Can you give a clear explanations of the introduction for this paper? Please explain as if your presenting a group presentation about this paper introduction. THE JOURNAL OF BIOLOGICAL CHEMISTRYⒸ2003 by The American Society for Biochemistry and Molecular Biology, Inc.Cell Signaling Microdomain with Na,K-ATPase and Inositol1,4,5-Trisphosphate Receptor Generates Calcium Oscillations*Received for publication, May 22, 2003, and in revised form, August 20, 2003Published, JBC Papers in Press, August 28, 2003, DOI 10.1074/jbc.M305378200Ayako Miyakawa-Naito‡, Per Uhlén, Mark Lal, Oleg Aizman, Katsuhiko Mikoshibas,Hjalmar Brismar, Sergey Zelenin, and Anita AperiaFrom the Department of Woman and Child Health, Karolinska Institutet, Astrid Lindgren Children's Hospital, S-171 76Stockholm, Sweden and the Department of Basic Medical Science, The Institute of Medical Science, The University ofTokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, JapanRecent studies indicate novel roles for the ubiquitousion pump, Na,K-ATPase, in addition to its function as akey regulator of intracellular sodium and potassiumconcentration. We have previously demonstrated thatouabain, the endogenous ligand of Na,K-ATPase, cantrigger intracellular Ca²+ oscillations, a versatile intra-cellular signal controlling a diverse range of cellularprocesses. Here we report that Na,K-ATPase and inosi-tol 1,4,5-trisphosphate (InsP3) receptor (InsP,R) form acell signaling microdomain that, in the presence of oua-bain, generates slow Ca²+ oscillations in renal cells. Us-ing fluorescent resonance energy transfer (FRET) meas-urements, we detected a close spatial proximity betweenNa,K-ATPase and InsP3R. Ouabain significantly en-hanced FRET between Na,K-ATPase TheFRET effect and ouabain-induced Ca²+ oscillations werenot observed following disruption of the actin cytoskel-eton. Partial truncation of the NH, terminus of Na,K-ATPase catalytic al-subunit abolished Ca2+ oscoscillationsand downstream activation of NF-kB. Ouabain-inducedCa²+ oscillations occurred in cells expressing an InsP;sponge and were hence independent of InsP, generation.Thus, we present a novel principle for a cell signalingmicrodomain where an ion pump serves as a receptor.TRVol. 278, No. 50, Issue of December 12, pp. 50355-50361, 2003Printed in U.S.A.Na,K-ATPase is an integral membrane protein expressed inall eukaryotic cells, where it functions as a key regulator ofintracellular Na+ and K+ concentrations (1). Recent studies,however, point to an additional role for Na,K-ATPase as asignal transducer (2-5). Importantly, Na,K-ATPase has an en-dogenous ligand, ouabain, a steroid hormone that dose-depend-ently inhibits the activity of Na,K-ATPase. The biological roleof ouabain is, despite extensive research, not well understood.Ouabain belongs to the family of cardiac glycosides, which havebeen used for centuries in the treatment of heart disease.Recently, several investigators have noted that cardiac glyco-sides may act as anticancer agents (6, 7).*This work was supported by grants from the Swedish ResearchCouncil, the Persson Family Foundation, the Märta and Gunnar V.Philipson Foundation (to A. A. and H. B.), Japan Science and Technol-ogy Corporation "Calcium Oscillation Project" (to K. M. and A. A.), andthe Ministry of Education, Science, Sports and Culture of Japan (toA. M.-N.). The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked "advertisement" in accordance with 18 U.S.C. Section 1734solely to indicate this fact.Both authors contributed equally to this work.To whom correspondence should be addressed: Dept. of Woman andChild Health, Karolinska Institutet, Astrid Lindgren Children's Hospi-tal, Q2:09, S-171 76 Stockholm, Sweden. Tel.: 46-8-51777326; Fax:46-8-51777328; E-mail: anita.aperia@ks.se.This paper is available on line http://www.jbc.orgWe have described a new cell signaling pathway triggered byouabain (2). Using rat renal proximal tubule (RPT)¹ cells, weshowed that exposure to concentrations of ouabain that causeonly partial inhibition of Na,K-ATPase activity induces slowintracellular Ca²+ oscillations and subsequent activation of thetranscription factors NF-kB and CAMP-response element-bind-ing protein. Our results from that study indicated that Ca²+oscillations occurred as an interplay between different Ca²+transporters and that Ca²+ release via the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) was involved in thisevent. Na,K-ATPase does not possess the characteristics of aG-protein-coupled receptor. Given the generality of Na,K-ATPase expression and its significant role in cell homeostasis,it is important to identify the molecular mechanisms by whichNa,K-ATPase can act as a signal transducer. Here we showthat the generation of Ca²+ oscillations by ouabain is depend-ent on the physical association of Na,K-ATPase and InsP3R ina signaling microdomain.EXPERIMENTAL PROCEDURESExpression Plasmids-A cDNA fragment encoding wild type ratNa,K-ATPase al-subunit (NKAa1) was amplified by AmpliTaq Gold(Applied Biosystems). The PCR product was digested by Apal/Xbalrestriction enzymes and cloned into pEGFP-C2 (Clontech) to obtainpGFP-NKAa1. A mutant NKAa1 with truncation of the first 32 aminoacids (NKAa1.M32) was generated using PCR. The region of truncationof the NH₂ terminus was decided on the basis of structure/functionanalysis reported elsewhere (8). The sense primer sequence was 5'-AAAGGGCCCATGGAAGTGTCTATGGACGAC-3', corresponding tonucleotide positions 349-366 of NKAa1 (GenBank™ accession numberNM_012504) with an additional Apal site and ATG codon on the 5'-endof the primer. The antisense primer was 5'-CTTGCCGTGGAGGAG-GATAGAACT-3', corresponding to nucleotide positions 1792-1815 ofNKAa1. The PCR product and pGFP-NKAa1 were hydrolyzed by Apal/AflII restriction enzymes and ligated for cloning pGFP-NKAa1.M32.A fusion protein with NH₂-terminal glutathione S-transferase (GST)and 95 amino acids of NKAa1 (GST-NKAa1.N95) was constructedusing Gateway Technology (Invitrogen). Briefly, a cDNA fragment en-coding 95 amino acids of the Na,K-ATPase al-subunit NH₂ terminuswas amplified by AmpliTaq Gold (Applied Biosystems). The PCR prod-uct was cloned in pENTR/D-TOPO vector using pENTR DirectionalTOPO cloning kit (Invitrogen) and subcloned into pDEST-15 usingGateway System (Invitrogen) to obtain pGST-NKAa1.N95. The nucle-otide sequences of all constructs were confirmed by automated sequenc-ing (KISEQ, Core Facilities of Karolinska Institutet, Stockholm, Swe-den) and subsequent bioinformatics analysis using Lasergene software(DNASTAR).¹The abbreviations used are: RPT, renal proximal tubule; InsP3,inositol 1,4,5-trisphosphate; InsP,R, inositol 1,4,5-trisphosphate recep-tor; FRET, fluorescent resonance energy transfer; PLC, phospholipaseC; ER, endoplasmic reticulum; SERCA, sarco-endoplasmic reticulumCa²+ ATPase; CPA, cyclopiazonic acid; 2-APB, 2-aminoethoxydiphenylborate; CytD, cytochalasin D; AQP4, aquaporin-4; GST, glutathioneS-transferase; GFP, green fluorescent protein; EGFP, enhanced GFP.50355

Question

Can you give a clear explanations of the introduction for this paper? Please explain as if your presenting a group presentation about this paper introduction. THE JOURNAL OF BIOLOGICAL CHEMISTRYⒸ2003 by The American Society for Biochemistry and Molecular Biology, Inc.Cell Signaling Microdomain with Na,K-ATPase and Inositol1,4,5-Trisphosphate Receptor Generates Calcium Oscillations*Received for publication, May 22, 2003, and in revised form, August 20, 2003Published, JBC Papers in Press, August 28, 2003, DOI 10.1074/jbc.M305378200Ayako Miyakawa-Naito‡, Per Uhlén, Mark Lal, Oleg Aizman, Katsuhiko Mikoshibas,Hjalmar Brismar, Sergey Zelenin, and Anita AperiaFrom the Department of Woman and Child Health, Karolinska Institutet, Astrid Lindgren Children's Hospital, S-171 76Stockholm, Sweden and the Department of Basic Medical Science, The Institute of Medical Science, The University ofTokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, JapanRecent studies indicate novel roles for the ubiquitousion pump, Na,K-ATPase, in addition to its function as akey regulator of intracellular sodium and potassiumconcentration. We have previously demonstrated thatouabain, the endogenous ligand of Na,K-ATPase, cantrigger intracellular Ca²+ oscillations, a versatile intra-cellular signal controlling a diverse range of cellularprocesses. Here we report that Na,K-ATPase and inosi-tol 1,4,5-trisphosphate (InsP3) receptor (InsP,R) form acell signaling microdomain that, in the presence of oua-bain, generates slow Ca²+ oscillations in renal cells. Us-ing fluorescent resonance energy transfer (FRET) meas-urements, we detected a close spatial proximity betweenNa,K-ATPase and InsP3R. Ouabain significantly en-hanced FRET between Na,K-ATPase TheFRET effect and ouabain-induced Ca²+ oscillations werenot observed following disruption of the actin cytoskel-eton. Partial truncation of the NH, terminus of Na,K-ATPase catalytic al-subunit abolished Ca2+ oscoscillationsand downstream activation of NF-kB. Ouabain-inducedCa²+ oscillations occurred in cells expressing an InsP;sponge and were hence independent of InsP, generation.Thus, we present a novel principle for a cell signalingmicrodomain where an ion pump serves as a receptor.TRVol. 278, No. 50, Issue of December 12, pp. 50355-50361, 2003Printed in U.S.A.Na,K-ATPase is an integral membrane protein expressed inall eukaryotic cells, where it functions as a key regulator ofintracellular Na+ and K+ concentrations (1). Recent studies,however, point to an additional role for Na,K-ATPase as asignal transducer (2-5). Importantly, Na,K-ATPase has an en-dogenous ligand, ouabain, a steroid hormone that dose-depend-ently inhibits the activity of Na,K-ATPase. The biological roleof ouabain is, despite extensive research, not well understood.Ouabain belongs to the family of cardiac glycosides, which havebeen used for centuries in the treatment of heart disease.Recently, several investigators have noted that cardiac glyco-sides may act as anticancer agents (6, 7).*This work was supported by grants from the Swedish ResearchCouncil, the Persson Family Foundation, the Märta and Gunnar V.Philipson Foundation (to A. A. and H. B.), Japan Science and Technol-ogy Corporation &#34;Calcium Oscillation Project&#34; (to K. M. and A. A.), andthe Ministry of Education, Science, Sports and Culture of Japan (toA. M.-N.). The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked &#34;advertisement&#34; in accordance with 18 U.S.C. Section 1734solely to indicate this fact.Both authors contributed equally to this work.To whom correspondence should be addressed: Dept. of Woman andChild Health, Karolinska Institutet, Astrid Lindgren Children's Hospi-tal, Q2:09, S-171 76 Stockholm, Sweden. Tel.: 46-8-51777326; Fax:46-8-51777328; E-mail: anita.aperia@ks.se.This paper is available on line http://www.jbc.orgWe have described a new cell signaling pathway triggered byouabain (2). Using rat renal proximal tubule (RPT)¹ cells, weshowed that exposure to concentrations of ouabain that causeonly partial inhibition of Na,K-ATPase activity induces slowintracellular Ca²+ oscillations and subsequent activation of thetranscription factors NF-kB and CAMP-response element-bind-ing protein. Our results from that study indicated that Ca²+oscillations occurred as an interplay between different Ca²+transporters and that Ca²+ release via the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) was involved in thisevent. Na,K-ATPase does not possess the characteristics of aG-protein-coupled receptor. Given the generality of Na,K-ATPase expression and its significant role in cell homeostasis,it is important to identify the molecular mechanisms by whichNa,K-ATPase can act as a signal transducer. Here we showthat the generation of Ca²+ oscillations by ouabain is depend-ent on the physical association of Na,K-ATPase and InsP3R ina signaling microdomain.EXPERIMENTAL PROCEDURESExpression Plasmids-A cDNA fragment encoding wild type ratNa,K-ATPase al-subunit (NKAa1) was amplified by AmpliTaq Gold(Applied Biosystems). The PCR product was digested by Apal/Xbalrestriction enzymes and cloned into pEGFP-C2 (Clontech) to obtainpGFP-NKAa1. A mutant NKAa1 with truncation of the first 32 aminoacids (NKAa1.M32) was generated using PCR. The region of truncationof the NH₂ terminus was decided on the basis of structure/functionanalysis reported elsewhere (8). The sense primer sequence was 5'-AAAGGGCCCATGGAAGTGTCTATGGACGAC-3', corresponding tonucleotide positions 349-366 of NKAa1 (GenBank™ accession numberNM_012504) with an additional Apal site and ATG codon on the 5'-endof the primer. The antisense primer was 5'-CTTGCCGTGGAGGAG-GATAGAACT-3', corresponding to nucleotide positions 1792-1815 ofNKAa1. The PCR product and pGFP-NKAa1 were hydrolyzed by Apal/AflII restriction enzymes and ligated for cloning pGFP-NKAa1.M32.A fusion protein with NH₂-terminal glutathione S-transferase (GST)and 95 amino acids of NKAa1 (GST-NKAa1.N95) was constructedusing Gateway Technology (Invitrogen). Briefly, a cDNA fragment en-coding 95 amino acids of the Na,K-ATPase al-subunit NH₂ terminuswas amplified by AmpliTaq Gold (Applied Biosystems). The PCR prod-uct was cloned in pENTR/D-TOPO vector using pENTR DirectionalTOPO cloning kit (Invitrogen) and subcloned into pDEST-15 usingGateway System (Invitrogen) to obtain pGST-NKAa1.N95. The nucle-otide sequences of all constructs were confirmed by automated sequenc-ing (KISEQ, Core Facilities of Karolinska Institutet, Stockholm, Swe-den) and subsequent bioinformatics analysis using Lasergene software(DNASTAR).¹The abbreviations used are: RPT, renal proximal tubule; InsP3,inositol 1,4,5-trisphosphate; InsP,R, inositol 1,4,5-trisphosphate recep-tor; FRET, fluorescent resonance energy transfer; PLC, phospholipaseC; ER, endoplasmic reticulum; SERCA, sarco-endoplasmic reticulumCa²+ ATPase; CPA, cyclopiazonic acid; 2-APB, 2-aminoethoxydiphenylborate; CytD, cytochalasin D; AQP4, aquaporin-4; GST, glutathioneS-transferase; GFP, green fluorescent protein; EGFP, enhanced GFP.50355

Accepted Answer

Background data: The introduction starts by citing recent research that have uncovered new roles for…