G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
ATPase, Ca++ transporting, plasma membrane 2
G00000072 (Mus musculus)

Databases (7)

ENSG00000157087 (Ensembl human gene)
491 (Entrez Gene)
411 (G2Cdb plasticity & disease)
ATP2B2 (GeneCards)
108733 (OMIM)
Marker Symbol
Protein Sequence
Q01814 (UniProt)

Synonyms (1)

  • PMCA2

Literature (32)

Pubmed - other

  • Identification of novel candidate genes for treatment response to risperidone and susceptibility for schizophrenia: integrated analysis among pharmacogenomics, mouse expression, and genetic case-control association approaches.

    Ikeda M, Tomita Y, Mouri A, Koga M, Okochi T, Yoshimura R, Yamanouchi Y, Kinoshita Y, Hashimoto R, Williams HJ, Takeda M, Nakamura J, Nabeshima T, Owen MJ, O'Donovan MC, Honda H, Arinami T, Ozaki N and Iwata N

    MRC, Centre for Neuropsychiatric Genetics and Genomics, Department of Psychological Medicine and Neurology, School of Medicine, Cardiff University, Cardiff, United Kingdom.

    Background: Pharmacogenomic approaches based on genomewide sets of single nucleotide polymorphisms (SNPs) are now feasible and offer the potential to uncover variants that influence drug response.

    Methods: To detect potential predictor gene variants for risperidone response in schizophrenic subjects, we performed a convergent analysis based on 1) a genomewide (100K SNP) SNP pharmacogenetic study of risperidone response and 2) a global transcriptome study of genes with mRNA levels influenced by risperidone exposure in mouse prefrontal cortex.

    Results: Fourteen genes were highlighted as of potential relevance to risperidone activity in both studies: ATP2B2, HS3ST2, UNC5C, BAG3, PDE7B, PAICS, PTGFRN, NR3C2, ZBTB20, ST6GAL2, PIP5K1B, EPHA6, KCNH5, and AJAP1. The SNPs related to these genes that were associated in the pharmacogenetic study were further assessed for evidence for association with schizophrenia in up to three case-control series comprising 1564 cases and 3862 controls in total (Japanese [JPN] 1st and 2nd samples and UK sample). Of 14 SNPs tested, one (rs9389370) in PDE7B showed significant evidence for association with schizophrenia in a discovery sample (p(allele) = .026 in JPN_1st, two-tailed). This finding replicated in a joint analysis of two independent case-control samples (p(JPN_2nd+UK) = .008, one-tailed, uncorrected) and in all combined data sets (p(all) = .0014, two-tailed, uncorrected and p(all) = .018, two-tailed, Bonferroni correction).

    Conclusions: We identified novel candidate genes for treatment response to risperidone and provide evidence that one of these additionally may confer susceptibility to schizophrenia. Specifically, PDE7B is an attractive candidate gene, although evidence from integrated methodology, including pharmacogenomics, pharmacotranscriptomic, and case-control association approaches.

    Funded by: Medical Research Council: G0800509

    Biological psychiatry 2010;67;3;263-9

  • Apical localization of PMCA2w/b is lipid raft-dependent.

    Xiong Y, Antalffy G, Enyedi A and Strehler EE

    Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA.

    Alternative splicing of the first intracellular loop differentially targets plasma membrane calcium ATPase (PMCA) isoform 2 to the apical or basolateral membrane in MDCK cells. To determine if the targeting is affected by lipid interactions, we stably expressed PMCA2w/b and PMCA2z/b in MDCK cells, and analyzed the PMCA distribution by confocal fluorescence microscopy and membrane fractionation. PMCA2w/b showed clear apical and lateral distribution, whereas PMCA2z/b was mainly localized to the basolateral membrane. A significant fraction of PMCA2w/b partitioned into low-density membranes associated with lipid rafts. Depletion of membrane cholesterol by methyl-beta-cyclodextrin resulted in reduced lipid raft association and a striking loss of PMCA2w/b from the apical membrane, whereas the lateral localization of PMCA2z/b remained unchanged. Our data indicate that alternative splicing differentially affects the lipid interactions of PMCA2w/b and PMCA2z/b and that the apical localization of PMCA2w/b is lipid raft-dependent and sensitive to cholesterol depletion.

    Funded by: NINDS NIH HHS: R01 NS051769, R01 NS051769-01A1, R01 NS051769-02, R01 NS051769-03, R01-NS51769

    Biochemical and biophysical research communications 2009;384;1;32-6

  • Life and death of sensory hair cells expressing constitutively active TRPML3.

    Grimm C, Jörs S and Heller S

    Departments of Otolaryngology-Head and Neck Surgery and Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5739, USA.

    The varitint-waddler mutation A419P renders TRPML3 constitutively active, resulting in cationic overload, particularly in sustained influx of Ca(2+). TRPML3 is expressed by inner ear sensory hair cells, and we were intrigued by the fact that hair cells are able to cope with expressing the TRPML3(A419P) isoform for weeks before they ultimately die. We hypothesized that the survival of varitint-waddler hair cells is linked to their ability to deal with Ca(2+) loads due to the abundance of plasma membrane calcium ATPases (PMCAs). Here, we show that PMCA2 significantly reduced [Ca(2+)](i) increase and apoptosis in HEK293 cells expressing TRPML3(A419P). The deaf-waddler isoform of PMCA2, operating at 30% efficacy, showed a significantly decreased ability to rescue the Ca(2+) loading of cells expressing TRPML3(A419P). When we combined mice heterozygous for the varitint-waddler mutant allele with mice heterozygous for the deaf-waddler mutant allele, we found severe hair bundle defects as well as increased hair cell loss compared with mice heterozygous for each mutant allele alone. Furthermore, 3-week-old double mutant mice lacked auditory brainstem responses, which were present in their respective littermates containing single mutant alleles. Likewise, heterozygous double mutant mice exhibited severe circling behavior, which was not observed in mice heterozygous for TRPML3(A419P) or PMCA2(G283S) alone. Our results provide a molecular rationale for the delayed hair cell loss in varitint-waddler mice. They also show that hair cells are able to survive for weeks with sustained Ca(2+) loading, which implies that Ca(2+) loading is an unlikely primary cause of hair cell death in ototoxic stress situations.

    Funded by: NIDCD NIH HHS: DC04563

    The Journal of biological chemistry 2009;284;20;13823-31

  • Genetics meets metabolomics: a genome-wide association study of metabolite profiles in human serum.

    Gieger C, Geistlinger L, Altmaier E, Hrabé de Angelis M, Kronenberg F, Meitinger T, Mewes HW, Wichmann HE, Weinberger KM, Adamski J, Illig T and Suhre K

    Institute of Epidemiology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany.

    The rapidly evolving field of metabolomics aims at a comprehensive measurement of ideally all endogenous metabolites in a cell or body fluid. It thereby provides a functional readout of the physiological state of the human body. Genetic variants that associate with changes in the homeostasis of key lipids, carbohydrates, or amino acids are not only expected to display much larger effect sizes due to their direct involvement in metabolite conversion modification, but should also provide access to the biochemical context of such variations, in particular when enzyme coding genes are concerned. To test this hypothesis, we conducted what is, to the best of our knowledge, the first GWA study with metabolomics based on the quantitative measurement of 363 metabolites in serum of 284 male participants of the KORA study. We found associations of frequent single nucleotide polymorphisms (SNPs) with considerable differences in the metabolic homeostasis of the human body, explaining up to 12% of the observed variance. Using ratios of certain metabolite concentrations as a proxy for enzymatic activity, up to 28% of the variance can be explained (p-values 10(-16) to 10(-21)). We identified four genetic variants in genes coding for enzymes (FADS1, LIPC, SCAD, MCAD) where the corresponding metabolic phenotype (metabotype) clearly matches the biochemical pathways in which these enzymes are active. Our results suggest that common genetic polymorphisms induce major differentiations in the metabolic make-up of the human population. This may lead to a novel approach to personalized health care based on a combination of genotyping and metabolic characterization. These genetically determined metabotypes may subscribe the risk for a certain medical phenotype, the response to a given drug treatment, or the reaction to a nutritional intervention or environmental challenge.

    PLoS genetics 2008;4;11;e1000282

  • RNA--induced silencing of the plasma membrane Ca2+-ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability.

    Fernandes D, Zaidi A, Bean J, Hui D and Michaelis ML

    Gene Logic, Inc., Cambridge, Massachusetts, USA.

    Intraneuronal calcium ([Ca(2+)](i)) regulation is altered in aging brain, possibly because of the changes in critical Ca(2+) transporters. We previously reported that the levels of the plasma membrane Ca(2+)-ATPase (PMCA) and the V(max) for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA(i) techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro--transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca(2+)](i) revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca(2+)](i), slower recovery from KCl-induced Ca(2+) transients, and incomplete return to pre-stimulation Ca(2+) levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca(2+) signaling and enhanced susceptibility to stresses that can alter the regulation of Ca(2+) homeostasis.

    Funded by: NCRR NIH HHS: P20RR017708; NIA NIH HHS: P01AG12993

    Journal of neurochemistry 2007;102;2;454-65

  • A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness.

    Ficarella R, Di Leva F, Bortolozzi M, Ortolano S, Donaudy F, Petrillo M, Melchionda S, Lelli A, Domi T, Fedrizzi L, Lim D, Shull GE, Gasparini P, Brini M, Mammano F and Carafoli E

    Telethon Institute of Genetics and Medicine, 80131 Naples, Italy.

    Ca2+ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca2+ pulse. The G293S and G283S mutations delayed the dissipation of Ca2+ transients induced in CHO cells by InsP3. In organotypic cultures, Ca2+ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca2+ transients induced by Ca2+ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.

    Funded by: Telethon: GGP04169

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;5;1516-21

  • Plasma membrane calcium-ATPase 2 and 4 in human breast cancer cell lines.

    Lee WJ, Roberts-Thomson SJ and Monteith GR

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

    There is evidence to suggest that plasma membrane Ca2+-ATPase (PMCA) isoforms are important mediators of mammary gland physiology. PMCA2 in particular is upregulated extensively during lactation. Expression of other isoforms such as PMCA4 may influence mammary gland epithelial cell proliferation and aberrant regulation of PMCA isoform expression may lead or contribute to mammary gland pathophysiology in the form of breast cancers. To explore whether PMCA2 and PMCA4 expression may be deregulated in breast cancer, we compared mRNA expression of these PMCA isoforms in tumorigenic and non-tumorigenic human breast epithelial cell lines using real time RT-PCR. PMCA2 mRNA has a higher level of expression in some breast cancer cell lines and is overexpressed more than 100-fold in ZR-75-1 cells, compared to non-tumorigenic 184B5 cells. Although differences in PMCA4 mRNA levels were observed between breast cell lines, they were not of the magnitude observed for PMCA2. We conclude that PMCA2 mRNA can be highly overexpressed in some breast cancer cells. The significance of PMCA2 overexpression on tumorigenicity and its possible correlation with other properties such as invasiveness requires further study.

    Biochemical and biophysical research communications 2005;337;3;779-83

  • Temporal pattern of plasma membrane calcium ATPase 2 expression in the spinal cord correlates with the course of clinical symptoms in two rodent models of autoimmune encephalomyelitis.

    Nicot A, Kurnellas M and Elkabes S

    University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Department of Neurology and Neuroscience, Newark, NJ, United States. nicot@st-antoine.inserm.fr

    Axonal/neuronal pathology is an important and early feature of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the underlying molecular mechanisms remain elusive. We have previously reported that the levels of an important neuronal calcium pump, plasma membrane calcium ATPase 2 and synaptic proteins, synapsin IIa and syntaxin 1B are decreased in the rat spinal cord at onset of acute EAE. Whether the expression of these genes is restored during neurological recovery and affected in other EAE models is currently unknown. The present study was undertaken to address these issues by use of validated multiplex quantitative real-time RT-PCR with fluoro-primers, western blot and immunocytochemistry. We report that plasma membrane calcium ATPase 2 (PMCA2) transcript and protein levels return to control values during recovery from acute disease in the Lewis rat, whereas they are reduced throughout the course of chronic, non-remitting EAE in the C57Bl/6 mouse. These results indicate a close correlation between PMCA2 levels and disease course as defined by clinical scores reflecting motor deficits. Decrease in synapsin IIa expression also correlated with the onset and progression of neurological symptoms, whereas the pattern of syntaxin 1B mRNA and protein expression suggested post-transcriptional regulation. The decrease in PMCA2 transcript and protein levels and the correlation between expression and disease course in two different EAE models further highlight the importance of this calcium pump in neuronal dysfunction during inflammation.

    Funded by: NINDS NIH HHS: NS 046363, R01 NS046363, R01 NS046363-04

    The European journal of neuroscience 2005;21;10;2660-70

  • Modification of human hearing loss by plasma-membrane calcium pump PMCA2.

    Schultz JM, Yang Y, Caride AJ, Filoteo AG, Penheiter AR, Lagziel A, Morell RJ, Mohiddin SA, Fananapazir L, Madeo AC, Penniston JT and Griffith AJ

    Section on Human Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Md 20850, USA.

    Five adult siblings presented with autosomal recessive sensorineural hearing loss: two had high-frequency loss, whereas the other three had severe-to-profound loss affecting all frequencies. Genetic evaluation revealed that a homozygous mutation in CDH23 (which encodes cadherin 23) caused the hearing loss in all five siblings and that a heterozygous, hypofunctional variant (V586M) in plasma-membrane calcium pump PMCA2, which is encoded by ATP2B2, was associated with increased loss in the three severely affected siblings. V586M was detected in two unrelated persons with increased sensorineural hearing loss, in the other caused by a mutation in MYO6 (which encodes myosin VI) in one and by noise exposure, suggesting that this variant may modify the severity of sensorineural hearing loss caused by a variety of factors.

    Funded by: NIDCD NIH HHS: 1 Z01 DC000039-05, 1 Z01 DC000060-02, 1 Z01 DC000064-02, DC04200; NIGMS NIH HHS: GM28825

    The New England journal of medicine 2005;352;15;1557-64

  • Expression and immunolocalization of plasma membrane calcium ATPase isoforms in human corneal epithelium.

    Talarico EF, Kennedy BG, Marfurt CF, Loeffler KU and Mangini NJ

    Department of Anatomy & Cell Biology, Indiana University School of Medicine-Northwest, Gary, IN 46408-1197, USA.

    Purpose: Plasma membrane Ca2+-ATPases (PMCAs) are integral membrane proteins essential to the control of intracellular Ca2+ ([Ca2+]i) concentration. Four genes encode PMCA proteins termed PMCA1-PMCA4. Little is known about the expression of these isoforms in corneal epithelium (CE). The purpose of this investigation is to characterize the expression and distribution of PMCAs in human CE (hCE).

    Methods: PMCA mRNA expression was examined by RT-PCR analysis of total RNA from native hCE using PMCA gene specific primers. PMCA isoform expression at the protein level in native hCE was examined by immunoblotting using isoform specific antibodies (Abs) and a panPMCA Ab that recognizes all PMCAs. Distribution of PMCAs in postmortem and surgical sections of hCE was determined by immunohistochemistry with the same Abs.

    Results: Immunoblot analysis with the panPMCA Ab yielded an intense band of approximately 135 kDa and several faintly staining bands above and below this major band. The isoform specific Abs labeled one or more bands that corresponded to bands detected with the panPMCA Ab. RT-PCR analysis of total RNA from hCE yielded PCR DNAs that were identified by sequencing as products of PMCA1, PMCA2, PMCA3, and PMCA4, thus confirming the immunoblot data. Immunohistochemistry demonstrated localization of PMCAs in all layers of hCE. PMCA4 was the predominant isoform, and was expressed along the plasma membrane of cells in all layers of CE, except with a notable absence along the basal cell membranes adjacent to the stroma. PMCA1 and PMCA2 were found mainly on basal and wing cells. In contrast to PMCA4, PMCA1 immunoreactivity (IR) was located on portions of basal cell plasma membranes adjacent to the stroma. PMCA2 IR was detected cytoplasmically within basal and wing cells in both central cornea and limbus. PMCA3 IR was located in basal cell nuclei in central cornea, but in a perinuclear location in the limbal, basal, and wing cells.

    Conclusions: Human CE expresses multiple PMCA isoforms that are differentially expressed and localized among the layers and cells that comprise the CE. We propose that the differential expression of multiple PMCA isoforms affords CE the requisite flexibility to respond to the demands for Ca2+ regulation required during renewal and regeneration of its multiple cell types.

    Funded by: NEI NIH HHS: R0I EY11308

    Molecular vision 2005;11;169-78

  • 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 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

  • Alternative splicing of the first intracellular loop of plasma membrane Ca2+-ATPase isoform 2 alters its membrane targeting.

    Chicka MC and Strehler EE

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

    Plasma membrane Ca(2+)-ATPases (PMCAs) are involved in local Ca(2+) signaling and in the spatial control of Ca(2+) extrusion, but how different PMCA isoforms are targeted to specific membrane domains is unknown. In polarized MDCK epithelial cells, a green fluorescent protein-tagged PMCA4b construct was targeted to the basolateral membrane, whereas a green fluorescent protein-tagged PMCA2b construct was localized to both the apical and basolateral domain. The PDZ protein-binding COOH-terminal tail of PMCA2b was not responsible for its apical membrane localization, as a chimeric pump made of an NH(2)-terminal portion from PMCA4 and a COOH-terminal tail from PMCA2b was targeted to the basolateral domain. Deletion of the last six residues of the COOH terminus of either PMCA2b or PMCA4b did not alter their membrane targeting, suggesting that PDZ protein interactions are not essential for proper membrane localization of the pumps. Instead, we found that alternative splicing affecting the first cytosolic loop determined apical membrane targeting of PMCA2. Only the "w" form, which contains a 45-amino acid residue insertion, showed prominent apical membrane localization. By contrast, the x and z splice variants containing insertions of 14 and 0 residues, respectively, localized to the basolateral membrane. The w splice insert was the crucial determinant of apical PMCA2 localization, and this was independent of the splice configuration at the COOH-terminal end of the pump; both PMCA2w/b and PMCA2w/a showed prominent apical targeting, whereas PMCA2x/b, PMCA2z/b, and PMCA2z/a were confined to the basolateral membrane. These data report the first differential effect of alternative splicing within the first cytosolic loop of PMCA2 and help explain the selective enrichment of specific PMCA2 isoforms in specialized membrane compartments such as stereocilia of auditory hair cells.

    Funded by: NIGMS NIH HHS: GM-58710

    The Journal of biological chemistry 2003;278;20;18464-70

  • Characterization of PISP, a novel single-PDZ protein that binds to all plasma membrane Ca2+-ATPase b-splice variants.

    Goellner GM, DeMarco SJ and Strehler EE

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

    Plasma membrane Ca(2+) ATPases (PMCAs) maintain intracellular Ca(2+) homeostasis and participate in the local regulation of Ca(2+) signaling. Spatially separate demands for Ca(2+) regulation require proper membrane targeting of PMCAs, but the mechanism of PMCA targeting is unknown. Using the PMCA2b carboxyl-terminal tail as yeast two-hybrid bait, we isolated a novel PDZ domain-containing protein from a human brain cDNA library. This protein, named PISP for PMCA-interacting single-PDZ protein, consists of 140 amino acids and contains little else besides a single PDZ domain. Pulldown experiments showed that PISP interacts with all PMCA b-splice forms. PISP was found to be ubiquitously expressed and, in MDCK cells, was present in a punctate pattern throughout the cytosol and at the basolateral membrane. When added to microsomal membranes expressing PMCA4b, PISP was unable to stimulate the PMCA-dependent ATPase activity. Our data suggest that PISP is a transiently interacting partner of the PMCA b-splice forms that may play a role in their sorting to or from the plasma membrane.

    Funded by: NIGMS NIH HHS: GM-58710

    Annals of the New York Academy of Sciences 2003;986;461-71

  • Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

    Wistow G, Bernstein SL, Wyatt MK, Fariss RN, Behal A, Touchman JW, Bouffard G, Smith D and Peterson K

    Section on Molecular Structure and Function, National Eye Institute, National Institutes of Health, Bethesda, MD 20892-2740, USA. graeme@helix.nih.gov

    Purpose: The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics.

    Methods: A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins.

    Results: Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping.

    Conclusions: The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

    Molecular vision 2002;8;205-20

  • 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

  • Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ (PSD95/Dlg/ZO-1) domain-containing proteins.

    DeMarco SJ and Strehler EE

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

    Spatial and temporal regulation of intracellular Ca(2+) signaling depends on localized Ca(2+) microdomains containing the requisite molecular components for Ca(2+) influx, efflux, and signal transmission. Plasma membrane Ca(2+)-ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains. The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen. The related MAGUKs SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b. Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95. These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca(2+) regulation.

    Funded by: NIGMS NIH HHS: GM-58710

    The Journal of biological chemistry 2001;276;24;21594-600

  • Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32.

    Usachev YM, Toutenhoofd SL, Goellner GM, Strehler EE and Thayer SA

    Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA. usach001@tc.umn.edu

    Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.

    Funded by: NIDA NIH HHS: DA07097, DA07304, DA09293, DA11806; NIGMS NIH HHS: GM58710

    Journal of neurochemistry 2001;76;6;1756-65

  • 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

  • Mutations in a plasma membrane Ca2+-ATPase gene cause deafness in deafwaddler mice.

    Street VA, McKee-Johnson JW, Fonseca RC, Tempel BL and Noben-Trauth K

    The Virginia Merrill Bloedel Hearing Research Center and Department of Otolaryngology-Head and Neck Surgery, University of Washington School of Medicine, Seattle 98195-7923, USA.

    Hearing loss is the most common sensory deficit in humans. Because the auditory systems of mice and humans are conserved, studies on mouse models have predicted several human deafness genes and identified new genes involved in hearing. The deafwaddler (dfw) mouse mutant is deaf and displays vestibular/motor imbalance. Here we report that the gene encoding a plasma membrane Ca2+-ATPase type 2 pump (Atp2b2, also known as Pmca2) is mutated in dfw. An A-->G nucleotide transition in dfw DNA causes a glycine-to-serine substitution at a highly conserved amino-acid position, whereas in a second allele, dfw2J, a 2-base-pair deletion causes a frameshift that predicts a truncated protein. In the cochlea, the protein Atp2b2 is localized to stereocilia and the basolateral wall of hair cells in wild-type mice, but is not detected in dfw2J mice. This indicates that mutation of Atp2b2 may cause deafness and imbalance by affecting sensory transduction in stereocilia as well as neurotransmitter release from the basolateral membrane. These mutations affecting Atp2b2 in dfw and dfw2J are the first to be found in a mammalian plasma membrane calcium pump and define a new class of deafness genes that directly affect hair-cell physiology.

    Nature genetics 1998;19;4;390-4

  • Balance and hearing deficits in mice with a null mutation in the gene encoding plasma membrane Ca2+-ATPase isoform 2.

    Kozel PJ, Friedman RA, Erway LC, Yamoah EN, Liu LH, Riddle T, Duffy JJ, Doetschman T, Miller ML, Cardell EL and Shull GE

    Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.

    Plasma membrane Ca2+-ATPase isoform 2 (PMCA2) exhibits a highly restricted tissue distribution, suggesting that it serves more specialized physiological functions than some of the other isoforms. A unique role in hearing is indicated by the high levels of PMCA2 expression in cochlear outer hair cells and spiral ganglion cells. To analyze the physiological role of PMCA2 we used gene targeting to produce PMCA2-deficient mice. Breeding of heterozygous mice yielded live homozygous mutant offspring. PMCA2-null mice grow more slowly than heterozygous and wild-type mice and exhibit an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice revealed that null mutants had slightly increased numbers of Purkinje neurons (in which PMCA2 is highly expressed), a decreased thickness of the molecular layer, an absence of otoconia in the vestibular system, and a range of abnormalities of the organ of Corti. Analysis of auditory evoked brainstem responses revealed that homozygous mutants were deaf and that heterozygous mice had a significant hearing loss. These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major source of the calcium used in the formation and maintenance of otoconia.

    Funded by: NHLBI NIH HHS: HL41496; NIDCD NIH HHS: K08 DC000119-06; NIDDK NIH HHS: DK50594; NIEHS NIH HHS: ES06096

    The Journal of biological chemistry 1998;273;30;18693-6

  • 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

  • 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

  • One-megabase yeast artificial chromosome and 400-kilobase cosmid-phage contigs containing the von Hippel-Lindau tumor suppressor and Ca(2+)-transporting adenosine triphosphatase isoform 2 genes.

    Kuzmin I, Stackhouse T, Latif F, Duh FM, Geil L, Gnarra J, Yao M, Orcutt ML, Li H, Tory K et al.

    Laboratory of Immunobiology, Research and Development Center, Maryland 21702-1201.

    We have isolated and ordered yeast artificial chromosomes (YACs) and cosmids surrounding the von Hippel-Lindau (VHL) tumor suppressor and plasma membrane Ca(2+)-transporting ATPase isoform 2 (PMCA-2) genes on chromosome 3p25-26. The YAC contig consists of six YACs and covers a region of 1 megabase. A cosmid-phage contig around VHL and PMCA-2 genes (400 kilobases) was established and integrated into the YAC map. Using these clones, we generated an EcoRI map of the 400-kilobase region. PMCA-2 and VHL complementary DNA were positioned entirely within the cosmid-phage contig as well as two polymorphic markers (D3S601 and D3S1317). This physical map of the cloned region will allow a detailed analysis of both the PMCA-2 and VHL genes. Some of the genomic clones may be useful for isolation of the full-length VHL complementary DNA.

    Cancer research 1994;54;9;2486-91

  • Localization of two genes encoding plasma membrane Ca2+ ATPases isoforms 2 (ATP2B2) and 3 (ATP2B3) to human chromosomes 3p26-->p25 and Xq28, respectively.

    Wang MG, Yi H, Hilfiker H, Carafoli E, Strehler EE and McBride OW

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

    The plasma membrane Ca2+ ATPases (PMCA) represent a highly conserved, widely dispersed, multigene family in eukaryotes consisting of at least four functional genes. The genes for PMCA isoforms 1 and 4 (ATP2B1 and ATP2B4) have been previously localized to human chromosomes 12q21-->q23 and 1q25-->q32, respectively. Based upon results of fluorescence in situ hybridization (FISH), analysis of somatic cell hybrids, and genetic linkage analyses, we now report localization of ATP2B3 (PMCA isoform 3) to human chromosome Xq28, and confirm the recent localization of ATP2B2 (PMCA isoform 2) to chromosome 3p26-->p25. In contrast to ATP2B1 and ATP2B4, recent studies have suggested tissue specific regulation of expression of both ATP2B2 and ATP2B3 particularly in the nervous system. The genes for several neurological and neuromuscular diseases have been assigned to the distal portion of Xq, and ATP2B3 is a candidate gene for these diseases.

    Cytogenetics and cell genetics 1994;67;1;41-5

  • 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

  • von Hippel-Lindau syndrome: cloning and identification of the plasma membrane Ca(++)-transporting ATPase isoform 2 gene that resides in the von Hippel-Lindau gene region.

    Latif F, Duh FM, Gnarra J, Tory K, Kuzmin I, Yao M, Stackhouse T, Modi W, Geil L, Schmidt L et al.

    Laboratory of Immunobiology, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702-1201.

    We have isolated and analyzed full-length complementary DNA clones encoded by a 200-kilobase gene encompassing the D3S601 locus that resides in the von Hippel-Lindau (VHL) gene region. The deduced amino acid sequence shows 99% identity with the published sequence of the rat plasma membrane Ca(++)-transporting ATPase isoform 2 complementary DNA, implying that we have cloned and positioned the human plasma membrane Ca(++)-transporting ATPase isoform 2 gene within the VHL critical region. The gene is expressed in VHL target tissues and should be considered a potential candidate gene for the VHL disease.

    Funded by: NCI NIH HHS: N01-CO-74102

    Cancer research 1993;53;4;861-7

  • Determination of the nucleotide sequence and chromosomal localization of the ATP2B2 gene encoding human Ca(2+)-pumping ATPase isoform PMCA2.

    Brandt P, Ibrahim E, Bruns GA and Neve RL

    Department of Psychobiology, University of California, Irvine 92717.

    The plasma membrane Ca(2+)-pumping ATPase (Ca(2+)-ATPase) is responsible for maintaining calcium homeostasis in eukaryotic cells. The Ca(2+)-ATPase is a family of pumps that are encoded by at least four genes. A cDNA for the human version of Ca(2+)-ATPase isoform PMCA2 was isolated and characterized. Comparison of the human and rat cDNA sequences showed that they were 95% homologous in the coding domain, and this homology was reflected in the deduced protein sequence where greater than 98% homology between the human and rat sequences was found. The amino acid differences that were found were almost all conservative. The PMCA2 cDNA was used to probe Southern blots of human-rodent somatic cell hybrid DNAs; the results indicated that the human PMCA2 gene was located on chromosome 3.

    Funded by: NICHD NIH HHS: HD18658; NINDS NIH HHS: NS28406

    Genomics 1992;14;2;484-7

  • Microdiversity of human-plasma-membrane calcium-pump isoform 2 generated by alternative RNA splicing in the N-terminal coding region.

    Heim R, Hug M, Iwata T, Strehler EE and Carafoli E

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

    cDNA species covering the entire coding sequence of the human homologue of the rat plasma membrane Ca(2+)-ATPase (PMCA) isoform 2 have been isolated and characterized. The deduced amino acid sequence shows 99% identity with that of the rat protein and can be aligned with the latter without gaps except for one 14-amino-acid-residue insert in the region immediately preceding the putative phospholipid-sensitive domain in the human pump. cDNA clones isolated by anchored polymerase-chain reaction revealed additional microheterogeneity in the same N-terminal PMCA2-coding region. Alternative RNA splicing involving a region of 135 nucleotides generates three types of cDNA. One does not contain any of the 135 bp, and the other two contain 42 bp or the entire 135 bp of the optional sequence. Analysis of genomic DNA indicates that this sequence is encoded by three separate exons of 33, 60 and 42 bp. Although each of these exons could be inserted into the mRNA without changing the reading frame, polymerase-chain amplifications using cDNA libraries from several human tissues show that the 33-bp and the 60-bp exons are never independently used during splicing. The unequal distribution of the splice variants suggests tissue-specific regulation of the alternative-splicing pathways and indicates a functional specialization of the encoded isoform subtypes.

    European journal of biochemistry 1992;205;1;333-40

  • 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

OMIM - other

Gene lists (8)

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
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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