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The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I.

Schuh K, Uldrijan S, Telkamp M, Rothlein N, Neyses L - J. Cell Biol. (2001)

Bottom Line: Biol.A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction.Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Wuerzburg, D-97080 Wuerzburg, Germany.

ABSTRACT
The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646-8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152-14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59-61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693-18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.

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Schematic overview of constructs used, expression of NOS-I constructs in HEK293 cells, and physical interaction of PMCA with NOS-I. (A) Plasmid constructs used in transfection assays. pCMV–PMCA 4b contains the wild-type human PMCA 4b cDNA under the control of the CMV promoter. pCMV–PMCA 4bmut carries a mutation Asp672→Glu, thereby reducing the ATPase activity to ∼10% (Adamo et al., 1995). In pCMV–hPMCA 4b cap the stop codon is mutated and the myc/6×His tag is added in frame, resulting in capping of the PDZ-binding COOH- terminal amino acids. NOS-I and NOS-III are also under the control of the CMV promoter, in pCMV-ΔNOS-I the interacting PDZ domain is deleted. (B) NOS-I was expressed in pCMV-NOS-I–transfected HEK293 cells, rat brain homogenate served as control. ΔNOS-I was expressed at comparable levels, the size difference is due to deletion of the PDZ domain. In untransfected HEK cells, NOS-I was undetectable. (C) GST pull-down assay (Brenman et al., 1995) demonstrating specific interaction of PMCA 4b with PDZ domain of NOS-I. Lysate of pCMV-hPMCA4b–transfected HEK293 cells was used as expression control (first lane), GST alone did not bind PMCA (second lane), whereas GST–PDZ fusion protein was able to bind PMCA 4b (last lane). (D) NOS-I–PMCA 4b interaction in cotransfected HEK293 cells. In the first lane NOS-I coprecipitation by a hPMCA 4b–specific antibody (JA3) from lysates of cotransfected cells is shown. In the third lane, lysates of NOS-I/PMCA–transfected cells were used as expression controls. Irrelevant antibody (anti-FLAG) or protein G beads alone showed no binding of NOS-I (second and fourth lane). Cotransfection with pCMV–hPMCA 4b cap, containing an additional COOH-terminal Myc/6×His epitope, prevented interaction of PMCA and NOS-I (penultimate lane), although NOS-I was present in the cells (last lane). (E) Coimmunoprecipitation using NOS-I–specific antibodies. The NOS-I–specific polyclonal antibody (ABR) coprecipitated the PMCA from lysates of cotransfected HEK293 cells (lane 4). Negative controls: unlabeled beads (lane 1), beads plus irrelevant antibodies (lane 2). Positive controls: calmodulin-coated beads (CaM beads, lane 3), anti-PMCA–specific coated beads (with polyclonal antibody “2A,” lane 5, and lysates of hPMCA 4b–transfected HEK293 cells (lane 6). (F) Immunoprecipitation using rat brain extracts and different PMCA-specific antibodies demonstrating interaction of PMCA and NOS-I in brain. Antibodies specific for NOS-I (lane 3), PMCA (monoclonal, clone 5F10, lane 4, rabbit polyclonal “2A”, lane 5), as well as calmodulin beads (CaM beads, lane 2) were capable of precipitating NOS-I from rat brain extracts. First lane, control using irrelevant antibodies; last lane, rat brain extract (input).
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fig1: Schematic overview of constructs used, expression of NOS-I constructs in HEK293 cells, and physical interaction of PMCA with NOS-I. (A) Plasmid constructs used in transfection assays. pCMV–PMCA 4b contains the wild-type human PMCA 4b cDNA under the control of the CMV promoter. pCMV–PMCA 4bmut carries a mutation Asp672→Glu, thereby reducing the ATPase activity to ∼10% (Adamo et al., 1995). In pCMV–hPMCA 4b cap the stop codon is mutated and the myc/6×His tag is added in frame, resulting in capping of the PDZ-binding COOH- terminal amino acids. NOS-I and NOS-III are also under the control of the CMV promoter, in pCMV-ΔNOS-I the interacting PDZ domain is deleted. (B) NOS-I was expressed in pCMV-NOS-I–transfected HEK293 cells, rat brain homogenate served as control. ΔNOS-I was expressed at comparable levels, the size difference is due to deletion of the PDZ domain. In untransfected HEK cells, NOS-I was undetectable. (C) GST pull-down assay (Brenman et al., 1995) demonstrating specific interaction of PMCA 4b with PDZ domain of NOS-I. Lysate of pCMV-hPMCA4b–transfected HEK293 cells was used as expression control (first lane), GST alone did not bind PMCA (second lane), whereas GST–PDZ fusion protein was able to bind PMCA 4b (last lane). (D) NOS-I–PMCA 4b interaction in cotransfected HEK293 cells. In the first lane NOS-I coprecipitation by a hPMCA 4b–specific antibody (JA3) from lysates of cotransfected cells is shown. In the third lane, lysates of NOS-I/PMCA–transfected cells were used as expression controls. Irrelevant antibody (anti-FLAG) or protein G beads alone showed no binding of NOS-I (second and fourth lane). Cotransfection with pCMV–hPMCA 4b cap, containing an additional COOH-terminal Myc/6×His epitope, prevented interaction of PMCA and NOS-I (penultimate lane), although NOS-I was present in the cells (last lane). (E) Coimmunoprecipitation using NOS-I–specific antibodies. The NOS-I–specific polyclonal antibody (ABR) coprecipitated the PMCA from lysates of cotransfected HEK293 cells (lane 4). Negative controls: unlabeled beads (lane 1), beads plus irrelevant antibodies (lane 2). Positive controls: calmodulin-coated beads (CaM beads, lane 3), anti-PMCA–specific coated beads (with polyclonal antibody “2A,” lane 5, and lysates of hPMCA 4b–transfected HEK293 cells (lane 6). (F) Immunoprecipitation using rat brain extracts and different PMCA-specific antibodies demonstrating interaction of PMCA and NOS-I in brain. Antibodies specific for NOS-I (lane 3), PMCA (monoclonal, clone 5F10, lane 4, rabbit polyclonal “2A”, lane 5), as well as calmodulin beads (CaM beads, lane 2) were capable of precipitating NOS-I from rat brain extracts. First lane, control using irrelevant antibodies; last lane, rat brain extract (input).

Mentions: Physical interaction of the PMCA and NOS-I was demonstrated by conventional immunoprecipitation (Fig. 1, D and E) as well as glutathione S-transferase (GST) pull-down assays, using the PDZ domain of NOS-I as a bait (Fig. 1 C). “Capping” the PMCA 4b COOH terminus (carrying the PDZ domain binding sequence) with an myc/6×His tag resulted in a loss of physical interaction (Fig. 1 D). Expression of wild-type NOS-I and deletion constructs (overview in Fig. 1 A) was confirmed by Western blotting (Fig. 1 B). This established that PMCA 4b and NOS-I interact physically, either directly or through unknown adapters. Full-length PMCA 4b interacted with purified GST–PDZ fusion protein, directly demonstrating specificity of interaction with the PDZ domain (Fig. 1 C). Similar interaction of PMCA and NOS-I was also observed in extracts of rat brain. Using PMCA-specific antibodies 5F10 and 2A, NOS-I was also coprecipitated from rat brain extracts (Fig. 1 F).


The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I.

Schuh K, Uldrijan S, Telkamp M, Rothlein N, Neyses L - J. Cell Biol. (2001)

Schematic overview of constructs used, expression of NOS-I constructs in HEK293 cells, and physical interaction of PMCA with NOS-I. (A) Plasmid constructs used in transfection assays. pCMV–PMCA 4b contains the wild-type human PMCA 4b cDNA under the control of the CMV promoter. pCMV–PMCA 4bmut carries a mutation Asp672→Glu, thereby reducing the ATPase activity to ∼10% (Adamo et al., 1995). In pCMV–hPMCA 4b cap the stop codon is mutated and the myc/6×His tag is added in frame, resulting in capping of the PDZ-binding COOH- terminal amino acids. NOS-I and NOS-III are also under the control of the CMV promoter, in pCMV-ΔNOS-I the interacting PDZ domain is deleted. (B) NOS-I was expressed in pCMV-NOS-I–transfected HEK293 cells, rat brain homogenate served as control. ΔNOS-I was expressed at comparable levels, the size difference is due to deletion of the PDZ domain. In untransfected HEK cells, NOS-I was undetectable. (C) GST pull-down assay (Brenman et al., 1995) demonstrating specific interaction of PMCA 4b with PDZ domain of NOS-I. Lysate of pCMV-hPMCA4b–transfected HEK293 cells was used as expression control (first lane), GST alone did not bind PMCA (second lane), whereas GST–PDZ fusion protein was able to bind PMCA 4b (last lane). (D) NOS-I–PMCA 4b interaction in cotransfected HEK293 cells. In the first lane NOS-I coprecipitation by a hPMCA 4b–specific antibody (JA3) from lysates of cotransfected cells is shown. In the third lane, lysates of NOS-I/PMCA–transfected cells were used as expression controls. Irrelevant antibody (anti-FLAG) or protein G beads alone showed no binding of NOS-I (second and fourth lane). Cotransfection with pCMV–hPMCA 4b cap, containing an additional COOH-terminal Myc/6×His epitope, prevented interaction of PMCA and NOS-I (penultimate lane), although NOS-I was present in the cells (last lane). (E) Coimmunoprecipitation using NOS-I–specific antibodies. The NOS-I–specific polyclonal antibody (ABR) coprecipitated the PMCA from lysates of cotransfected HEK293 cells (lane 4). Negative controls: unlabeled beads (lane 1), beads plus irrelevant antibodies (lane 2). Positive controls: calmodulin-coated beads (CaM beads, lane 3), anti-PMCA–specific coated beads (with polyclonal antibody “2A,” lane 5, and lysates of hPMCA 4b–transfected HEK293 cells (lane 6). (F) Immunoprecipitation using rat brain extracts and different PMCA-specific antibodies demonstrating interaction of PMCA and NOS-I in brain. Antibodies specific for NOS-I (lane 3), PMCA (monoclonal, clone 5F10, lane 4, rabbit polyclonal “2A”, lane 5), as well as calmodulin beads (CaM beads, lane 2) were capable of precipitating NOS-I from rat brain extracts. First lane, control using irrelevant antibodies; last lane, rat brain extract (input).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198825&req=5

fig1: Schematic overview of constructs used, expression of NOS-I constructs in HEK293 cells, and physical interaction of PMCA with NOS-I. (A) Plasmid constructs used in transfection assays. pCMV–PMCA 4b contains the wild-type human PMCA 4b cDNA under the control of the CMV promoter. pCMV–PMCA 4bmut carries a mutation Asp672→Glu, thereby reducing the ATPase activity to ∼10% (Adamo et al., 1995). In pCMV–hPMCA 4b cap the stop codon is mutated and the myc/6×His tag is added in frame, resulting in capping of the PDZ-binding COOH- terminal amino acids. NOS-I and NOS-III are also under the control of the CMV promoter, in pCMV-ΔNOS-I the interacting PDZ domain is deleted. (B) NOS-I was expressed in pCMV-NOS-I–transfected HEK293 cells, rat brain homogenate served as control. ΔNOS-I was expressed at comparable levels, the size difference is due to deletion of the PDZ domain. In untransfected HEK cells, NOS-I was undetectable. (C) GST pull-down assay (Brenman et al., 1995) demonstrating specific interaction of PMCA 4b with PDZ domain of NOS-I. Lysate of pCMV-hPMCA4b–transfected HEK293 cells was used as expression control (first lane), GST alone did not bind PMCA (second lane), whereas GST–PDZ fusion protein was able to bind PMCA 4b (last lane). (D) NOS-I–PMCA 4b interaction in cotransfected HEK293 cells. In the first lane NOS-I coprecipitation by a hPMCA 4b–specific antibody (JA3) from lysates of cotransfected cells is shown. In the third lane, lysates of NOS-I/PMCA–transfected cells were used as expression controls. Irrelevant antibody (anti-FLAG) or protein G beads alone showed no binding of NOS-I (second and fourth lane). Cotransfection with pCMV–hPMCA 4b cap, containing an additional COOH-terminal Myc/6×His epitope, prevented interaction of PMCA and NOS-I (penultimate lane), although NOS-I was present in the cells (last lane). (E) Coimmunoprecipitation using NOS-I–specific antibodies. The NOS-I–specific polyclonal antibody (ABR) coprecipitated the PMCA from lysates of cotransfected HEK293 cells (lane 4). Negative controls: unlabeled beads (lane 1), beads plus irrelevant antibodies (lane 2). Positive controls: calmodulin-coated beads (CaM beads, lane 3), anti-PMCA–specific coated beads (with polyclonal antibody “2A,” lane 5, and lysates of hPMCA 4b–transfected HEK293 cells (lane 6). (F) Immunoprecipitation using rat brain extracts and different PMCA-specific antibodies demonstrating interaction of PMCA and NOS-I in brain. Antibodies specific for NOS-I (lane 3), PMCA (monoclonal, clone 5F10, lane 4, rabbit polyclonal “2A”, lane 5), as well as calmodulin beads (CaM beads, lane 2) were capable of precipitating NOS-I from rat brain extracts. First lane, control using irrelevant antibodies; last lane, rat brain extract (input).
Mentions: Physical interaction of the PMCA and NOS-I was demonstrated by conventional immunoprecipitation (Fig. 1, D and E) as well as glutathione S-transferase (GST) pull-down assays, using the PDZ domain of NOS-I as a bait (Fig. 1 C). “Capping” the PMCA 4b COOH terminus (carrying the PDZ domain binding sequence) with an myc/6×His tag resulted in a loss of physical interaction (Fig. 1 D). Expression of wild-type NOS-I and deletion constructs (overview in Fig. 1 A) was confirmed by Western blotting (Fig. 1 B). This established that PMCA 4b and NOS-I interact physically, either directly or through unknown adapters. Full-length PMCA 4b interacted with purified GST–PDZ fusion protein, directly demonstrating specificity of interaction with the PDZ domain (Fig. 1 C). Similar interaction of PMCA and NOS-I was also observed in extracts of rat brain. Using PMCA-specific antibodies 5F10 and 2A, NOS-I was also coprecipitated from rat brain extracts (Fig. 1 F).

Bottom Line: Biol.A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction.Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Wuerzburg, D-97080 Wuerzburg, Germany.

ABSTRACT
The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646-8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152-14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59-61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693-18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.

Show MeSH
Related in: MedlinePlus