J. Biol. Chem. 269, 26178-26183 (1994)[PubMed:7929331]
Full-length cDNAs for the three human plasma membrane Ca2+ pump isoforms 2 (PMCA2) differently spliced at the A site were constructed and transferred to baculovirus. The corresponding proteins were expressed after infection in Sf9 insect cells. The proteins were expressed at high levels and retained the canonical properties of the plasma membrane Ca2+ pump. The alternative splicing process failed to produce functional differences detectable with the methods used. The Ca(2+)-dependent ATPase activity of the PMCA2 pumps had a 5-10-fold higher affinity for calmodulin than the PMCA4 pump expressed in the same system. Experiments on the formation of the phosphoenzyme intermediate from ATP revealed that the PMCA2 pumps had higher affinity for ATP than did the PMCA4 counterpart. The response of the two pump types to activating acidic phospholipids was the same.
J. Biol. Chem. 269, 26178-26183 (1994)[PubMed:7929331]
Full-length cDNAs for the three human plasma membrane Ca2+ pump isoforms 2 (PMCA2) differently spliced at the A site were constructed and transferred to baculovirus. The corresponding proteins were expressed after infection in Sf9 insect cells. The proteins were expressed at high levels and retained the canonical properties of the plasma membrane Ca2+ pump. The alternative splicing process failed to produce functional differences detectable with the methods used. The Ca(2+)-dependent ATPase activity of the PMCA2 pumps had a 5-10-fold higher affinity for calmodulin than the PMCA4 pump expressed in the same system. Experiments on the formation of the phosphoenzyme intermediate from ATP revealed that the PMCA2 pumps had higher affinity for ATP than did the PMCA4 counterpart. The response of the two pump types to activating acidic phospholipids was the same.
Catalysis of the transfer of a solute or solutes from one side of a membrane to the other according to the reaction: ATP + H2O + Ca2+(cis) = ADP + phosphate + Ca2+(trans).
Eur. J. Biochem. 205, 333-340 (1992)[PubMed:1313367]
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.
Evidence
2:
Inferred from Direct AssayUniProtKB
Evidence for ZB and YB
J. Biol. Chem. 269, 26178-26183 (1994)[PubMed:7929331]
Full-length cDNAs for the three human plasma membrane Ca2+ pump isoforms 2 (PMCA2) differently spliced at the A site were constructed and transferred to baculovirus. The corresponding proteins were expressed after infection in Sf9 insect cells. The proteins were expressed at high levels and retained the canonical properties of the plasma membrane Ca2+ pump. The alternative splicing process failed to produce functional differences detectable with the methods used. The Ca(2+)-dependent ATPase activity of the PMCA2 pumps had a 5-10-fold higher affinity for calmodulin than the PMCA4 pump expressed in the same system. Experiments on the formation of the phosphoenzyme intermediate from ATP revealed that the PMCA2 pumps had higher affinity for ATP than did the PMCA4 counterpart. The response of the two pump types to activating acidic phospholipids was the same.
Interacting selectively and non-covalently with calmodulin, a calcium-binding protein with many roles, both in the calcium-bound and calcium-free states.
Evidence
2:
Inferred from Direct AssayUniProtKB
Evidence for ZB and YB
J. Biol. Chem. 269, 26178-26183 (1994)[PubMed:7929331]
Full-length cDNAs for the three human plasma membrane Ca2+ pump isoforms 2 (PMCA2) differently spliced at the A site were constructed and transferred to baculovirus. The corresponding proteins were expressed after infection in Sf9 insect cells. The proteins were expressed at high levels and retained the canonical properties of the plasma membrane Ca2+ pump. The alternative splicing process failed to produce functional differences detectable with the methods used. The Ca(2+)-dependent ATPase activity of the PMCA2 pumps had a 5-10-fold higher affinity for calmodulin than the PMCA4 pump expressed in the same system. Experiments on the formation of the phosphoenzyme intermediate from ATP revealed that the PMCA2 pumps had higher affinity for ATP than did the PMCA4 counterpart. The response of the two pump types to activating acidic phospholipids was the same.
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.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
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.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Ann. N. Y. Acad. Sci. 986, 461-471 (2003)[PubMed:12763866]
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.
Evidence
3:
Inferred from Physical InteractionIntAct
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.
Interacting selectively and non-covalently with a protein C-terminus, the end of any peptide chain at which the 1-carboxy function of a constituent amino acid is not attached in peptide linkage to another amino-acid residue.
Evidence
1:
Inferred from Physical InteractionUniProtKB
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.
The chemical reactions and pathways resulting in the formation of ATP, adenosine 5'-triphosphate, a universally important coenzyme and enzyme regulator.
A process that is carried out at the cellular level which results in the assembly, arrangement of constituent parts, or disassembly of a stereocilium. A stereocilium is an actin-based protrusion from the apical surface of auditory hair cells.
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.
The process in which neuroblasts acquire specialized structural and/or functional features that characterize the mature cerebellar granule cell. Differentiation includes the processes involved in commitment of a neuroblast to a granule cell fate. A granule cell is a glutamatergic interneuron found in the cerebellar cortex.
The process in which neuroblasts acquire specialized structural and/or functional features that characterize the mature cerebellar Purkinje cell. Differentiation includes the processes involved in commitment of a neuroblast to a Purkinje cell fate. A Purkinje cell is an inhibitory GABAergic neuron found in the cerebellar cortex that projects to the deep cerebellar nuclei and brain stem.
The progression of the cochlea over time from its formation to the mature structure. The cochlea is the snail-shaped portion of the inner ear that is responsible for the detection of sound.
Any process involved in the maintenance of an internal steady state of calcium ions within the cytosol of a cell or between the cytosol and its surroundings.
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.
The specific movement from place to place of an organism in response to external or internal stimuli. Locomotion of a whole organism in a manner dependent upon some combination of that organism's internal state and external conditions.
Any process that an organism uses to control its balance, the orientation of the organism (or the head of the organism) in relation to the source of gravity. In humans and animals, balance is perceived through visual cues, the labyrinth system of the inner ears and information from skin pressure receptors and muscle and joint receptors.
J. Neurochem. 76, 1756-1765 (2001)[PubMed:11259493]
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.
A process that is carried out at the cellular level which results in the assembly, arrangement of constituent parts, or disassembly of an organelle within a cell. An organelle is an organized structure of distinctive morphology and function. Includes the nucleus, mitochondria, plastids, vacuoles, vesicles, ribosomes and the cytoskeleton. Excludes the plasma membrane.
Any process that activates or increases the frequency, rate or extent of the directed movement of calcium ions into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore.
A process that modulates synaptic plasticity, the ability of synapses to change as circumstances require. They may alter function, such as increasing or decreasing their sensitivity, or they may increase or decrease in actual numbers.
The series of events required for an organism to receive an auditory stimulus, convert it to a molecular signal, and recognize and characterize the signal. Sonic stimuli are detected in the form of vibrations and are processed to form a sound.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
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.
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.
The chemical reactions and pathways involving serotonin (5-hydroxytryptamine), a monoamine neurotransmitter occurring in the peripheral and central nervous systems, also having hormonal properties.
A process that is carried out at the cellular level which results in the assembly, arrangement of constituent parts, or disassembly of a synapse, the junction between a neuron and a target (neuron, muscle, or secretory cell).
The directed movement of substances (such as macromolecules, small molecules, ions) into, out of or within a cell, or between cells, or within a multicellular organism by means of some agent such as a transporter or pore.
Eur. J. Biochem. 205, 333-340 (1992)[PubMed:1313367]
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.
Protein involved in the transport of calcium ions. Calcium is essential for a variety of bodily functions, such as neurotransmission, muscle contraction and proper heart function.
Protein involved in the transport of ions. Such proteins are usually transmembrane and mediate a movement of ions across cell membranes. Transport may be passive (facilitated diffusion; down the electrochemical gradient), or active (against the electrochemical gradient). Active transport requires energy which may come from light, oxidation reactions, ATP hydrolysis, or cotransport of other ions or molecules.
Protein involved in the transport of a molecule (metabolite, protein, etc), a ion or an electron across cell membranes, inside the cell or in a tissue fluid.
Enzyme which catalyzes hydrolysis reaction, i.e. the addition of the hydrogen and hydroxyl ions of water to a molecule with its consequent splitting into two or more simpler molecules.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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