Katalin Pászty

All three splice variants of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene are translated to proteins: a study of their co-expression in platelets and lymphoid cells.

The molecular cloning of two previously unknown human sarco/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3) 3-end transcripts, 3b and 3c, has been recently published. Data were lacking, however, for the presence of these SERCA3 variants in different tissue or cell types at the protein level. Here we report the co-expression of three human SERCA3 protein isoforms in platelets and T lymphoid Jurkat cells. Isoform-specific polyclonal anti-peptide antibodies have been generated that recognize specifically the SERCA3a, 3b or 3c splice variants at their C-termini, and this has been confirmed by peptide-competition experiments as well. None of these antibodies cross-reacted with the housekeeping SERCA2b isoform co-expressed endogenously with SERCA3 proteins in non-muscle cells. Although all three SERCA3 isoforms could be detected in platelets, the 3a form was the most abundantly expressed species. Its size matched the apparent size of SERCA3a over-expressed in HEK-293 cells. Immunoprecipitation of the SERCA3 variants from platelet membranes using a PL/IM 430-affinity matrix provided evidence that the putative pan-anti-SERCA3 antibody, PL/IM 430, recognizes all SERCA3 protein isoforms. The epitope for the PL/IM 430 antibody could be localized in a 40 kDa N-terminal tryptic fragment common to all three SERCA3 variants. Comparative Western-blot analysis showed that the expression level of the SERCA3a, 3b and 3c isoforms was more than 10 times lower in Jurkat cells than in platelets, whereas expression of the ubiquitous SERCA2b was nearly identical. This work highlights new Ca(2+)-transporting proteins of haematopoietic cells and provides specific antibodies for their detection.

Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ ATPase system in human platelets

We recently identified a multi‐SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti‐Ca2+ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti‐SERCA 2b, anti‐SERCA 3, anti‐SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some α‐granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes. u2003Thus, the multi‐SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub‐compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca2+ stores in these cells.

Expression of hPMCA4b, the major form of the plasma membrane calcium pump in megakaryoblastoid cells is greatly reduced in mature human platelets.

Antibodies 5F10 and JA3 (raised against the erythrocyte Ca2+ pump) were used to identify hPMCA4b as the major form of the plasma membrane Ca2+ pump in human platelets and in three human megakaryoblastoid cell lines, MEG 01, DAMI and CHRF 288-11. 5F10 was used because it has been shown to recognize all known isoforms of the hPMCA and JA3 because it reacts exclusively with hPMCA4b [Caride A.J., Filoteo A.G., Enyedi A., Verma A.K., Penniston J.T. Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies. Biochem J 1996; 316: 353-359]. In addition to hPMCA4b, hPMCA1b was also detected in the megakaryoblastoid cells by using isoform-specific polyclonal antibodies. The apparent size of this isoform, however, was smaller than that seen in HeLa and COS-7 cell membranes indicating the presence of a modified form of hPMCA1b. In platelets, no evidence of the expression of hPMCA1b could be found. The amount of PMCA in these cells was compared with that of the constitutive form of the sarco/endoplasmic reticulum Ca2+ pump in non-muscle cells (SERCA2b) and also with the amount of PMCA in human erythrocytes. A very low level of the plasma membrane Ca2+ pump was found in platelets while in their precursor cells the expression of this Ca2+ pump was much more abundant. Whereas the expression level of PMCA decreased dramatically in mature human platelets, the expression of SERCA2b did not change substantially upon megakaryocytic differentiation.

Apical scaffolding protein NHERF2 modulates the localization of alternatively spliced plasma membrane Ca2+ pump 2B variants in polarized epithelial cells.

The membrane localization of the plasma membrane Ca2+-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na+/H+ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.

Plasma membrane Ca2+-ATPases can shape the pattern of Ca2+ transients induced by store-operated Ca2+ entry

Plasma membrane calcium pumps differentially affect the pattern of calcium signals. Shaping the calcium signal Cells use spatially and locally controlled calcium signals to regulate specific responses and have multiple mechanisms for regulating cytosolic calcium and for removing cytosolic calcium after stimuli that trigger its increase. Pászty et al. investigated how the Ca2+-ATPases that pump calcium out of the cell (PMCAs) contribute to the shape of calcium signals under conditions in which the internal calcium stores have been depleted and cannot contribute to clearing of the calcium from the cytosol. The pattern of calcium signals (oscillating, sustained elevation, or rapid return to baseline) produced in multiple cultured cell lines depended on the abundance of the specific PMCA isoforms, which have different kinetic and regulatory properties. The authors developed a mathematical model that recapitulated the calcium signaling patterns observed in the cultured cells and used the model to identify which properties of PMCA were critical for producing an oscillating Ca2+ pattern. Calcium (Ca2+) is a critical cofactor and signaling mediator in cells, and the concentration of cytosolic Ca2+ is regulated by multiple proteins, including the plasma membrane Ca2+–ATPases (adenosine triphosphatases) (PMCAs), which use ATP to transport Ca2+ out of cells. PMCA isoforms exhibit different kinetic and regulatory properties; thus, the presence and relative abundance of individual isoforms may help shape Ca2+ transients and cellular responses. We studied the effects of three PMCA isoforms (PMCA4a, PMCA4b, and PMCA2b) on Ca2+ transients elicited by conditions that trigger store-operated Ca2+ entry (SOCE) and that blocked Ca2+ uptake into the endoplasmic reticulum in HeLa cells, human embryonic kidney (HEK) 293 cells, or primary endothelial cell isolated from human umbilical cord veins (HUVECs). The slowly activating PMCA4b isoform produced long-lasting Ca2+ oscillations in response to SOCE. The fast-activating isoforms PMCA2b and PMCA4a produced different effects. PMCA2b resulted in rapid and highly PMCA abundance–sensitive clearance of SOCE-mediated Ca2+ transients, whereas PMCA4a reduced cytosolic Ca2+, resulting in the establishment of a higher than baseline cytosolic Ca2+ concentration. Mathematical modeling showed that slow activation was critical to the sustained oscillation induced by the “slow” PMCA4b pump. The modeling and experimental results indicated that the distinct properties of PMCA isoforms differentially regulate the pattern of SOCE-mediated Ca2+ transients, which would thus affect the activation of downstream signaling pathways.

PSD-95 mediates membrane clustering of the human plasma membrane Ca2+ pump isoform 4b

Besides the control of global calcium changes, specific plasma membrane calcium ATPase (PMCA) isoforms are involved in the regulation of local calcium signals. Although local calcium signaling requires the confinement of signaling molecules into microdomains, little is known about the specific organization of PMCA molecules within the plasma membrane. Here we show that co-expression with the postsynaptic density-95 (PSD-95) scaffolding protein increased the plasma membrane expression of PMCA4b and redistributed the pump into clusters. The clustering of PMCA4b was fully dependent on the presence of its PDZ-binding sequence. Using the fluorescence recovery after photobleaching (FRAP) technique, we show that the lateral membrane mobility of the clustered PMCA4b is significantly lower than that of the non-clustered molecules. Disruption of the actin-based cytoskeleton by cytochalasin D resulted in increased cluster size. Our results suggest that PSD-95 promotes the formation of high-density PMCA4b microdomains in the plasma membrane and that the membrane cytoskeleton plays an important role in the regulation of this process.

Plasma membrane calcium pump (PMCA) isoform 4 is targeted to the apical membrane by the w-splice insert from PMCA2

Local Ca(2+) signaling requires proper targeting of the Ca(2+) signaling toolkit to specific cellular locales. Different isoforms of the plasma membrane Ca(2+) pump (PMCA) are responsible for Ca(2+) extrusion at the apical and basolateral membrane of polarized epithelial cells, but the mechanisms and signals for differential targeting of the PMCAs are not well understood. Recent work demonstrated that the alternatively spliced w-insert in PMCA2 directs this pump to the apical membrane. We now show that inserting the w-insert into the corresponding location of the PMCA4 isoform confers apical targeting to this normally basolateral pump. Mutation of a di-leucine motif in the C-tail thought to be important for basolateral targeting did not enhance apical localization of the chimeric PMCA4(2w)/b. In contrast, replacing the C-terminal Val residue by Leu to optimize the PDZ ligand site for interaction with the scaffolding protein NHERF2 enhanced the apical localization of PMCA4(2w)/b, but not of PMCA4x/b. Functional studies showed that both apical PMCA4(2w)/b and basolateral PMCA4x/b handled ATP-induced Ca(2+) signals with similar kinetics, suggesting that isoform-specific functional characteristics are retained irrespective of membrane targeting. Our results demonstrate that the alternatively spliced w-insert provides autonomous apical targeting information in the PMCA without altering its functional characteristics.

Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells

The w splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.

Chimaeras reveal the role of the catalytic core in the activation of the plasma membrane Ca2+ pump

Isoform 2b of the plasma membrane calcium pump differs from the ubiquitous isoform 4b in the following: (a) higher basal activity in the absence of calmodulin; (b) higher affinity for calmodulin; and (c) higher affinity for Ca(2+) in the presence of calmodulin [Elwess, Filoteo, Enyedi and Penniston (1997) J. Biol. Chem. 272, 17981-17986]. To investigate which parts of the molecule determine these kinetic differences, we made four chimaeric constructs in which portions of isoform 2b were grafted into isoform 4b: chimaera I contains only the C-terminal regulatory region of isoform 2b; chimaera II contains the N-terminal moiety of isoform 2b, including both cytoplasmic loops; chimaera III contains the sequence of isoform 2b starting from the N-terminus to after the end of the first (small) cytoplasmic loop; and chimaera IV contains only the second (large) cytoplasmic loop. Surprisingly, chimaera I showed low basal activity in the absence of calmodulin and low affinity for calmodulin, unlike isoform 2b. In contrast, the chimaera containing both loops showed high basal activity, and Ca(2+) activation curves (both in the absence and in the presence of calmodulin) similar to those of isoform 2b. The rates of activation by calmodulin and of inactivation by calmodulin removal were measured, and the apparent K(d) for calmodulin was calculated from the ratio between these rate constants. The order of affinity was: 2b=II>4b=IV>III=I. From these results it is clear that the construct that most closely resembles isoform 2b is chimaera II. This shows that, in order to obtain an enzyme with properties similar to those of isoform 2b, both cytoplasmic loops are needed.

Analysis of Intracellular Calcium Signaling in Human Embryonic Stem Cells

Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well.