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Originally published In Press as doi:10.1074/jbc.M601578200 on June 1, 2006 Originally published In Press as doi:10.1074/jbc.M601578200 on June 1, 2006 Originally published In Press as doi:10.1074/jbc.M601578200 on May 31, 2006 Originally published In Press as doi:10.1074/jbc.M601578200 on May 24, 2006

J. Biol. Chem., Vol. 281, Issue 31, 21954-21962, August 4, 2006
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Distinct Role of the N-terminal Tail of the Na,K-ATPase Catalytic Subunit as a Signal Transducer*

Songbai Zhang{ddagger}§12, Seth Malmersjö{ddagger}1, Juan Li{ddagger}, Hideaki Ando§, Oleg Aizman{ddagger}, Per Uhlén{ddagger}, Katsuhiko Mikoshiba§, and Anita Aperia{ddagger}3

From the {ddagger}Department of Woman and Child Health, Karolinska Institutet, Astrid Lindgren Children's Hospital, Q2:09, SE-171 76 Stockholm, Sweden, the §Division of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan, and the Laboratory for Developmental Neurobiology, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Received for publication, February 17, 2006 , and in revised form, May 16, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mounting evidence suggests that the ion pump, Na,K-ATPase, can, in the presence of ouabain, act as a signal transducer. A prominent binding motif linking the Na,K-ATPase to intracellular signaling effectors has, however, not yet been identified. Here we report that the N-terminal tail of the Na,K-ATPase catalytic {alpha}-subunit ({alpha}NT-t) binds directly to the N terminus of the inositol 1,4,5-trisphosphate receptor. Three amino acid residues, LKK, conserved in most species and most {alpha}-isoforms, are essential for the binding to occur. In wild-type cells, low concentrations of ouabain trigger low frequency calcium oscillations that activate NF-{kappa}B and protect from apoptosis. All of these effects are suppressed in cells overexpressing a peptide corresponding to {alpha}NT-t but not in cells overexpressing a peptide corresponding to {alpha}NT-t{Delta}LKK. Thus we have identified a well conserved Na,K-ATPase motif that binds to the inositol 1,4,5-trisphosphate receptor and can trigger an anti-apoptotic calcium signal.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Na,K-ATPase is an integral plasma membrane protein that establishes the electrochemical gradient across the plasma membrane in all mammalian cells. Ouabain is a steroid derivative that binds specifically to Na,K-ATPase. Several recent studies suggest that the ouabain·Na,K-ATPase complex may act as a signal transducer and transcription activator (1-4) modulating cell growth (5, 6), apoptosis (7), and cell motility (8). These effects have been ascribed to the activation of a number of intracellular signaling pathways (for review see Refs. 9 and 10). Most, if not all, of these pathways involve the release of calcium (Ca2+) from intracellular stores via the inositol 1,4,5-trisphosphate receptor (InsP3R),4 and results from recent studies indicate that Na,K-ATPase tethers the InsP3R into a Ca2+ regulatory complex (3, 4). The exact mechanisms by which Na,K-ATPase activates the InsP3R remains to be elucidated. Here we report that the N-terminal tail of the Na,K-ATPase catalytic {alpha}-subunit binds to the N terminus of the InsP3R. Interaction between Na,K-ATPase and InsP3R modulates the Ca2+ oscillatory signal, which serves to protect the cell from apoptosis (11). The identification of a distinct motif in the Na,K-ATPase that via protein-protein interaction transmits this signal to the InsP3R has important implications for the many vital cell functions that are regulated by ouabain·Na,K-ATPase signaling.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Tissue—Two types of cells were used. COS-7 cells, a cell line derived from fetal monkey kidney, were used in most protocols. Because transformed cells are not suitable for serum deprivation-induced apoptosis, rat proximal tubule (RPT) cells in primary culture were used in these protocols. COS-7 cells were purchased from the European Collection of Cell Cultures and were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum and 2 mM L-glutamine. RPT cells were prepared as described previously (1). Briefly, the kidneys of 20-day-old male Sprague-Dawley rats were used, and the cells were cultured in supplemented Dulbecco's modified Eagle's medium on glass coverslips for 24 h. Lysates from whole brain and kidney cortex from male adolescent Sprague-Dawley rats were used in co-immunoprecipitation studies.

Plasmids and Transfection—All of the plasmids were propagated in Escherichia coli strain DH5{alpha}. All of the PCR products of cDNA fragments were generated in frame using Platinum® Pfx DNA polymerase (Invitrogen) and were verified by nucleotide sequencing. GFP-Na,K-ATPase {alpha}1, and GFP-Na,K-ATPase{alpha}1{Delta}NT-t were prepared as described previously (3). The Na,K-ATPase {alpha}2 and {alpha}3 plasmids were kind gifts from Dr. Thomas Pressly (Texas Tech University) and Dr. Jerry Lingrel (University of Cincinnati). The PCR products of the GFP cDNA fragment were subcloned into the site of HindIII of the Na,K-ATPase {alpha}2 plasmid to generate GFP-Na,K-ATPase {alpha}2 and into the site of HindIII and SacII of the Na,K-ATPase {alpha}3 plasmid to generate GFP-Na,K-ATPase {alpha}3. The PCR product of mRFP was subcloned into the site of EcoRI and EcoRV of pcDNA4/MyC-His B (Invitrogen) to generate pcDNA4-mRFP (the original mRFP cDNA was a kind gift from Dr. Roger Tsien, University of California, San Diego). Truncated cDNA fragments corresponding to different lengths of the N-terminal fragments of rat Na,K-ATPase {alpha}1 were subcloned into the site of HindIII and BamHI of pEGFP-C1 (Clontech) to generate GFP-{alpha}NT-t and GFP-{alpha}NT-t{Delta}LKK, and into the site of EcoRV and XhoI of pcDNA4-mRFP to generate mRFP-{alpha}NT-t and mRFP-{alpha}NT-t{Delta}LKK (both of these two plasmids contain a stop codon before the XhoI site). The PCR products corresponding to different lengths of the N-terminal fragments of human Na,K-ATPase {alpha}1 or rat Na,K-ATPase {alpha}3 were subcloned into the site of HindIII and BamHI of pEGFP-C1 (Clontech) to generate GFP-h{alpha}NT-t, GFP-{alpha}3NT-t, and GFP-{alpha}3NT-t{Delta}LKK (Table 1).


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  		TABLE 1
Description of the plasmids used in this study

 
The PCR products corresponding to the suppressor domain and the InsP3-binding domain (amino acids 1-604 of mouse InsP3R type-1) were subcloned into the site of BamHI and EcoRI of pGEX-KG (12) to generate GST-InsP3R (1-604) and into BamHI and XhoI of pET-23a(+) (Novagen) to generate InsP3R (1-604)-His. GST-InsP3R (1-225), GST-InsP3R (1-343), and GST-InsP3R (226-604) were generated as previously described (13).

Transfections were performed using Effectene transfection reagent (Qiagen) according to the manufacturer's protocol. Transfected COS-7 cells were harvested for binding assay or used for Ca2+ imaging experiments 1-2 days after transfection.

Purification of Recombinant Proteins and GST Pulldown Assays—The plasmids encoding GST or His fusion proteins were transformed into E. coli BL21 (DE3) pLysS (Stratagene). Harvested E. coli expressed with GST fusion proteins or GST alone was sonicated in buffer A (10 mM Hepes, pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol, 0.5% Triton X-100). Adult whole rat brains were homogenized in buffer A. Harvested E. coli expressed with InsP3R (1-604)-His was sonicated in buffer B (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM 2-mercaptoethanol, 0.1% Triton X-100). The lysates of E. coli or rat brains were centrifuged at 800 x g for 10 min at 4 °C. The supernatants were then subjected to another centrifugation at 20,000 x g for 30 min at 4 °C, and the supernatants from the second centrifugation were used for pulldown binding assay. The strategy for GST pulldown assay was modified from previous description (14). For each reaction, 200 µg of protein of solubilized lysates of E. coli expressed with GST fusion proteins or GST alone were incubated with 30 µl of 1:1 slurry of glutathione-Sepharose 4B (Amersham Biosciences) at 4 °C for 1 h and then washed with Buffer C (4 mM Hepes, 100 mM NaCl, 0.1% Triton X-100) three times. The spun down complexes of glutathione-Sepharose 4B, including the fusion proteins, were incubated with 200 µg of protein of solubilized lysates of rat brain for 2 h or overnight at 4 °C. The complexes were then spun down and washed with Buffer D (Buffer C + 50 mM NaCl) three times. The proteins were eluted by boiling in 60 µl of 2x SDS-PAGE sample buffer for 3 min, separated by SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were probed with anti-GFP antibody or anti-Na,K-ATPase {alpha}1 antibody. For in vitro binding assays, GST or GST-{alpha}NT-t were eluted with reduced glutathione (Sigma). InsP3R (1-604)-His was purified with ProBond resin (Invitrogen). All three proteins were dialyzed in Buffer C. 15 µg of dialyzed GST, GST-{alpha}NT-t were incubated with 5-15 µg of InsP3R (1-604)-His for 2 h or overnight at 4 °C. The total volume for each reaction was 1 ml. The complexes were spun down and washed with Buffer D three times and then subjected to SDS-PAGE for Western blotting assay or Coomassie Brilliant Blue staining.

Calcium Imaging and Power Spectrum Analysis—COS-7 cells were loaded with 5 µM Fura2/AM (Molecular Probes) at room temperature for 1 h. Calcium measurements were performed at ~37 °C in a heated chamber (QE-1; Warner Instruments) with a cooled CCD camera (ORCA-ERG; Hamamatsu) mounted on an upright microscope (Axioskop 2 FS; Zeiss) with a40 x 0.8 NA water dipping lens. Excitation at 340 and 380 nm was performed with a monochromator (Polychrome IV; TILL Photonics). All of the devices were controlled, and the data were analyzed with computer software (MetaFluor; Molecular Devices). Sampling frequencies were between 0.05 and 0.1 Hz. All of the experiments were performed in physiological buffer (100 mM NaCl, 4 mM KCl, 20 mM Hepes, 25 mM NaHCO3, 1.5 mM CaCl2, 1.1 mM MgCl2, 1 mM NaH2PO4, 10 mM D-glucose, pH 7.4). To determine the number of oscillating cells, an oscillating cell was defined as a cell that displayed at least two well defined Ca2+ peaks, where each peak value was an increase in Ca2+ of more than 10% compared with the base line. Spectral analysis of Ca2+ oscillations was performed with MATLAB software as described previously (15). The red fluorescent protein mRFP was used, because its spectral properties are better in combination with Fura2/AM compared with GFP. The Ca2+ imaging studies were preformed by an investigator blind to the transfection protocol.

Detection and Quantification of Apoptotic Cells (TUNEL Staining)—We have previously found that ouabain protects from serum deprivation-induced apoptosis, but not from apoptosis triggered by 0.5 µM staurosporin (11). Because cell lines are resistant to serum deprivation-induced apoptosis, we used RPT cells in primary culture in this protocol. This cell type has previously been shown to respond to ouabain with Ca2+ oscillations and downstream NF-{kappa}B activation (1, 3). Cells were cultured with 10 or 0.2% fetal bovine serum for 24 h and treated with 10 nM ouabain. To obtain a semiquantitative estimation of the level of apoptosis, the TUNEL assay (ApopTag Red in situ apoptosis detection kit; Chemicon Int.) was carried out according to the manufacturer's instructions. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; 1.5 µg/ml).


Figure 1
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  		FIGURE 1.
Binding of InsP3R (1-604) to mammalian Na,K-ATPase {alpha}-subunits. A, schematic representation of mouse InsP3R type-1 and the peptide fragments corresponding to various lengths of the N terminus of the InsP3R fused with GST. B, the GST fusion proteins or GST alone attached to GSH-Sepharose were incubated with the lysates of whole rat brain or rat kidney cortex and subjected to Western blotting with anti-Na,K-ATPase {alpha}1 antibody. GST-InsP3R (1-343) and GST-InsP3R (1-604) bind to Na,K-ATPase {alpha}1, whereas GST-InsP3R (1-225), GST-InsP3R (226-604), and GST does not. C, the GST fusion proteins or GST alone attached to GSH-Sepharose were incubated with the lysates of COS-7 cells expressing GFP-Na,K-ATPase {alpha}1, GFP-Na,K-ATPase {alpha}2, or GFP-Na,K-ATPase {alpha}3. The proteins were subjected to Western blotting with anti-GFP antibody. GST-InsP3R (1-343) and GST-InsP3R (1-604) bind to GFP-Na,K-ATPase {alpha}1, GFP-Na,K-ATPase {alpha}2, and GFP-Na,K-ATPase {alpha}3, whereas GST-InsP3R (1-225), GST-InsP3R (226-604), and GST do not.

 
The cells were examined in a Leica TCS SP inverted confocal scanning laser microscope using a 40x/1.4 N.A. oilimmersion objective. ApopTag Red fluorescence was excited at 543 nm and detected with a 560-620-nm band pass filter. DAPI-stained cells were viewed with UV light. The apoptotic index (AI), i.e. number of apoptotic cells/total number of cells counted x 100, was determined by counting the number of ApopTag-positive cells over total number of cells, determined by DAPI staining. In each preparation 8-10 randomly selected areas were examined, and in each area 100-200 DAPI-stained cells were counted. Approximately 1000 DAPI-stained cells were counted in each preparation.

NF-{kappa}B Activity Assay—NF-{kappa}B translocation to nucleus was used as an index of NF-{kappa}B activation and studied with immunocytochemistry. Immunocytochemistry was performed as previously described (1).

Rubidium Uptake—The dose-dependent effect of ouabain on Na,K-ATPase activity was determined by measuring ouabain-sensitive 86Rb+ uptake as described previously (16).

Antibodies and Chemicals—The following antibodies and chemicals were purchased: mouse anti-GFP monoclonal antibody (Clontech), anti-Na,K-ATPase {alpha}1 monoclonal antibody (Upstate%20Biotechnology">Upstate Biotechnology), mouse anti-His monoclonal antibody (Invitrogen), Rabbit anti-NF-{kappa}B polyclonal antibody (Santa Cruz Biotechnology), ouabain and bradykinin (Sigma), Fura2/AM (Invitrogen), and thapsigargin (Calbiochem).

Statistics—The data are presented as the means ± S.E. Student's t test and one-way analysis of variance with a Bonferroni post hoc test was used, and significance was accepted at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Na,K-ATPase {alpha}1, {alpha}2, and {alpha}3-Subunits Interact with the N Terminus of the InsP3R—The InsP3R is a protein of 2749 residues. There are three functionally distinct regions within the InsP3R, the long N-terminal portion of the receptor, the channel-forming region, and the short C-terminal regulatory segment (Fig. 1A). To screen for an interaction between the Na,K-ATPase and the InsP3R, we first incubated glutathione S-transferase (GST)-fused proteins encoding for various lengths of the N terminus of the InsP3R type 1 with lysates from whole rat brain or with lysates from COS-7 cells transfected with GFP-fused rat Na,K-ATPase {alpha}-subunits. Consistent interaction was found between the Na,K-ATPase {alpha}1-subunit and peptide fragments corresponding to residues 1-604 of InsP3R type 1. This fragment of InsP3R type 1 consists of a suppressor domain and an InsP3-binding domain (17) and shares high homology with other subtypes of InsP3R. Because the main goal of this study was to identify an InsP3R-binding site in the Na,K-ATPase molecule, all of the subsequent studies were performed with peptide fragments corresponding to various lengths of the 1-604 portion of the InsP3R type 1.

As shown in Fig. 1, GST fusion proteins encoding for various lengths of InsP3R (1-604) were incubated with lysates of whole rat brain and rat kidney cortex and pulled down with GSH-Sepharose (Fig. 1B). GST-InsP3R (1-604) and GST-InsP3R (1-343), which encodes the suppressor domain and the first 118 residues of the InsP3-binding domain, did pull down Na,K-ATPase {alpha}1 from lysates of whole rat brain and rat kidney cortex, whereas GST-InsP3R (1-225), which encodes only the suppressor domain, and GST-InsP3R (226-604), which encodes only the InsP3-binding domain, did not (Fig. 1B). To examine whether InsP3R (1-604) also assembles with other Na,K-ATPase {alpha}-isoforms, the GST fusion proteins were incubated with lysates of COS-7 cells transfected with GFP-fused rat Na,K-ATPase {alpha}1, {alpha}2, and {alpha}3. As shown in Fig. 1C, GST-InsP3R (1-343) and GST-InsP3R (1-604) were found to assemble with GFP-Na,K-ATPase {alpha}1, {alpha}2, and {alpha}3. GST-InsP3R (1-225), GST-InsP3R (226-604) and GST alone did not assemble with any of the {alpha}-isoforms.


Figure 2
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  		FIGURE 2.
The N-terminal tail of Na,K-ATPase {alpha}-subunits is necessary and sufficient for binding to InsP3R (1-604). A, schematic structure of the rat Na,K-ATPase {alpha}1 and the amino acids sequence of the N-terminal cytoplasmic tail. B and C, GST-InsP3R (1-604) attached to GSH-Sepharose was incubated with the lysates of COS-7 cells expressing GFP-Na,K-ATPase {alpha}1 or its truncations (B). The proteins were subjected to Western blotting with anti-GFP antibody (C). GFP-Na,K-ATPase {alpha}1 and GFP-{alpha}NT-t bind to GST-InsP3R (1-604), whereas GFP-Na,K-ATPase{alpha}1{Delta}NT-t does not.

 
The N-terminal Tail of Na,K-ATPase {alpha}-Subunit Is Responsible for Binding to InsP3R (1-604)—Because it was suggested from previous studies that the N-terminal tail of the Na,K-ATPase {alpha}-subunit plays an important role for Na,K-ATPase signaling (3, 4), we examined whether GST-InsP3R (1-604) would assemble with a truncated form of the Na,K-ATPase {alpha}-subunit, where the first 32 residues of the N-terminal tail had been deleted (GFP-Na,K-ATPase{alpha}1{Delta}NT-t) and/or with a peptide fragment, corresponding to the N-terminal tail of Na,K-ATPase {alpha}1 (GFP-{alpha}NT-t) (Fig. 2, A and B). These constructs were expressed in COS-7 cells, and pulldown assays were performed. As shown in Fig. 2C, GST-InsP3R (1-604) assembled with GFP-{alpha}NT-t. In contrast, GST-InsP3R (1-604) did not assemble with GFP-Na,K-ATPase{alpha}1{Delta}NT-t.

The Well Conserved Amino Acid Residues LKK in the N-terminal Tail of Na,K-ATPase {alpha}-Subunit Are Essential for the Interaction with InsP3R—Because the studies shown in Fig. 1C demonstrated that GST-InsP3R (1-604) also assembled with the Na,K-ATPase {alpha}2- and {alpha}3-isoforms, we next examined the homology of the N-terminal tail. The N-terminal tail of different isoforms and species displayed little homology, except for 3 amino acid residues, LKK, which are conserved in all of the Na,K-ATPase {alpha}-subunit isoforms examined (Fig. 3A).

To study the role of the LKK residues for the Na,K-ATPase-InsP3R interaction, a peptide fragment corresponding to the N-terminal tail of the rat Na,K-ATPase {alpha}1-subunit in which the LKK residues were deleted was generated (GFP-{alpha}NT-t{Delta}LKK). This fragment did not assemble with GST-InsP3R (1-604) (Fig. 3, B and C). In contrast, a peptide fragment corresponding to the human {alpha}1 N-terminal tail, which has little homology with the rat {alpha}1 N-terminal tail, except for the LKK motif, did assemble with GST-InsP3R (1-604) (Fig. 3, D and E). To further examine the role of the conserved LKK for the interaction, two different lengths of the N-terminal tail of rat Na,K-ATPase {alpha}3-subunit, with or without the conserved LKK, were constructed and used for pulldown assay. As shown in Fig. 3 (F and G), the fragment of the N-terminal tail of rat Na,K-ATPase {alpha}3-subunit with the conserved LKK did assemble with GST-InsP3R (1-604), whereas the fragment of the N-terminal tail of rat Na,K-ATPase {alpha}3-subunit lacking the conserved LKK did not.

To determine whether the Na,K-ATPase {alpha}-subunit can bind directly to the InsP3R (1-604), InsP3R (1-604) was tagged with His (InsP3R (1-604)-His) (Fig. 4A) and expressed in E. coli. Purified InsP3R (1-604)-His was incubated with purified GST-{alpha}NT-t and then precipitated with GSH-Sepharose. As depicted in Fig. 4 (B and C), GST-{alpha}NT-t did bind to InsP3R (1-604)-His.


Figure 3
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  		FIGURE 3.
The three residues, LKK, conserved in the Na,K-ATPase {alpha}-subunit of all species, are essential for the interaction with the InsP3R (1-604). A, sequence alignment of the N-terminal tails of Na,K-ATPase {alpha}-subunits. Conserved residues are highlighted in gray. B and C, GST-InsP3R (1-604) attached to GSH-Sepharose was incubated with the lysate of COS-7 cells expressing GFP-{alpha}NT-t or GFP-{alpha}NT-t{Delta}LKK (B). The proteins were subjected to Western blotting with anti-GFP antibody (C). GFP-{alpha}NT-t binds to GST-InsP3R (1-604), whereas GFP-{alpha}NT-t{Delta}LKK does not. D and E, GST-InsP3R (1-604) or GST attached to GSH-Sepharose were incubated with the lysate of COS-7 cells expressing GFP-h{alpha}NT (D). The proteins were subjected to Western blotting with anti-GFP antibody (E). GFP-h{alpha}NT-t binds to GST-InsP3R (1-604) but not to GST. F and G, GST-InsP3R (1-604) attached to GSH-Sepharose was incubated with the lysates of COS-7 cells expressing GFP-{alpha}3NT-t or GFP-{alpha}3NT-t{Delta}LKK (F). The proteins were subjected to Western blotting with anti-GFP antibody (G). GFP-{alpha}3NT-t binds to GST-InsP3R (1-604), whereas GFP-{alpha}3NT-t{Delta}LKK does not.

 
The First Five Residues, Which Are Not Present in the Mature Na,K-ATPase {alpha}1-Subunit Protein, May Block Na,K-ATPase-InsP3R Interaction—The first five residues of rat Na,K-ATPase {alpha}1-subunit, MGKGV, are considered to be absent in the mature protein (18). The physiological significance of these five residues is unknown. To examine whether these five residues have an effect on Na,K-ATPase-InsP3R interaction, GFP-MGKGV-{alpha}NT-t were expressed in COS-7 cells, and pulldown assays were performed. The GFP fusion prevents the cleavage of MGKGV (18). As shown in Fig. 5 (A and B), GFP-MGKGV-{alpha}NT-t did not assemble with GST-InsP3R (1-604), suggesting that these five residues of the Na,K-ATPase {alpha}1-subunit may inhibit the Na,K-ATPase-InsP3R interaction. To further test this possibility, we recombined these first five residues of the {alpha}1-subunit with the N-terminal tail of the {alpha}3-subunit. GFP-MGKGV-{alpha}3NT-t expressed in COS-7 cells was subjected to GST-InsP3R (1-604) for pulldown assay. As shown in Fig. 5 (C and D), GFP-MGKGV-{alpha}3NT-t did not assemble with GST-InsP3R (1-604). These findings indicate that these five amino acid residues of the Na,K-ATPase block the Na,K-ATPase-InsP3R interaction and interfere with the signaling function of Na,K-ATPase. Thus it will be an important topic for further studies to identify the mechanisms that regulate the translational modification of the Na,K-ATPase {alpha}1.

Role of the Na,K-ATPase N Terminus for Ouabain-induced Ca2+ Oscillations—Next we examined the impact of the Na,K-ATPase N terminus for the functional interaction between Na,K-ATPase and the InsP3R. We have previously shown that ouabain enhances the interaction between Na,K-ATPase and the InsP3R and triggers an oscillatory Ca2+ response that is dependent on release of Ca2+ via the InsP3R. Those studies were performed mainly on rat renal cells in primary culture. A similar oscillatory response to ouabain is also observed in COS-7 cells (Fig. 6, A and B). The majority of ouabain-exposed cells displayed regular Ca2+ oscillations with an initial stable base line. The Ca2+ oscillations were analyzed using power spectral analysis and revealed an oscillatory period of 3.9 ± 0.2 min for 0.2 µM ouabain (66 cells from seven experiments) and 3.3 ± 0.1 min for 1 µM ouabain (95 cells from nine experiments) treatment (Fig. 6, C and D). The number of cells responding with Ca2+ oscillations increased dose-dependently with increasing ouabain concentration (Fig. 6E, open circles). The threshold concentration of ouabain required for triggering Ca2+ oscillations gave less than 10% inhibition of Rb+ uptake, used as an index of Na,K-ATPase dependent ion flux (Fig. 6E, filled circles). The off rate for ouabain to Na,K-ATPase is much slower than the on rate (19), and if cells were exposed to 10-50 nM ouabain for several hours, Ca2+ oscillations were observed in 5-30% of COS-7 cells. Ouabain failed to induce Ca2+ oscillations in cells that had been treated with thapsigargin to deplete the endoplasmic stores of Ca2+ (Fig. 6F). The ouabain response was then studied in cells transfected with truncated form of the {alpha}-subunit, where the first 32 amino acid residues were deleted. The truncated {alpha}-subunit was fused to GFP. The number of GFP-positive cells responding with Ca2+ oscillations was significantly lower than the number of GFP-negative cells responding to ouabain (44.8 ± 7.9 and 70.1 ± 6.7%, respectively) (Fig. 6G). GFP-positive and GFP-negative cells on the same plate were compared. Cells expressing GFP alone showed a similar ouabain response as GFP-negative cells (data not shown).


Figure 4
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  		FIGURE 4.
Na,K-ATPase {alpha}-subunits bind directly to the InsP3R (1-604). Purified InsP3R (1-604)-His was incubated with purified GST-{alpha}NT-t (A) or GST and then precipitated with GSH-Sepharose. The proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB)(B) or probed with anti-His antibody (C). GST-{alpha}NT-t binds to InsP3R (1-604)-His, whereas GST does not. The arrows indicate the size for InsP3R (1-604)-His. WB, Western blotting.

 
To study whether overexpression of {alpha}NT-t would result in a constitutive activation of InsP3R, leading to lower Ca2+ levels in ER, the Ca2+ content in ER was evaluated by estimating the release of Ca2+ from the ER following inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase by thapsigargin. As shown in Fig. 7A, the Ca2+ release from the ER was similar in cells overexpressing mRFP-{alpha}NT-t and mRFP-{alpha}NT-t{Delta}LKK.

Thereafter we examined whether the {alpha}NT-t peptide might act as an inhibitor of ouabain-induced Ca2+ oscillations. Overexpression of the fusion peptide mRFP-{alpha}NT-t resulted in a robust decline of the number of cells responding to 0.2 µM ouabain (37.3 ± 8.0% for mRFP-positive cells and 63.2 ± 4.7% for mRFP-negative cells) (Fig. 7B). In contrast, the number of ouabain responding cells was unaltered following overexpression of mRFP-{alpha}NT-t{Delta}LKK (73.5 ± 7.9% for mRFP-positive cells and 73.6 ± 4.8% for mRFP-negative cells).

To test whether the {alpha}NT-t peptide might act as an inhibitor of InsP3 activation of the InsP3R Ca2+ release channel, cells were exposed to bradykinin to stimulate the generation of endogenous InsP3. The number of responding cells (Fig. 7C) as well as the amplitude and full duration at half maximum of the intracellular Ca2+ peak (data not shown) was similar in wild-type cells and cells overexpressing mRFP-{alpha}NT-t and mRFP-{alpha}NT-t{Delta}LKK.

Ouabain Protection from Serum Deprivation-induced Apoptosis and Activation of NF-{kappa}B Is Mediated via the Na,K-ATPase {alpha}1-Subunit N-terminal Tail—Slow Ca2+ oscillations have been shown to activate the Ca2+-dependent transcription factor NF-{kappa}B (20). NF-{kappa}B has in many systems an anti-apoptotic effect (21, 22), and we have, in a recently completed study, shown that noninhibitory doses of ouabain protect from serum deprivation-induced apoptosis (11). Here we have examined whether expression of the {alpha}NT-t may attenuate the ouabain-induced protection from apoptosis. Because cell lines are fairly independent of growth factors, we used RPT cells in primary culture to provoke serum deprivation-induced apoptosis. This cell type has previously been shown to respond to low doses of ouabain with Ca2+ oscillations (3). RPT cells expressing GFP-{alpha}NT-t, GFP-{alpha}NT-t{Delta}LKK, or GFP only were incubated with 10 nM ouabain for 24 h. NF-{kappa}B activation was estimated by measuring the ratio of NF-{kappa}B nuclear to cytosolic signal. In cells expressing GFP only or GFP-{alpha}NT-t{Delta}LKK, exposure to ouabain caused a significant increase in NF-{kappa}B nuclear to cytosolic ratio (154.8 ± 3.0% and 129.3 ± 7.3%, respectively). In contrast, the ratio was not affected by ouabain in cells expressing GFP-{alpha}NT-t (93.0 ± 4.1%).


Figure 5
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  		FIGURE 5.
The first five residues, which are generally considered to be cleaved in the mature Na,K-ATPase {alpha}1-subunit, can block the Na,K-ATPase-InsP3R interaction. A and B, GST-InsP3R (1-604) attached to GSH-Sepharose was incubated with the lysates of COS-7 cells expressing GFP-MGKGV-{alpha}NT-t or GFP-{alpha}NT-t (A). The proteins were subjected to Western blotting (WB) with anti-GFP antibody (B). GFP-{alpha}NT-t binds to GST-InsP3R (1-604), but GFP-MGKGV-{alpha}NT-t does not. C and D, GST-InsP3R (1-604) attached to GSH-Sepharose was incubated with the lysates of COS-7 cells expressing GFP-MGKGV-{alpha}3NT-t or GFP-{alpha}3NT-t (C). The proteins were subjected to Western blotting with anti-GFP antibody (D). GFP-{alpha}3NT-t binds to GST-InsP3R (1-604), whereas GFP-MGKGV-{alpha}3NT-t does not.

 
To explore the ouabain-induced protection from apoptosis, the AI was measured. In each protocol AI was measured from more than 1000 cells. During control conditions AI ranges between 1 and 4%. In serum-deprived cells, the AI ranged between 10 and 15%. Ouabain failed to protect from serum deprivation-induced apoptosis in RPT cells expressing GFP-{alpha}NT-t, but not in cells expressing GFP-{alpha}NT-t{Delta}LKK (Fig. 7D). Representative confocal images of RPT cells treated with 10 nM ouabain and transfected with either GFP-{alpha}NT-t{Delta}LKK or GFP-{alpha}NT-t and stained using TUNEL assay for apoptosis are shown in Fig. 7 (E-J). RPT cells were grown in 0.2% fetal bovine serum for 24 h in the presence or absence of 10 nM ouabain.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Na,K-ATPase is a well studied molecule in its role as the main determinant of the sodium/potassium gradient across the plasma membrane in most eukaryotic cells (23-25). The minimal functional unit is a heterodimer of a catalytic {alpha}-subunit and a beta-subunit that is required for proper Na,K-ATPase plasma membrane insertion (26). Recent work from many laboratories has demonstrated that Na,K-ATPase is an important signaling receptor. Ouabain, a steroid hormone generally considered to be produced in the adrenals and hypothalamus (27), is a highly specific ligand of the Na,K-ATPase {alpha}-subunit. Many recent studies have indicated that ouabain-bound Na,K-ATPase can, independent of its ion transporting function, activate a number of signaling pathways (9, 10) and modulate cell growth (5, 6), migration (8), and programmed cell death (7). Low doses of ouabain have in several cell types been shown to activate InsP3R and trigger intracellular Ca2+ oscillation (1, 3). Recent work from many laboratories has begun to define the Na,K-ATPase signalosome and to map the functional domains that are involved in the organization of the individual signaling. The present study provides the first documentation that Na,K-ATPase can regulate cellular functions via direct interaction with an intracellular signaling molecule. We show here that the N-terminal tail of the Na,K-ATPase {alpha}-subunit binds to InsP3R and that a peptide fragment, corresponding to the N-terminal tail of Na,K-ATPase {alpha}-subunit, will attenuate ouabain-induced Ca2+ oscillations as well as the downstream effects on NF-{kappa}B activation and apoptosis. The Na,K-ATPase {alpha}-subunit can tolerate extensive mutation within the N-terminal tail without compromising the pump function, and a number of studies have shown that the first 32 amino acid residues of the rat Na,K-ATPase catalytic {alpha}1-subunit, the {alpha}-isoform most extensively used in this study, can be deleted without changes in overall Na,K-ATPase function (28-30). Thus the Ca2+ signaling function of Na,K-ATPase resides in a segment of the molecule that is not essential for pump function.

The mammalian catalytic subunit of Na,K-ATPase exists in at least four isoforms (31). The {alpha}1-isoform is ubiquitously expressed, whereas the other isoforms, such as the neuron-specific {alpha}3-isoform, are more tissue-specific (32). All of the isoforms of rat Na,K-ATPase {alpha}-subunit interacted with the InsP3R. Furthermore the N-terminal tail of both human and rat {alpha}1- and {alpha}3-subunit were found to interact with InsP3R (1-604) (Fig. 3). Although all Na,K-ATPase {alpha}-isoforms share more than 80% homology, the N-terminal tail shows little homology between the different isoforms (33). One exception is the lysine-rich motif (LKK) in the N-terminal tail that is conserved in almost all species. This well conserved motif was found to be essential for the binding between the Na,K-ATPase {alpha}-subunit and the InsP3R, indicating the universal importance of this protein-protein interaction.

The N terminus of the InsP3R consists of two distinct functional units: the suppressor domain (amino acids 1-225) and the InsP3-binding domain (amino acids 226-579). Na,K-ATPase {alpha}1-subunit did not interact with either functional unit alone but did interact with the suppressor domain and the first 118 amino acids of the InsP3-binding domain (InsP3R (1-343)). The crystal structure of the suppressor domain of the InsP3R and the InsP3-binding domain have recently been solved (17, 34). As discussed by Bosanac et al. (34), a highly conserved surface on the InsP3-binding domain, including amino acids Glu283, Val286, Lys306, and Tyr313, is a likely site for interaction with other proteins, such as the Na,K-ATPase {alpha}-subunit. Functional data from this and previous study imply that the binding sites for the {alpha}NT-t and the binding site for the InsP3 molecule do not overlap. Bradykinin, a well known trigger of intracellular InsP3 generation, produced the same Ca2+ response in cells overexpressing the {alpha}NT-t as in wild-type cells. Ouabain-induced Ca2+ oscillations are still observed in the presence of phospholipase C inhibitor, as well as in cells expressing a peptide that binds InsP3 with much higher affinity than the InsP3R (3).


Figure 6
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  		FIGURE 6.
Ouabain-induced Ca2+ oscillations in COS-7 cells. A and B, slow regular Ca2+ oscillations were observed in cells loaded with a Ca2+-sensitive dye (Fura2/AM) and treated with 0.2 µM or 1.0 µM ouabain. [Ca2+]i (a.u.) represents the ratio of the Fura2/AM images (340/380 nm), corresponding to changes in intracellular Ca2+ levels. C and D, power spectral analysis of the two ouabain-evoked Ca2+ oscillations depicted in A and B, respectively. For each concentration the mean period (time ± S.E.) of the oscillations was calculated from more than 65 cells from at least seven different experiments. E, dose-dependent effect of ouabain. Filled circles show the percentage of inhibition of Rb+-uptake, used as an index of K+ uptake for different ouabain concentrations. Open circles show the percentage of oscillating cells in response to different ouabain concentrations. F, ouabain does not trigger Ca2+ oscillations following depletion of intracellular Ca2+ stores with thapsigargin. G, overexpression of a truncated Na,K-ATPase {alpha}1, where 32 amino acids from the N terminus were deleted (GFP-Na,K-ATPase{alpha}1{Delta}NT-t), significantly suppressed the ouabain-induced Ca2+ oscillations.

 


Figure 7
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  		FIGURE 7.
Overexpression of {alpha}NT-t attenuates ouabain-induced Ca2+ oscillations, NF-{kappa}B activation, and the anti-apoptotic effect. A, Ca2+ release from the endoplasmic stores in response to thapsigargin was similar in COS-7 cells overexpressing mRFP-{alpha}NT-t and mRFP-{alpha}NT-t{Delta}LKK. B, overexpression of mRFP-{alpha}NT-t resulted in inhibition of the ouabain-induced Ca2+ oscillations in COS-7 cells, whereas overexpression of mRFP-{alpha}NT-t{Delta}LKK had no effect. C, the percentage of cells responding to bradykinin was similar in COS-7 cells overexpressing mRFP-{alpha}NT-t and mRFP-{alpha}NT-t{Delta}LKK. D-J, ouabain failed to protect from serum deprivation-induced apoptosis in RPT cells expressing GFP-{alpha}NT-t, but not in cells expressing GFP-{alpha}NT-t{Delta}LKK (D). Representative confocal images of RPT cells treated with 10 nM ouabain and transfected with GFP-{alpha}NT-t{Delta}LKK (E) or GFP-{alpha}NT-t (H) and stained using TUNEL assay for apoptosis (F and I) and merged images (G and J).

 
The exact mechanism by which the ouabain·Na,K-ATPase complex triggers repetitive release of Ca2+ from the InsP3R channel remains to be determined, but we speculate that the {alpha}NT-t acts as an allosteric modulator of the InsP3R, altering its channel properties. Based on the knowledge of the crystal structure of the sarco/endoplasmic reticulum Ca2+-ATPase, the cytoplasmic domains of the Na,K-ATPase {alpha}-subunit has been organized into three domains: the nucleotide-binding domain, the phosphorylation domain, and the actuator domain (A domain) (35). The A domain contains the N terminus and is involved in the transmission of conformational changes. Ouabain shifts the conformational equilibrium of Na,K-ATPase, and this should affect the position of the A domain. Because the N terminus of the A domain is associated with the InsP3R, it is likely that this will also lead to an allosteric modification of the InsP3R molecule.

Release of Ca2+ from the intracellular stores via the InsP3Ris the major determinant of ouabain-induced Ca2+ oscillations (3). The InsP3R is a modulator of a wide variety of functions, such as cell proliferation, differentiation, apoptosis, fertilization, behavior, and memory (36, 37). Activation of the InsP3R can result in single or repeated transient increases in intracellular Ca2+. The periodicity of the repeated, oscillatory, Ca2+ response can, depending on the activator, vary from seconds to hours. This temporal diversity is of utmost importance for the versatility of the Ca2+ signaling, because most cells have tools to encode the frequency of Ca2+ oscillations (36). Ouabain-induced Ca2+ oscillations in COS-7 cells revealed a periodicity of ~4 min, whereas in RPT cells the periodicity was ~5 min (1). Calcium oscillations with a periodicity in this range will typically activate the transcription factor NF-{kappa}B (20), which in many systems has an anti-apoptotic effect (21, 22).

We have in a recently completed study demonstrated that ouabain fails to protect from serum deprivation-induced apoptosis in cells where the ER stores have been Ca2+ depleted (11). We also demonstrated that long term exposure to a low dose of ouabain (10 nM for 24 h) triggered a nuclear translocation of NF-{kappa}B and that protection from apoptosis was abolished in cells exposed to an inhibitor of NF-{kappa}B. These results were confirmed in the present study. Overexpression of {alpha}NT-t significantly blocked the effects of ouabain on intracellular Ca2+ oscillation and NF-{kappa}B activation. Ouabain-dependent protection from apoptosis was completely abolished in cells overexpressing {alpha}NT-t. These data indicate that the {alpha}NT-t peptide acts as a dominant negative inhibitor of the downstream effects of the Na,K-ATPase-InsP3R interaction.

A comparison between the effect of ouabain-bound Na,K-ATPase and the effect of the pro-apoptotic protein cytochrome c, which binds to the C terminus of the InsP3 R, on Ca2+ release from the InsP3R, illustrates the versatility of InsP3R dependent Ca2+ signaling. The periodicity of ouabain·Na,K-ATPase triggered oscillations is lower than the cytochrome c triggered oscillations, which are in the range of 20 min (38). Thus a Ca2+ oscillatory signal, triggered by the direct interaction between the InsP3R and another protein, may be pro-apoptotic or anti-apoptotic depending on the periodicity of the resulting Ca2+ oscillation. Given this intriguing role of the InsP3R as one of the modulators of the threshold for apoptosis, the question of whether Na,K-ATPase binding to InsP3R suppresses the apoptotic effects of cytochrome c interaction with InsP3R will be an interesting possibility for future studies.


   FOOTNOTES
 
* This work was supported by the Swedish Research Council, the Persson Family Foundation, the Märta and Gunnar V. Philipson Foundation, the Foundation for Strategic Research, the Japan Science and Technology Agency, and the Ministry of Education, Science, Sports, Culture, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this work. Back

2 Supported by the Japan Science and Technology Agency (Calcium Oscillation, International Cooperative Research Project). Back

3 To whom correspondence should be addressed. Tel.: 46-8-51777326; Fax: 46-8-51777328; E-mail: anita.aperia{at}ki.se.

4 The abbreviations used are: InsP3, inositol 1,4,5-trisphosphate; InsP3R, InsP3 receptor; ER, endoplasmic reticulum; GST, glutathione S-transferase; RPT, rat proximal tubule; GFP, green fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole; AI, apoptotic index; A domain, actuator domain; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aizman, O., Uhlen, P., Lal, M., Brismar, H., and Aperia, A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13420-13424[Abstract/Free Full Text]
  2. Harwood, S., and Yaqoob, M. M. (2005) Front. Biosci. 10, 2011-2017[Medline] [Order article via Infotrieve]
  3. Miyakawa-Naito, A., Uhlen, P., Lal, M., Aizman, O., Mikoshiba, K., Brismar, H., Zelenin, S., and Aperia, A. (2003) J. Biol. Chem. 278, 50355-50361[Abstract/Free Full Text]
  4. Yuan, Z., Cai, T., Tian, J., Ivanov, A. V., Giovannucci, D. R., and Xie, Z. (2005) Mol. Biol. Cell 16, 4034-4045[Abstract/Free Full Text]
  5. Abramowitz, J., Dai, C., Hirschi, K. K., Dmitrieva, R. I., Doris, P. A., Liu, L., and Allen, J. C. (2003) Circulation 108, 3048-3053[Abstract/Free Full Text]
  6. Liu, L., Abramowitz, J., Askari, A., and Allen, J. C. (2004) Am. J. Physiol. 287, H2173-H2182
  7. Wang, X. Q., and Yu, S. P. (2005) J. Neurochem. 93, 1515-1523[CrossRef][Medline] [Order article via Infotrieve]
  8. Barwe, S. P., Anilkumar, G., Moon, S. Y., Zheng, Y., Whitelegge, J. P., Rajasekaran, S. A., and Rajasekaran, A. K. (2005) Mol. Biol. Cell 16, 1082-1094[Abstract/Free Full Text]
  9. Scheiner-Bobis, G., and Schoner, W. (2001) Nat. Med. 7, 1288-1289[CrossRef][Medline] [Order article via Infotrieve]
  10. Xie, Z., and Cai, T. (2003) Mol. Interv. 3, 157-168[Abstract/Free Full Text]
  11. Li, J., Zelenin, S., Aperia, A., and Aizman, O. (2006) J. Am. Soc. Nephrol. 17, 1848-1857[Abstract/Free Full Text]
  12. Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem. 192, 262-267[CrossRef][Medline] [Order article via Infotrieve]
  13. Ando, H., Mizutani, A., Matsu-ura, T., and Mikoshiba, K. (2003) J. Biol. Chem. 278, 10602-10612[Abstract/Free Full Text]
  14. Zhang, S., Mizutani, A., Hisatsune, C., Higo, T., Bannai, H., Nakayama, T., Hattori, M., and Mikoshiba, K. (2003) J. Biol. Chem. 278, 4048-4056[Abstract/Free Full Text]
  15. Uhlen, P. (2004) Sci. STKE 2004, P115
  16. Cheng, X. J., Fisone, G., Aizman, O., Aizman, R., Levenson, R., Greengard, P., and Aperia, A. (1997) Am. J. Physiol. 273, C893-C901[Medline] [Order article via Infotrieve]
  17. Bosanac, I., Yamazaki, H., Matsu-Ura, T., Michikawa, T., Mikoshiba, K., and Ikura, M. (2005) Mol. Cell 17, 193-203[CrossRef][Medline] [Order article via Infotrieve]
  18. Pressley, T. A., Allen, J. C., Clarke, C. H., Odebunmi, T., and Higham, S. C. (1996) Am. J. Physiol. 271, C825-C832[Medline] [Order article via Infotrieve]
  19. Kawamura, A., Abrell, L. M., Maggiali, F., Berova, N., Nakanishi, K., Labutti, J., Magil, S., Haupert, G. T., Jr., and Hamlyn, J. M. (2001) Biochemistry 40, 5835-5844[CrossRef][Medline] [Order article via Infotrieve]
  20. Dolmetsch, R. E., Xu, K., and Lewis, R. S. (1998) Nature 392, 933-936[CrossRef][Medline] [Order article via Infotrieve]
  21. Mogi, M., Ozeki, N., Nakamura, H., and Togari, A. (2004) Bone 35, 507-516[Medline] [Order article via Infotrieve]
  22. Nakanishi, C., and Toi, M. (2005) Nat. Rev. Cancer 5, 297-309[CrossRef][Medline] [Order article via Infotrieve]
  23. Kaplan, J. H. (2002) Annu. Rev. Biochem. 71, 511-535[CrossRef][Medline] [Order article via Infotrieve]
  24. Lingrel, J. B., and Kuntzweiler, T. (1994) J. Biol. Chem. 269, 19659-19662[Free Full Text]
  25. Skou, J. C. (1998) Biosci. Rep. 18, 155-169[CrossRef][Medline] [Order article via Infotrieve]
  26. Beguin, P., Hasler, U., Beggah, A., Horisberger, J. D., and Geering, K. (1998) J. Biol. Chem. 273, 24921-24931[Abstract/Free Full Text]
  27. Schoner, W. (2002) Eur. J. Biochem. 269, 2440-2448[Medline] [Order article via Infotrieve]
  28. Daly, S. E., Lane, L. K., and Blostein, R. (1994) J. Biol. Chem. 269, 23944-23948[Abstract/Free Full Text]
  29. Segall, L., Javaid, Z. Z., Carl, S. L., Lane, L. K., and Blostein, R. (2003) J. Biol. Chem. 278, 9027-9034[Abstract/Free Full Text]
  30. Shanbaky, N. M., and Pressley, T. A. (1994) Am. J. Physiol. 267, C590-C597[Medline] [Order article via Infotrieve]
  31. Lingrel, J., Moseley, A., Dostanic, I., Cougnon, M., He, S., James, P., Woo, A., O'Connor, K., and Neumann, J. (2003) Ann. N. Y. Acad. Sci. 986, 354-359[Abstract/Free Full Text]
  32. Moseley, A. E., Lieske, S. P., Wetzel, R. K., James, P. F., He, S., Shelly, D. A., Paul, R. J., Boivin, G. P., Witte, D. P., Ramirez, J. M., Sweadner, K. J., and Lingrel, J. B. (2003) J. Biol. Chem. 278, 5317-5324[Abstract/Free Full Text]
  33. Ohta, T., Noguchi, S., Nakanishi, M., Mutoh, Y., Hirata, H., Kagawa, Y., and Kawamura, M. (1991) Biochim. Biophys. Acta 1059, 157-164[Medline] [Order article via Infotrieve]
  34. Bosanac, I., Alattia, J. R., Mal, T. K., Chan, J., Talarico, S., Tong, F. K., Tong, K. I., Yoshikawa, F., Furuichi, T., Iwai, M., Michikawa, T., Mikoshiba, K., and Ikura, M. (2002) Nature 420, 696-700[CrossRef][Medline] [Order article via Infotrieve]
  35. Sweadner, K. J., and Donnet, C. (2001) Biochem. J. 356, 685-704[CrossRef][Medline] [Order article via Infotrieve]
  36. Berridge, M. J., Bootman, M. D., and Roderick, H. L. (2003) Nat. Rev. Mol. Cell. Biol. 4, 517-529[CrossRef][Medline] [Order article via Infotrieve]
  37. Furuichi, T., and Mikoshiba, K. (1995) J. Neurochem. 64, 953-960[Medline] [Order article via Infotrieve]
  38. Boehning, D., Patterson, R. L., Sedaghat, L., Glebova, N. O., Kurosaki, T., and Snyder, S. H. (2003) Nat. Cell Biol. 5, 1051-1061[CrossRef][Medline] [Order article via Infotrieve]

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