- Email: [email protected]
Zo-1 scaffolds
The International Journal of Biochemistry & Cell Biology 42 (2010) 805–808
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecules in focus
The plasma membrane Ca2+ -ATPase: Regulation by PSD-95/Dlg/Zo-1 scaffolds Wade A. Kruger a , Gregory R. Monteith b , Philip Poronnik a,∗ a b
Health Innovations Research Institute, School of Medical Sciences, RMIT University, Bundoora, Melbourne, PO Box 71, VIC 3083, Australia Faculty of Health Sciences, School of Pharmacy, The University of Queensland, Brisbane, QLD 4072, Australia
a r t i c l e
i n f o
Article history: Received 16 December 2009 Received in revised form 18 January 2010 Accepted 18 January 2010 Available online 25 January 2010 Keywords: PMCA PDZ Scaffold Calcium
a b s t r a c t Since its first characterization in the erythrocyte membrane the plasma membrane Ca2+ -ATPase has been well-defined as a ubiquitous mechanism for the efflux of Ca2+ from eukaryotic cells. With 4 isoforms and potentially 30 splice variants, defining the absolute physiological role of plasma membrane Ca2+ -ATPase has been difficult and very limited due to the lack of effective blockers/antibodies and difficulties in measuring the activity of individual isoforms. This review highlights recent developments showing that specific plasma membrane Ca2+ -ATPase isoforms are subject to dynamic regulation by PSD-95/Dlg/Zo-1 scaffold proteins. Such interactions support a new paradigm, that by serving as key players in multifunctional protein complexes, transporters can regulate other signalling processes independent of their primary ion pumping function. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction The spatial and temporal regulation of the ubiquitous second messenger, intracellular free Ca2+ ([Ca2+ ]i ), is fundamental for normal cellular function. Changes in [Ca2+ ]i trigger a myriad of cellular events from fertilization to gene transcription, neurotransmitter release and cell death. Many of these changes in [Ca2+ ]i are mediated by G-protein coupled receptors (GPCRs) to effect physiological responses (Carafoli, 2003). These GPCR-mediated increases in [Ca2+ ]i are typically biphasic, starting with mobilization of Ca2+ from intracellular stores which in turn triggers Ca2+ influx via plasma membrane ion channels. Following termination of receptor signalling, [Ca2+ ]i must return rapidly to its nanomolar resting levels. This is achieved by the reuptake of Ca2+ into intracellular stores and Ca2+ efflux across the plasma membrane mediated by the primarily ubiquitous plasma membrane Ca2+ -ATPase (PMCA) as well as the Na+ –Ca2+ exchanger in some cell types. Hence the dynamic interplay between influx, efflux and reuptake governs the magnitude, duration and location of the increase in [Ca2+ ]i to determine the physiological outcome. How then do cells produce specific responses in the face of a plethora of Ca2+ mobilizing agents in the extracellular milieu? One strategy to direct functional specificity is by locally constraining discrete molecular Ca2+ signalling complexes using protein–protein interactions and trafficking mechanisms to maintain spatial heterogeneity of Ca2+ signalling. There has been increasing interest in the trafficking of
∗ Corresponding author. Tel.: +61 3 9925 7071; fax: +61 7 9925 1766. E-mail address: [email protected] (P. Poronnik). 1357-2725/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2010.01.023
channels and associated proteins that are involved in regulating Ca2+ influx and release pathways. In this review we highlight the dynamic regulation of PMCA-mediated Ca2+ by PSD-95/Dlg/Zo-1 (PDZ) scaffold proteins. 2. Structure PMCAs belong to the P2 (subtype 2B) subfamily of P-type primary ion transport ATPases, which are characterized by the formation of an aspartyl phosphate intermediate upon ATP hydrolysis during their reaction cycle (Strehler and Zacharias, 2001). All four PMCA isoforms are encoded by the genes Atp2b1-4. These genes have a number of splice variants caused by the insertion of alternative exons at site A in the cytosolic loop connecting transmembrane domains 2 and 3, or at site C in the C-terminus of the pump (Ficarella et al., 2007). PMCA is predicted to contain 10 membrane spanning domains, a structure closely related to other P-type ATPases such as the sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) (Fig. 1). The first intracellular loop, between domains 2 and 3 corresponds to the “transduction domain” and is thought to play the key role in the conformational changes occurring during the Ca2+ transport cycle. The very large loop between domains 4 and 5 is the major catalytic domain and includes the ATP binding site (Strehler et al., 2007). Both the N- and C-termini are located at the cytosolic face of the membrane, enabling the association with accessory/scaffold proteins that can directly regulate the transporter. The C-terminus represents one of the major regulatory sites of the protein and contains many consensus binding/interaction sites for regulatory molecules (Brini, 2009). Importantly, the b-splice variants have a highly conserved Class 1 PDZ binding motif at the C-terminus (Table 1).
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Fig. 1. Predicted structure of PMCA in active and inactive states. PMCA is predicted to contain 10 membrane spanning domains, a structure closely related to other P-type ATPases. Both the N- and C-termini are located at the cytosolic face of the membrane, enabling the association with accessory/scaffold proteins that can directly regulate the transporter. In the unstimulated state, the calmodulin-binding domain binds to two sites within the transporter resulting in autoinhibition. However, when [Ca2+ ]i rises and binds to calmodulin, the Ca2+ -bound calmodulin interacts with the PMCA calmodulin-binding domain, thereby reversing the autoinhibition activating the pump to remove Ca2+ from the cytosol.
3. Biological function PMCA was first described in the erythrocyte membrane in the mid-1960s (Schatzmann, 1966) and has since been well-defined as a primary mechanism for Ca2+ efflux from eukaryotic cells (Fig. 1(ii)). Defining the absolute physiological roles of PMCAs is difficult due to the lack of effective blockers/antibodies and difficulties in measuring the activity of individual isoforms. Studies using transgenic animals have been equally challenging. Mice in which PMCA1 was ablated resulted in embryonic lethality. In contrast, despite its apparent ubiquity, Pmca4−/− male mice are infertile but have normal spermatogenesis and mating behaviour (Okunade et al., 2004). These findings challenge the “housekeeping” role proposed for PMCA4 or may simply reflect functional compensation by other PMCA isoforms. PMCA2 knockout mice (also known as “deafwaddler” and “wriggle mouse Sagami”) exhibit very specific phenotypes of unsteady gait, difficulties maintaining balance and hearing defects (Penheiter et al., 2001). Studies extrapolating from these knockout models have proven a link between loss of PMCA2 activity and degenerative hearing loss in humans (Schultz et al., 2005). Further studies into the role of specific PMCA isoforms will require strategies such as tissue specific knockdown. 4. Expression, activation and turnover As mentioned above, the four PMCAs isoforms differ in their tissue distribution. PMCA1 and 4 are ubiquitous while PMCA2 and 3 are more restricted, primarily in brain, nervous tissue and some types of muscle (Brini, 2009). A large number of splice variants are theoretically possible and to date approximately 30 have been detected at the RNA or protein level (Strehler and Zacharias, 2001). The diversity of isoform and splice variants is thought to provide the structural flexibility to enable the formation of the different functional complexes required for the dynamic, spatio-temporal regulation of cellular Ca2+ handling (Strehler and Zacharias, 2001). Each PMCA isoform has specific biophysical/biochemical properties. For example, the most extensively studied regulator of PMCA activity is calmodulin. In the unstimulated state, the calmodulin-
binding domain located at the C-terminus of PMCA binds to two intramolecular “receptor” sites within the transporter resulting in autoinhibition. However, when [Ca2+ ]i rises and binds to calmodulin, the Ca2+ -bound calmodulin subsequently interacts with the PMCA calmodulin-binding domain, thereby reversing the autoinhibition activating the pump (Strehler and Zacharias, 2001). The different PMCA isoforms and their splice variants have different affinities for calmodulin (PMCA2 having the highest and PMCA4 having the lowest). This in turn results in different levels of pump activity in response to changes in [Ca2+ ]i (Brini et al., 2003). PMCAs are activated by a variety of other mechanisms that include self-association (oligomerization), acidic phospholipids as well as regulatory proteins including protein kinase C (PKC), protein kinase A (PKA) and proteases such as calpain (Strehler et al., 2007). Interestingly, many of these regulatory proteins require the presence of molecular scaffolds to recruit them into the proximity of the transporter in order to act. It is therefore clear that trafficking plays a significant role in determining PMCA levels at the plasma membrane (Kruger et al., 2009). 4.1. Regulation of PMCA by PDZ scaffolds Ion channels, transporters and receptors form dynamic macromolecular complexes at the plasma membrane that direct functional specificity within cellular microdomains. The assembly of these complexes requires molecular scaffolds such as PDZ domain containing proteins. These PDZ domains bind to PDZ binding motifs contained within the membrane protein thereby allowing the assembly of heterologous protein complexes. In this context, the C-termini of all PMCA-b splice variants have a highly conserved Class 1 PDZ binding motif (refer Table 1) that has been reported to interact with a number of PDZ containing proteins such as nitric oxide synthase I (NOS-I), members of the membraneassociated guanylate kinase family, and the Na+ /H+ exchanger regulatory factor-2 (NHERF-2) (DeMarco and Strehler, 2001; Zabe and Dean, 2001; DeMarco et al., 2002). However, despite several reports of PMCA–PDZ interactions, the physiological outcomes of these interactions remain largely undefined.
Table 1 All PMCA-b splice variants contain a class 1 PDZ consensus binding motif. Alignment of the C-termini tail of all PMCA-b splice variants reveals a highly conserved class 1 PDZ binding motif (X–S/T–X–ϕ; where ϕ = hydrophobic residue). Table adapted from Strehler and Zacharias (2001). hPMCA 1b hPMCA 2b hPMCA 3b hPMCA 4b Class 1 PDZ ligands
... ... ... ...
S S S S
P P P S
L I L L
H H H Q
S S S S
L L V L ...
E E E E ED
T T T T ST
S S S S X
L L L V ˚
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PSD-95 is responsible for membrane clustering and retention of a number of transporters and receptors including PMCA4b (Padanyi et al., 2009). This is of interest because it has been shown that the susceptibility of NMDARs to calpain cleavage is controlled by interactions with PSD-95 (Yuen et al., 2008). Thus a possible regulatory mechanism may involve PSD-95 recruiting calpain into complexes containing PMCA4b. 5. Medical/industrial applications
Fig. 2. Macromolecular complexing of PMCA1b and NHERF-2 in epithelial cells. In HT-29 epithelial cells, PMCA1b interacts with the second PDZ binding module (PDZ-2) of NHERF-2 via its C-terminal PDZ binding domain. Immediately following muscarinic activation a PMCA1b-NHERF-2 efflux complex is rapidly formed at the plasma membrane that corresponds to the peak of the Ca2+ transient. In this model we propose that (1) NHERF-2 binds to ezrin and the actin cytoskeleton to stabilise the complex at the plasma membrane and (2) ezrin may also act to recruit other known regulators of PMCAs such as PKA.
PMCA–PDZ interactions were first reported in platelets where the filopodia play a key role in clotting. In response to thrombin, PMCA was shown to translocate to the filopodia, where it is thought to maintain a low [Ca2+ ]i thereby regulating the rate of clot retraction (Dean and Whiteheart, 2004). This translocation was inhibited by the incorporation of a peptide encoding the last 10 amino acids of PMCA4b, the predominant isoform in these cells, by blocking PDZ interactions with the cytoskeleton. Subsequently it was found that this involved interactions with the PDZ proteins CLIP36 and alpha-actinin (Bozulic et al., 2007). The cardiac myocyte was also a focus of earlier studies on PMCA–PDZ interactions. PMCAs have a relatively low pumping efficiency and hence are not thought to be major modulators of myocyte contractile cycle. However, it was found that over expression of PMCA4b and alpha-1 syntrophin resulted in strong inhibition of nitric oxide (NO) production in myocytes, an effect due to regulation of NOS-1 by the PMCA4b-syntrophin complex (Williams et al., 2006). These data highlight an important emerging paradigm, that transporters in macromolecular complexes can regulate other signalling processes independent of their primary pumping function. An example of this is the Na+ –K+ ATPase, which plays a role in so-called signalomes, where it can act as a signalling molecule, mediate cross-talk with other receptors (such as the EGF receptor) or act via CREB to modulate dendritic growth in neurons (Desfrere et al., 2009). It is likely that such functions will also be identified for PMCA as we learn more about its protein–protein interactions. Our group has recently investigated the role of PDZ interactions in regulating PMCA-mediated Ca2+ efflux following GPCR activation. Using endogenous PMCA and NHERF-2 in HT-29 cells, we demonstrated the dynamic assembly of a transient PMCA1bNHERF-2 complex at the membrane in response to muscarinic activation (Kruger et al., 2009). This complex involves NHERF-2 moving to the membrane within 30 s of activation followed by PMCA1b within 60 s. The complex is then removed from the membrane within 120 s, despite [Ca2+ ]i remaining high. These data suggest that a specific PMCA efflux complex is nucleated by NHERF2 during the peak Ca2+ phase, presumably to protect against a Ca2+ overshoot (Fig. 2). This is an example of how PDZ mediated interactions can underlie the diversity of cellular responses mediated by increases in [Ca2+ ]i in response to GPCR activation. One place where PMCA modulation via these mechanisms could be particularly important is the synapse. The synaptic PDZ protein
There are a number of reviews that discuss changes in Ca2+ transporter activity in disease. In particular, some types of cancer are associated with remodelling of Ca2+ channel and PMCA expression (Monteith et al., 2007). Increased expression of PMCAs may contribute to apoptotic resistance and down-regulation may augment responses to proliferative stimuli which act through increases in [Ca2+ ]i . Indeed down-regulation of PMCA1 has been reported in human oral cancers (Saito et al., 2006). Changes in the expression profiles of PMCA isoforms are also reported in breast cancer cell lines, with modest upregulation of PMCA1 (Lee et al., 2002) and pronounced upregulation of PMCA2 (Lee et al., 2005a). This is of interest because PMCA2 is the isoform most upregulated in the mammary gland during lactation (Reinhardt et al., 2000), a time at which there is increased proliferation of the epithelial cells. More indirect studies also implicate remodelling of PMCA-mediated Ca2+ efflux as a feature of aberrant cell growth. For example, SV40mediated transformation of human skin fibroblasts is associated with a down-regulation of PMCA1b and PMCA4b (Reisner et al., 1997), whereas the differentiation of colon cancer cell lines is associated with upregulation of PMCA4 alone (Aung et al., 2007). The multiple roles of PMCAs and the macromolecular complexes responsible for Ca2+ signalling/efflux suggest that these interactions may well be potential drug targets for specific types of cancers and other diseases. In terms of PMCA inhibitors, other members of the P-type ATPase family have pharmacological modulators, such as thapsigargin for SERCA and ouabain for the Na+ –K+ -ATPase. Thus it stands to reason that PMCA should also be amenable to chemogenomic approaches to drug development (Monteith et al., 2007). Peptide inhibitors of PMCA, namely caloxins have been reported, with a PMCA4 isoform specific inhibitor recently developed (Szewczyk et al., 2008). Indeed, inhibition of PMCA expression dramatically inhibits the proliferation of MCF-7 breast cancer cells lines, through alterations in cell cycle kinetics (Lee et al., 2005b). As our understanding of the cellular physiology of PMCA and accessory proteins increases it is clear that further key roles for Ca2+ in disease will emerge. The fact that PMCA expression and activity is highly variable in different types of tumours highlights the need to target specific PMCA interactions rather than using a less specific pharmacological approach. From this perspective, PDZ interactions themselves are currently being explored as potential drug targets (Dev, 2004). Thus a detailed understanding of the exact protein composition and function of specific functional PMCA complexes may well pave the way for new drug strategies. References Aung CS, Kruger WA, Poronnik P, Roberts-Thomson SJ, Monteith GR. Plasma membrane Ca2+ -ATPase expression during colon cancer cell line differentiation. Biochem Biophys Res Commun 2007;355:932–6. Bozulic LD, Malik MT, Powell DW, Nanez A, Link AJ, Ramos KS, et al. Plasma membrane Ca2+ -ATPase associates with CLP36, alpha-actinin and actin in human platelets. Thromb Haemost 2007;97:587–97. Brini M. Plasma membrane Ca2+ -ATPase: from a housekeeping function to a versatile signalling role. Pflugers Arch 2009;457:657–64. Brini M, Coletto L, Pierobon N, Kraev N, Guerini D, Carafoli E. A comparative functional analysis of plasma membrane Ca2+ pump isoforms in intact cells. J Biol Chem 2003;278:24500–8. Carafoli E. The calcium-signalling saga: tap water and protein crystals. Nat Rev Mol Cell Biol 2003;4:326–32.
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Dean WL, Whiteheart SW. Plasma membrane Ca2+ -ATPase (PMCA) translocates to filopodia during platelet activation. Thromb Haemost 2004;91:325–33. DeMarco SJ, Chicka MC, Strehler EE. Plasma membrane Ca2+ ATPase isoform 2b interacts preferentially with Na+ /H+ exchanger regulatory factor 2 in apical plasma membranes. J Biol Chem 2002;277:10506–11. DeMarco SJ, Strehler EE. 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. J Biol Chem 2001;276:21594–600. Desfrere L, Karlsson M, Hiyoshi H, Malmersjo S, Nanou E, Estrada M, et al. Na+ K+ ATPase signal transduction triggers CREB activation and dendritic growth. Proc Natl Acad Sci USA 2009;106:2212–7. Dev KK. Making protein interactions druggable: targeting PDZ domains. Nat Rev Drug Discov 2004;3:1047–56. Ficarella R, Di Leva F, Bortolozzi M, Ortolano S, Donaudy F, Petrillo M, et al. A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness. Proc Natl Acad Sci USA 2007;104:1516–21. Kruger WA, Yun CC, Monteith GR, Poronnik P. Muscarinic-induced recruitment of plasma membrane Ca2+ -ATPase involves PSD-95/Dlg/Zo-1-mediated interactions. J Biol Chem 2009;284:1820–30. Lee WJ, Roberts-Thomson SJ, Holman NA, May FJ, Lehrbach GM, Monteith GR. Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines. Cell Signal 2002;14:1015–22. Lee WJ, Roberts-Thomson SJ, Monteith GR. Plasma membrane calcium-ATPase 2 and 4 in human breast cancer cell lines. Biochem Biophys Res Commun 2005a;337:779–83. Lee WJ, Robinson JA, Holman NA, McCall MN, Roberts-Thomson SJ, Monteith GR. Antisense-mediated Inhibition of the plasma membrane calciumATPase suppresses proliferation of MCF-7 cells. J Biol Chem 2005b;280: 27076–84. Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. Calcium and cancer: targeting Ca2+ transport. Nat Rev Cancer 2007;7:519–30. Okunade GW, Miller ML, Pyne GJ, Sutliff RL, O’Connor KT, Neumann JC, et al. Targeted ablation of plasma membrane Ca2+ -ATPase (PMCA) 1 and 4 indicates a major housekeeping function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4. J Biol Chem 2004;279: 33742–50.
Padanyi R, Paszty K, Strehler EE, Enyedi A. PSD-95 mediates membrane clustering of the human plasma membrane Ca2+ pump isoform 4b. Biochim Biophys Acta 2009;1793:1023–32. Penheiter AR, Filoteo AG, Croy CL, Penniston JT. Characterization of the deafwaddler mutant of the rat plasma membrane calcium-ATPase 2. Hear Res 2001;162:19–28. Reinhardt TA, Filoteo AG, Penniston JT, Horst RL. Ca2+ -ATPase protein expression in mammary tissue. Am J Physiol Cell Physiol 2000;279:C1595–602. Reisner PD, Brandt PC, Vanaman TC. Analysis of plasma membrane Ca2+ -ATPase expression in control and SV40-transformed human fibroblasts. Cell Calcium 1997;21:53–62. Saito K, Uzawa K, Endo Y, Kato Y, Nakashima D, Ogawara K, et al. Plasma membrane Ca2+ -ATPase isoform 1 down-regulated in human oral cancer. Oncol Rep 2006;15:49–55. Schatzmann HJ. ATP-dependent Ca2+ -extrusion from human red cells. Experientia 1966;22:364–5. Schultz JM, Yang Y, Caride AJ, Filoteo AG, Penheiter AR, Lagziel A, et al. Modification of human hearing loss by plasma-membrane calcium pump PMCA2. N Engl J Med 2005;352:1557–64. Strehler EE, Filoteo AG, Penniston JT, Caride AJ. Plasma-membrane Ca2+ pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 2007;35:919–22. Strehler EE, Zacharias DA. Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 2001;81:21–50. Szewczyk MM, Pande J, Grover AK. Caloxins: a novel class of selective plasma membrane Ca2+ pump inhibitors obtained using biotechnology. Pflugers Arch 2008;456:255–66. Williams JC, Armesilla AL, Mohamed TM, Hagarty CL, McIntyre FH, Schomburg S, et al. The sarcolemmal calcium pump, alpha-1 syntrophin, and neuronal nitricoxide synthase are parts of a macromolecular protein complex. J Biol Chem 2006;281:23341–8. Yuen EY, Ren Y, Yan Z. Postsynaptic density-95 (PSD-95) and calcineurin control the sensitivity of N-methyl-d-aspartate receptors to calpain cleavage in cortical neurons. Mol Pharmacol 2008;74:360–70. Zabe M, Dean WL. Plasma membrane Ca2+ -ATPase associates with the cytoskeleton in activated platelets through a PDZ-binding domain. J Biol Chem 2001;276: 14704–9.
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecules in focus
The plasma membrane Ca2+ -ATPase: Regulation by PSD-95/Dlg/Zo-1 scaffolds Wade A. Kruger a , Gregory R. Monteith b , Philip Poronnik a,∗ a b
Health Innovations Research Institute, School of Medical Sciences, RMIT University, Bundoora, Melbourne, PO Box 71, VIC 3083, Australia Faculty of Health Sciences, School of Pharmacy, The University of Queensland, Brisbane, QLD 4072, Australia
a r t i c l e
i n f o
Article history: Received 16 December 2009 Received in revised form 18 January 2010 Accepted 18 January 2010 Available online 25 January 2010 Keywords: PMCA PDZ Scaffold Calcium
a b s t r a c t Since its first characterization in the erythrocyte membrane the plasma membrane Ca2+ -ATPase has been well-defined as a ubiquitous mechanism for the efflux of Ca2+ from eukaryotic cells. With 4 isoforms and potentially 30 splice variants, defining the absolute physiological role of plasma membrane Ca2+ -ATPase has been difficult and very limited due to the lack of effective blockers/antibodies and difficulties in measuring the activity of individual isoforms. This review highlights recent developments showing that specific plasma membrane Ca2+ -ATPase isoforms are subject to dynamic regulation by PSD-95/Dlg/Zo-1 scaffold proteins. Such interactions support a new paradigm, that by serving as key players in multifunctional protein complexes, transporters can regulate other signalling processes independent of their primary ion pumping function. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction The spatial and temporal regulation of the ubiquitous second messenger, intracellular free Ca2+ ([Ca2+ ]i ), is fundamental for normal cellular function. Changes in [Ca2+ ]i trigger a myriad of cellular events from fertilization to gene transcription, neurotransmitter release and cell death. Many of these changes in [Ca2+ ]i are mediated by G-protein coupled receptors (GPCRs) to effect physiological responses (Carafoli, 2003). These GPCR-mediated increases in [Ca2+ ]i are typically biphasic, starting with mobilization of Ca2+ from intracellular stores which in turn triggers Ca2+ influx via plasma membrane ion channels. Following termination of receptor signalling, [Ca2+ ]i must return rapidly to its nanomolar resting levels. This is achieved by the reuptake of Ca2+ into intracellular stores and Ca2+ efflux across the plasma membrane mediated by the primarily ubiquitous plasma membrane Ca2+ -ATPase (PMCA) as well as the Na+ –Ca2+ exchanger in some cell types. Hence the dynamic interplay between influx, efflux and reuptake governs the magnitude, duration and location of the increase in [Ca2+ ]i to determine the physiological outcome. How then do cells produce specific responses in the face of a plethora of Ca2+ mobilizing agents in the extracellular milieu? One strategy to direct functional specificity is by locally constraining discrete molecular Ca2+ signalling complexes using protein–protein interactions and trafficking mechanisms to maintain spatial heterogeneity of Ca2+ signalling. There has been increasing interest in the trafficking of
∗ Corresponding author. Tel.: +61 3 9925 7071; fax: +61 7 9925 1766. E-mail address: [email protected] (P. Poronnik). 1357-2725/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2010.01.023
channels and associated proteins that are involved in regulating Ca2+ influx and release pathways. In this review we highlight the dynamic regulation of PMCA-mediated Ca2+ by PSD-95/Dlg/Zo-1 (PDZ) scaffold proteins. 2. Structure PMCAs belong to the P2 (subtype 2B) subfamily of P-type primary ion transport ATPases, which are characterized by the formation of an aspartyl phosphate intermediate upon ATP hydrolysis during their reaction cycle (Strehler and Zacharias, 2001). All four PMCA isoforms are encoded by the genes Atp2b1-4. These genes have a number of splice variants caused by the insertion of alternative exons at site A in the cytosolic loop connecting transmembrane domains 2 and 3, or at site C in the C-terminus of the pump (Ficarella et al., 2007). PMCA is predicted to contain 10 membrane spanning domains, a structure closely related to other P-type ATPases such as the sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) (Fig. 1). The first intracellular loop, between domains 2 and 3 corresponds to the “transduction domain” and is thought to play the key role in the conformational changes occurring during the Ca2+ transport cycle. The very large loop between domains 4 and 5 is the major catalytic domain and includes the ATP binding site (Strehler et al., 2007). Both the N- and C-termini are located at the cytosolic face of the membrane, enabling the association with accessory/scaffold proteins that can directly regulate the transporter. The C-terminus represents one of the major regulatory sites of the protein and contains many consensus binding/interaction sites for regulatory molecules (Brini, 2009). Importantly, the b-splice variants have a highly conserved Class 1 PDZ binding motif at the C-terminus (Table 1).
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Fig. 1. Predicted structure of PMCA in active and inactive states. PMCA is predicted to contain 10 membrane spanning domains, a structure closely related to other P-type ATPases. Both the N- and C-termini are located at the cytosolic face of the membrane, enabling the association with accessory/scaffold proteins that can directly regulate the transporter. In the unstimulated state, the calmodulin-binding domain binds to two sites within the transporter resulting in autoinhibition. However, when [Ca2+ ]i rises and binds to calmodulin, the Ca2+ -bound calmodulin interacts with the PMCA calmodulin-binding domain, thereby reversing the autoinhibition activating the pump to remove Ca2+ from the cytosol.
3. Biological function PMCA was first described in the erythrocyte membrane in the mid-1960s (Schatzmann, 1966) and has since been well-defined as a primary mechanism for Ca2+ efflux from eukaryotic cells (Fig. 1(ii)). Defining the absolute physiological roles of PMCAs is difficult due to the lack of effective blockers/antibodies and difficulties in measuring the activity of individual isoforms. Studies using transgenic animals have been equally challenging. Mice in which PMCA1 was ablated resulted in embryonic lethality. In contrast, despite its apparent ubiquity, Pmca4−/− male mice are infertile but have normal spermatogenesis and mating behaviour (Okunade et al., 2004). These findings challenge the “housekeeping” role proposed for PMCA4 or may simply reflect functional compensation by other PMCA isoforms. PMCA2 knockout mice (also known as “deafwaddler” and “wriggle mouse Sagami”) exhibit very specific phenotypes of unsteady gait, difficulties maintaining balance and hearing defects (Penheiter et al., 2001). Studies extrapolating from these knockout models have proven a link between loss of PMCA2 activity and degenerative hearing loss in humans (Schultz et al., 2005). Further studies into the role of specific PMCA isoforms will require strategies such as tissue specific knockdown. 4. Expression, activation and turnover As mentioned above, the four PMCAs isoforms differ in their tissue distribution. PMCA1 and 4 are ubiquitous while PMCA2 and 3 are more restricted, primarily in brain, nervous tissue and some types of muscle (Brini, 2009). A large number of splice variants are theoretically possible and to date approximately 30 have been detected at the RNA or protein level (Strehler and Zacharias, 2001). The diversity of isoform and splice variants is thought to provide the structural flexibility to enable the formation of the different functional complexes required for the dynamic, spatio-temporal regulation of cellular Ca2+ handling (Strehler and Zacharias, 2001). Each PMCA isoform has specific biophysical/biochemical properties. For example, the most extensively studied regulator of PMCA activity is calmodulin. In the unstimulated state, the calmodulin-
binding domain located at the C-terminus of PMCA binds to two intramolecular “receptor” sites within the transporter resulting in autoinhibition. However, when [Ca2+ ]i rises and binds to calmodulin, the Ca2+ -bound calmodulin subsequently interacts with the PMCA calmodulin-binding domain, thereby reversing the autoinhibition activating the pump (Strehler and Zacharias, 2001). The different PMCA isoforms and their splice variants have different affinities for calmodulin (PMCA2 having the highest and PMCA4 having the lowest). This in turn results in different levels of pump activity in response to changes in [Ca2+ ]i (Brini et al., 2003). PMCAs are activated by a variety of other mechanisms that include self-association (oligomerization), acidic phospholipids as well as regulatory proteins including protein kinase C (PKC), protein kinase A (PKA) and proteases such as calpain (Strehler et al., 2007). Interestingly, many of these regulatory proteins require the presence of molecular scaffolds to recruit them into the proximity of the transporter in order to act. It is therefore clear that trafficking plays a significant role in determining PMCA levels at the plasma membrane (Kruger et al., 2009). 4.1. Regulation of PMCA by PDZ scaffolds Ion channels, transporters and receptors form dynamic macromolecular complexes at the plasma membrane that direct functional specificity within cellular microdomains. The assembly of these complexes requires molecular scaffolds such as PDZ domain containing proteins. These PDZ domains bind to PDZ binding motifs contained within the membrane protein thereby allowing the assembly of heterologous protein complexes. In this context, the C-termini of all PMCA-b splice variants have a highly conserved Class 1 PDZ binding motif (refer Table 1) that has been reported to interact with a number of PDZ containing proteins such as nitric oxide synthase I (NOS-I), members of the membraneassociated guanylate kinase family, and the Na+ /H+ exchanger regulatory factor-2 (NHERF-2) (DeMarco and Strehler, 2001; Zabe and Dean, 2001; DeMarco et al., 2002). However, despite several reports of PMCA–PDZ interactions, the physiological outcomes of these interactions remain largely undefined.
Table 1 All PMCA-b splice variants contain a class 1 PDZ consensus binding motif. Alignment of the C-termini tail of all PMCA-b splice variants reveals a highly conserved class 1 PDZ binding motif (X–S/T–X–ϕ; where ϕ = hydrophobic residue). Table adapted from Strehler and Zacharias (2001). hPMCA 1b hPMCA 2b hPMCA 3b hPMCA 4b Class 1 PDZ ligands
... ... ... ...
S S S S
P P P S
L I L L
H H H Q
S S S S
L L V L ...
E E E E ED
T T T T ST
S S S S X
L L L V ˚
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PSD-95 is responsible for membrane clustering and retention of a number of transporters and receptors including PMCA4b (Padanyi et al., 2009). This is of interest because it has been shown that the susceptibility of NMDARs to calpain cleavage is controlled by interactions with PSD-95 (Yuen et al., 2008). Thus a possible regulatory mechanism may involve PSD-95 recruiting calpain into complexes containing PMCA4b. 5. Medical/industrial applications
Fig. 2. Macromolecular complexing of PMCA1b and NHERF-2 in epithelial cells. In HT-29 epithelial cells, PMCA1b interacts with the second PDZ binding module (PDZ-2) of NHERF-2 via its C-terminal PDZ binding domain. Immediately following muscarinic activation a PMCA1b-NHERF-2 efflux complex is rapidly formed at the plasma membrane that corresponds to the peak of the Ca2+ transient. In this model we propose that (1) NHERF-2 binds to ezrin and the actin cytoskeleton to stabilise the complex at the plasma membrane and (2) ezrin may also act to recruit other known regulators of PMCAs such as PKA.
PMCA–PDZ interactions were first reported in platelets where the filopodia play a key role in clotting. In response to thrombin, PMCA was shown to translocate to the filopodia, where it is thought to maintain a low [Ca2+ ]i thereby regulating the rate of clot retraction (Dean and Whiteheart, 2004). This translocation was inhibited by the incorporation of a peptide encoding the last 10 amino acids of PMCA4b, the predominant isoform in these cells, by blocking PDZ interactions with the cytoskeleton. Subsequently it was found that this involved interactions with the PDZ proteins CLIP36 and alpha-actinin (Bozulic et al., 2007). The cardiac myocyte was also a focus of earlier studies on PMCA–PDZ interactions. PMCAs have a relatively low pumping efficiency and hence are not thought to be major modulators of myocyte contractile cycle. However, it was found that over expression of PMCA4b and alpha-1 syntrophin resulted in strong inhibition of nitric oxide (NO) production in myocytes, an effect due to regulation of NOS-1 by the PMCA4b-syntrophin complex (Williams et al., 2006). These data highlight an important emerging paradigm, that transporters in macromolecular complexes can regulate other signalling processes independent of their primary pumping function. An example of this is the Na+ –K+ ATPase, which plays a role in so-called signalomes, where it can act as a signalling molecule, mediate cross-talk with other receptors (such as the EGF receptor) or act via CREB to modulate dendritic growth in neurons (Desfrere et al., 2009). It is likely that such functions will also be identified for PMCA as we learn more about its protein–protein interactions. Our group has recently investigated the role of PDZ interactions in regulating PMCA-mediated Ca2+ efflux following GPCR activation. Using endogenous PMCA and NHERF-2 in HT-29 cells, we demonstrated the dynamic assembly of a transient PMCA1bNHERF-2 complex at the membrane in response to muscarinic activation (Kruger et al., 2009). This complex involves NHERF-2 moving to the membrane within 30 s of activation followed by PMCA1b within 60 s. The complex is then removed from the membrane within 120 s, despite [Ca2+ ]i remaining high. These data suggest that a specific PMCA efflux complex is nucleated by NHERF2 during the peak Ca2+ phase, presumably to protect against a Ca2+ overshoot (Fig. 2). This is an example of how PDZ mediated interactions can underlie the diversity of cellular responses mediated by increases in [Ca2+ ]i in response to GPCR activation. One place where PMCA modulation via these mechanisms could be particularly important is the synapse. The synaptic PDZ protein
There are a number of reviews that discuss changes in Ca2+ transporter activity in disease. In particular, some types of cancer are associated with remodelling of Ca2+ channel and PMCA expression (Monteith et al., 2007). Increased expression of PMCAs may contribute to apoptotic resistance and down-regulation may augment responses to proliferative stimuli which act through increases in [Ca2+ ]i . Indeed down-regulation of PMCA1 has been reported in human oral cancers (Saito et al., 2006). Changes in the expression profiles of PMCA isoforms are also reported in breast cancer cell lines, with modest upregulation of PMCA1 (Lee et al., 2002) and pronounced upregulation of PMCA2 (Lee et al., 2005a). This is of interest because PMCA2 is the isoform most upregulated in the mammary gland during lactation (Reinhardt et al., 2000), a time at which there is increased proliferation of the epithelial cells. More indirect studies also implicate remodelling of PMCA-mediated Ca2+ efflux as a feature of aberrant cell growth. For example, SV40mediated transformation of human skin fibroblasts is associated with a down-regulation of PMCA1b and PMCA4b (Reisner et al., 1997), whereas the differentiation of colon cancer cell lines is associated with upregulation of PMCA4 alone (Aung et al., 2007). The multiple roles of PMCAs and the macromolecular complexes responsible for Ca2+ signalling/efflux suggest that these interactions may well be potential drug targets for specific types of cancers and other diseases. In terms of PMCA inhibitors, other members of the P-type ATPase family have pharmacological modulators, such as thapsigargin for SERCA and ouabain for the Na+ –K+ -ATPase. Thus it stands to reason that PMCA should also be amenable to chemogenomic approaches to drug development (Monteith et al., 2007). Peptide inhibitors of PMCA, namely caloxins have been reported, with a PMCA4 isoform specific inhibitor recently developed (Szewczyk et al., 2008). Indeed, inhibition of PMCA expression dramatically inhibits the proliferation of MCF-7 breast cancer cells lines, through alterations in cell cycle kinetics (Lee et al., 2005b). As our understanding of the cellular physiology of PMCA and accessory proteins increases it is clear that further key roles for Ca2+ in disease will emerge. The fact that PMCA expression and activity is highly variable in different types of tumours highlights the need to target specific PMCA interactions rather than using a less specific pharmacological approach. From this perspective, PDZ interactions themselves are currently being explored as potential drug targets (Dev, 2004). Thus a detailed understanding of the exact protein composition and function of specific functional PMCA complexes may well pave the way for new drug strategies. References Aung CS, Kruger WA, Poronnik P, Roberts-Thomson SJ, Monteith GR. Plasma membrane Ca2+ -ATPase expression during colon cancer cell line differentiation. Biochem Biophys Res Commun 2007;355:932–6. Bozulic LD, Malik MT, Powell DW, Nanez A, Link AJ, Ramos KS, et al. Plasma membrane Ca2+ -ATPase associates with CLP36, alpha-actinin and actin in human platelets. Thromb Haemost 2007;97:587–97. Brini M. Plasma membrane Ca2+ -ATPase: from a housekeeping function to a versatile signalling role. Pflugers Arch 2009;457:657–64. Brini M, Coletto L, Pierobon N, Kraev N, Guerini D, Carafoli E. A comparative functional analysis of plasma membrane Ca2+ pump isoforms in intact cells. J Biol Chem 2003;278:24500–8. Carafoli E. The calcium-signalling saga: tap water and protein crystals. Nat Rev Mol Cell Biol 2003;4:326–32.
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Dean WL, Whiteheart SW. Plasma membrane Ca2+ -ATPase (PMCA) translocates to filopodia during platelet activation. Thromb Haemost 2004;91:325–33. DeMarco SJ, Chicka MC, Strehler EE. Plasma membrane Ca2+ ATPase isoform 2b interacts preferentially with Na+ /H+ exchanger regulatory factor 2 in apical plasma membranes. J Biol Chem 2002;277:10506–11. DeMarco SJ, Strehler EE. 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. J Biol Chem 2001;276:21594–600. Desfrere L, Karlsson M, Hiyoshi H, Malmersjo S, Nanou E, Estrada M, et al. Na+ K+ ATPase signal transduction triggers CREB activation and dendritic growth. Proc Natl Acad Sci USA 2009;106:2212–7. Dev KK. Making protein interactions druggable: targeting PDZ domains. Nat Rev Drug Discov 2004;3:1047–56. Ficarella R, Di Leva F, Bortolozzi M, Ortolano S, Donaudy F, Petrillo M, et al. A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness. Proc Natl Acad Sci USA 2007;104:1516–21. Kruger WA, Yun CC, Monteith GR, Poronnik P. Muscarinic-induced recruitment of plasma membrane Ca2+ -ATPase involves PSD-95/Dlg/Zo-1-mediated interactions. J Biol Chem 2009;284:1820–30. Lee WJ, Roberts-Thomson SJ, Holman NA, May FJ, Lehrbach GM, Monteith GR. Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines. Cell Signal 2002;14:1015–22. Lee WJ, Roberts-Thomson SJ, Monteith GR. Plasma membrane calcium-ATPase 2 and 4 in human breast cancer cell lines. Biochem Biophys Res Commun 2005a;337:779–83. Lee WJ, Robinson JA, Holman NA, McCall MN, Roberts-Thomson SJ, Monteith GR. Antisense-mediated Inhibition of the plasma membrane calciumATPase suppresses proliferation of MCF-7 cells. J Biol Chem 2005b;280: 27076–84. Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. Calcium and cancer: targeting Ca2+ transport. Nat Rev Cancer 2007;7:519–30. Okunade GW, Miller ML, Pyne GJ, Sutliff RL, O’Connor KT, Neumann JC, et al. Targeted ablation of plasma membrane Ca2+ -ATPase (PMCA) 1 and 4 indicates a major housekeeping function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4. J Biol Chem 2004;279: 33742–50.
Padanyi R, Paszty K, Strehler EE, Enyedi A. PSD-95 mediates membrane clustering of the human plasma membrane Ca2+ pump isoform 4b. Biochim Biophys Acta 2009;1793:1023–32. Penheiter AR, Filoteo AG, Croy CL, Penniston JT. Characterization of the deafwaddler mutant of the rat plasma membrane calcium-ATPase 2. Hear Res 2001;162:19–28. Reinhardt TA, Filoteo AG, Penniston JT, Horst RL. Ca2+ -ATPase protein expression in mammary tissue. Am J Physiol Cell Physiol 2000;279:C1595–602. Reisner PD, Brandt PC, Vanaman TC. Analysis of plasma membrane Ca2+ -ATPase expression in control and SV40-transformed human fibroblasts. Cell Calcium 1997;21:53–62. Saito K, Uzawa K, Endo Y, Kato Y, Nakashima D, Ogawara K, et al. Plasma membrane Ca2+ -ATPase isoform 1 down-regulated in human oral cancer. Oncol Rep 2006;15:49–55. Schatzmann HJ. ATP-dependent Ca2+ -extrusion from human red cells. Experientia 1966;22:364–5. Schultz JM, Yang Y, Caride AJ, Filoteo AG, Penheiter AR, Lagziel A, et al. Modification of human hearing loss by plasma-membrane calcium pump PMCA2. N Engl J Med 2005;352:1557–64. Strehler EE, Filoteo AG, Penniston JT, Caride AJ. Plasma-membrane Ca2+ pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 2007;35:919–22. Strehler EE, Zacharias DA. Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 2001;81:21–50. Szewczyk MM, Pande J, Grover AK. Caloxins: a novel class of selective plasma membrane Ca2+ pump inhibitors obtained using biotechnology. Pflugers Arch 2008;456:255–66. Williams JC, Armesilla AL, Mohamed TM, Hagarty CL, McIntyre FH, Schomburg S, et al. The sarcolemmal calcium pump, alpha-1 syntrophin, and neuronal nitricoxide synthase are parts of a macromolecular protein complex. J Biol Chem 2006;281:23341–8. Yuen EY, Ren Y, Yan Z. Postsynaptic density-95 (PSD-95) and calcineurin control the sensitivity of N-methyl-d-aspartate receptors to calpain cleavage in cortical neurons. Mol Pharmacol 2008;74:360–70. Zabe M, Dean WL. Plasma membrane Ca2+ -ATPase associates with the cytoskeleton in activated platelets through a PDZ-binding domain. J Biol Chem 2001;276: 14704–9.