantitative analysis of alternative splicing options of human plasma membrane calcium pump genes.

    Stauffer TP, Hilfiker H, Carafoli E and Strehler EE

    Laboratory for Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich.

    The alternative splicing options and the quantitative tissue distribution of the transcripts of the four currently known human plasma membrane calcium pump (PMCA) genes have been analyzed in seven tissues (cerebral cortex, skeletal and heart muscle, stomach, liver, lung, and kidney) by quantitative polymerase chain reaction on reverse transcribed mRNA with glyceraldehyde-3-phosphate dehydrogenase as the internal standard. The mRNAs of genes 1 and 4 were found to be present in similar amounts in all tissues, whereas the transcripts of genes 2 and 3 were expressed in a tissue-specific manner, i.e. their amounts were highest in fetal skeletal muscle and brain. Alternative splicing was found to occur in the PMCA transcripts at two major regulatory sites (sites A and C), adjacent to the amino-terminal phospholipid-responsive region and within the carboxyl-terminal calmodulin binding domain, respectively. Novel splicing variants not described previously for human genes were detected for hPMCA3 and 4 at site A and for hPMCA1, 2, and 3 at site C. For all genes a common splice variant was found at both splice sites. The common splice variant at site A was characterized by the inclusion of a small exon (hPMCA1, 39 base pairs (bp); hPMCA2, 42 bp; hPMCA3, 42 bp; hPMCA4, 36 bp). In the common splice variant at site C, an exon (hPMCA1, 154 bp; hPMCA2, 227 bp; hPMCA3, 154 bp; hPMCA4, 178 bp) was excluded in the mRNA. All genes normally express these main splice variants in all tissues in which the corresponding isoform is present. The splicing complexity at site C was found to be augmented in the transcripts of PMCA2 and PMCA3 through the use of additional exons, and in PMCA1 and 3 through the use of additional internal splice sites in the single alternatively spliced 154-base pair exon.

    The Journal of biological chemistry 1993;268;34;25993-6003

  • Human and rat intestinal plasma membrane calcium pump isoforms.

    Howard A, Legon S and Walters JR

    Department of Medicine, Royal Postgraduate Medical School, London, United Kingdom.

    The intestinal basolateral membrane Ca(2+)-transporting adenosinetriphosphatase is the energy-dependent step in the absorption of dietary Ca2+ by the vitamin D-dependent transcellular pathway. Multiple plasma membrane Ca(2+)-pump isoforms are produced from four genes (PMCA1 to 4) and alternative mRNA splicing. We have studied which isoforms are detectable in adult human and rat gastrointestinal tissues by polymerase chain reaction (PCR) amplification, sequencing, and blotting. PMCA1 was the predominant gene product amplified from human small intestinal mucosa, although a minor additional variant lacking the exon at splice site B was detected, which resembled that described for PMCA4. Of the variants described at site C, only the shortest transcript of PMCA1 was amplified; both previously described forms of PMCA4 were found, particularly in colon where PMCA4 predominated. From rat intestinal cDNA, mixed primer PCR amplified PMCA1 and a novel sequence, the rat PMCA4 homologue, which was expressed in many tissues including small intestinal muscle and colon. However, PMCA1 was overwhelmingly predominant in the mucosa of the small intestine, being most abundant in duodenum. These results suggest the involvement of the Ca(2+)-pump isoform PMCA1b in intestinal Ca2+ absorption.

    The American journal of physiology 1993;265;5 Pt 1;G917-25

  • Structure of the gene encoding the human plasma membrane calcium pump isoform 1.

    Hilfiker H, Strehler-Page MA, Stauffer TP, Carafoli E and Strehler EE

    Laboratory for Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.

    The complete structure of the gene for the human plasma membrane calcium ATPase isoform 1 (hPMCA1) has been elucidated. The protein is encoded by 21 exons present on overlapping clones covering more than 100 kilobases (kb) of DNA. An intron of over 35 kb separates the 5'-untranslated exon 1 from the exon containing the translational start codon. The entire putative promoter and 5'-flanking region is embedded in a CpG island and is characterized by the presence of numerous Sp1 factor-binding sequences and by the absence of a TATA box. In accordance with the ubiquitous tissue distribution of its mRNA these results suggest that the hPMCA1 gene is of the housekeeping type. No alternative splicing comparable to that identified in PMCA2 RNAs at site "A" and in PMCA3 RNAs close to site "C" seems to occur in hPMCA1 transcripts; however, a region in intron 6 shows significant resemblance to the site "A" alternatively spliced exons in PMCA2 and may represent a pseudoexon or a functional exon not yet detected in any PMCA1 mRNA. At six positions, intron interruptions in the hPMCA1 gene correlate with the boundaries of putative transmembrane domains in the protein, whereas most of the remaining intron positions do not show an obvious correlation with the proposed pump domain structure. The limited conservation of intron positions in different P-type pump genes indicates their early separation from a common ancestor.

    The Journal of biological chemistry 1993;268;26;19717-25

  • Molecular cloning of a plasma membrane calcium pump from human osteoblasts.

    Kumar R, Haugen JD and Penniston JT

    Nephrology Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota.

    The osteoblast plays a critical role in bone formation, bone remodelling, bone matrix formation, and matrix calcification. To better understand the process of osteoblast-controlled bone formation, we determined the structure and isoform types of the plasma membrane calcium pump from normal human osteoblasts. A complementary DNA library from normal human osteoblasts was screened for plasma membrane calcium pump clones. Sequencing and analysis of cDNA clones revealed the presence of a 3986 base pair cDNA that encoded a 1220 amino acid protein that was similar to the human plasma membrane calcium pump isoform 1. Polyadenylated RNA from human osteoblast cells contains bands of RNA approximately 5050 and 6750 bases long. Reverse transcription of polyadenylated RNA from human osteoblasts followed by amplification of the RNA-DNA duplex with calcium pump isoform-specific primers revealed the presence of isoforms 1 and 2 of the calcium pump. Isoform 4 was not detected. We conclude that normal adult human osteoblasts contain a plasma membrane calcium pump that is similar to the human plasma membrane calcium pump isoform 1. It is likely that this pump plays an important role in the cell biology of the human osteoblast.

    Funded by: NIDDK NIH HHS: DK 25409, DK 42971, R01 DK025409, R01 DK058546

    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 1993;8;4;505-13

  • Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

    Kessler F, Falchetto R, Heim R, Meili R, Vorherr T, Strehler EE and Carafoli E

    Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich.

    The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.

    Biochemistry 1992;31;47;11785-92

  • Protein kinase C phosphorylates the carboxyl terminus of the plasma membrane Ca(2+)-ATPase from human erythrocytes.

    Wang KK, Wright LC, Machan CL, Allen BG, Conigrave AD and Roufogalis BD

    Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.

    Purified Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase) from human erythrocytes was phosphorylated with a stoichiometry of about 1 mol of phosphate/mol of ATPase at both threonine and serine residues by purified rat brain type III protein kinase C. In the presence of calmodulin, the phosphorylation was markedly reduced. Labeled phosphate from [gamma-32P]ATP was retained on an 86-kDa calmodulin-binding tryptic fragment of Ca(2+)-ATPase but not on 82- and 77-kDa non-calmodulin-binding fragments. Similarly, fragmentation of the phosphorylated Ca(2+)-ATPase by calpain I revealed that calmodulin-binding fragments (127 and 125 kDa) retained phosphate label whereas a non-calmodulin-binding fragment (124 kDa) did not. The calmodulin-binding domain, located about 12 kDa from the carboxyl terminus of the Ca(2+)-ATPase, was thus located as a site of protein kinase C phosphorylation. A synthetic peptide corresponding to a segment of the calmodulin-binding domain (H2 N-R-G-L-N-R-I-Q-T-Q-I-K-V-V-N-COOH) was indeed phosphorylated at the single threonine residue within this sequence. The additional serine phosphorylation site was carboxyl terminal to the calmodulin domain. Phosphorylation by purified type III protein kinase C (canine heart) antagonized the calmodulin activation of the Ca(2+)-ATPase, particularly at lower Ca2+ concentrations (0.2-1.0 microM). By contrast, a purified but unresolved protein kinase C isoenzyme mixture from rat brain stimulated the activity of Ca(2+)-ATPase prepared in asolectin, but not glycerol, by more than 2-fold in the presence of the ionophore A23187, without increasing its Ca2+ sensitivity. The results clearly indicate that human erythrocyte Ca(2+)-ATPase is a substrate of protein kinase C, but the effect of phosphorylation on the activity of the enzyme depends on the isoenzyme form of protein kinase C used and on the lipid associated with the Ca(2+)-ATPase.

    The Journal of biological chemistry 1991;266;14;9078-85

  • Localization of two genes encoding plasma membrane Ca2(+)-transporting ATPases to human chromosomes 1q25-32 and 12q21-23.

    Olson S, Wang MG, Carafoli E, Strehler EE and McBride OW

    Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.

    Human plasma membrane Ca2(+)-ATPase (PMCA) isoforms are encoded by at least four separate genes and the diversity of these enzymes is further increased by alternative splicing of transcripts. Cloned cDNAs for two of these isoforms have been used as probes to localize chromosomally the human PMCA1 (ATP2B1) gene to 12q21-q23 and PMCA4 (ATP2B2) to 1q25-q32. These results were obtained by three independent methods, including Southern analysis of human-rodent somatic cell hybrids, in situ hybridization of human metaphase spreads, and genetic linkage analysis in the CEPH pedigrees. High-frequency RFLPs detected at each locus were used in these linkage analyses. No evidence was obtained for multiple copies of the gene at either locus. A cross-hybridizing sequence was detected with PMCA4 probes on Xq13-qter at low stringency. Further studies are required to determine whether this X-chromosomal sequence represents a third member of the PMCA gene family.

    Genomics 1991;9;4;629-41

  • mRNAs for plasma membrane calcium pump isoforms differing in their regulatory domain are generated by alternative splicing that involves two internal donor sites in a single exon.

    Strehler EE, Strehler-Page MA, Vogel G and Carafoli E

    Laboratory for Biochemistry, Swiss Federal Institute of Technology, Zurich.

    cDNA clones coding for human plasma membrane Ca2+ pump isoforms have been isolated from a fetal skeletal muscle cDNA library. Compared with the sequence of a teratoma cDNA-encoded pump these clones specify isoforms that contain either 29- or 38-amino acid insertions within the calmodulin-binding region. Replacement of two basic arginine residues by an aspartic acid and a glutamine residue could influence the binding of calmodulin to these isoforms. RNase mapping shows that RNA species containing the 29-residue-encoding insertion are particularly abundant in skeletal muscles. The sequences coding for the insertions are present on a single 154-base-pair exon, as demonstrated by an analysis of the corresponding genomic region, and they are included in their respective mRNAs by alternative splicing involving the differential usage of two internal "cryptic" donor splice sites in the presence of a nearby canonical one. Inclusion of the complete 154-base-pair exon results in an mRNA coding for a pump protein with a shorter C-terminal amino acid sequence that lacks a consensus site for phosphorylation by the cAMP-dependent kinase. Exclusion, inclusion, or partial inclusion of the same exon can thus lead to the production of four different mRNAs from a single gene. When expressed as protein, these mRNAs encode Ca2+ pump isoforms that differ in their C-terminal regulatory domains.

    Proceedings of the National Academy of Sciences of the United States of America 1989;86;18;6908-12

  • Primary structure of the cAMP-dependent phosphorylation site of the plasma membrane calcium pump.

    James PH, Pruschy M, Vorherr TE, Penniston JT and Carafoli E

    Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.

    The primary structure of a region of the erythrocyte plasma membrane calcium pump which is phosphorylated by the cAMP-dependent protein kinase has been determined. The sequence is A-P-T-K-R-N-S-S(P)-P-P-P-S-P-D. The site is located between the calmodulin binding domain and the C-terminus of the ATPase. The ATPase is phosphorylated only at this site by the cAMP-dependent protein kinase, and the phosphorylation is inhibited by calmodulin. The effect of the phosphorylation is to decrease the Km for Ca2+ of the purified ATPase from about 10 microM to about 1.4 microM and to increase the Vmax of ATP hydrolysis about 2-fold.

    Funded by: NIGMS NIH HHS: GM 28835

    Biochemistry 1989;28;10;4253-8

  • Complete primary structure of a human plasma membrane Ca2+ pump.

    Verma AK, Filoteo AG, Stanford DR, Wieben ED, Penniston JT, Strehler EE, Fischer R, Heim R, Vogel G, Mathews S et al.

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905.

    cDNAs coding for a plasma membrane Ca2+ pump were isolated from a human teratoma library and sequenced. The translated sequence contained 1,220 amino acids with a calculated molecular weight of 134,683. All regions of functional importance known from other ion-transporting ATPases could be identified. The translated sequence also contained, near the carboxyl terminus, the calmodulin-binding domain and two domains which are very rich in glutamic acid and aspartic acid. These two domains resemble calmodulin somewhat and one of them may play a role in the binding of Ca2+. The enzyme also contains domains rich in serine and threonine, one of which has a sequence matching those of good cAMP-dependent protein kinase substrates. The carboxyl-terminal region is important for regulation by calmodulin, proteolysis, and phosphorylation. Near the amino terminus are two domains which are very rich in lysine and glutamic acid, as well as two domains resembling EF hands, one of which also has some resemblance to calmodulin. Comparison of the cloned sequence with peptide sequences from the erythrocyte Ca2+ pump showed that the two proteins have a very high proportion of identical residues but are not 100% identical, indicating that they represent different isozymes.

    Funded by: NIGMS NIH HHS: GM 28835

    The Journal of biological chemistry 1988;263;28;14152-9

Gene lists (9)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000013 G2C Homo sapiens Human mGluR5 Human orthologues of mouse mGluR5 complex adapted from Collins et al (2006) 52
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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