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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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(A) Dose-dependent inhibition of NOS-I activity in neuro-2a cells. Coexpression of increasing amounts of PMCA 4b and constant levels of NOS-I resulted in a dose-dependent inhibition of NOS-I activity, comparable to the effects observed in HEK293 cells. In this cellular system smaller amounts of PMCA (∼0.5 μg transfected plasmid) were sufficient to obtain maximum inhibition of NOS-I, suggesting that an upper limit of PMCA expression is reached earlier in this cellular system (n = 10, mean ±SEM, asterisk indicates P < 0.01). (B) Representative Western blot demonstrating constant NOS-I expression despite dose-dependent expression of PMCA 4b in transfected neuro-2a cells.
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fig3: (A) Dose-dependent inhibition of NOS-I activity in neuro-2a cells. Coexpression of increasing amounts of PMCA 4b and constant levels of NOS-I resulted in a dose-dependent inhibition of NOS-I activity, comparable to the effects observed in HEK293 cells. In this cellular system smaller amounts of PMCA (∼0.5 μg transfected plasmid) were sufficient to obtain maximum inhibition of NOS-I, suggesting that an upper limit of PMCA expression is reached earlier in this cellular system (n = 10, mean ±SEM, asterisk indicates P < 0.01). (B) Representative Western blot demonstrating constant NOS-I expression despite dose-dependent expression of PMCA 4b in transfected neuro-2a cells.

Mentions: The potential physiological relevance of NOS-I regulation by PMCA 4b was tested in neuro-2a neuroblastoma cells, a commonly used model system in neuronal biology (Olmsted et al., 1970). Similarly to HEK293 cells, tight functional coupling of PMCA4b and NOS-I was observed: NOS-I activity was strongly downregulated by the PMCA4b (Fig. 3 A). This effect was reversed by the NO donor NOC-18 (2,2′-[hydroxynitrosohydrazino]bis-ethanamine), suggesting that interaction of these proteins not only occurs in HEK293 cells, but also in a neuroblastoma-derived cell line.


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)

(A) Dose-dependent inhibition of NOS-I activity in neuro-2a cells. Coexpression of increasing amounts of PMCA 4b and constant levels of NOS-I resulted in a dose-dependent inhibition of NOS-I activity, comparable to the effects observed in HEK293 cells. In this cellular system smaller amounts of PMCA (∼0.5 μg transfected plasmid) were sufficient to obtain maximum inhibition of NOS-I, suggesting that an upper limit of PMCA expression is reached earlier in this cellular system (n = 10, mean ±SEM, asterisk indicates P < 0.01). (B) Representative Western blot demonstrating constant NOS-I expression despite dose-dependent expression of PMCA 4b in transfected neuro-2a cells.
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Related In: Results  -  Collection

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

fig3: (A) Dose-dependent inhibition of NOS-I activity in neuro-2a cells. Coexpression of increasing amounts of PMCA 4b and constant levels of NOS-I resulted in a dose-dependent inhibition of NOS-I activity, comparable to the effects observed in HEK293 cells. In this cellular system smaller amounts of PMCA (∼0.5 μg transfected plasmid) were sufficient to obtain maximum inhibition of NOS-I, suggesting that an upper limit of PMCA expression is reached earlier in this cellular system (n = 10, mean ±SEM, asterisk indicates P < 0.01). (B) Representative Western blot demonstrating constant NOS-I expression despite dose-dependent expression of PMCA 4b in transfected neuro-2a cells.
Mentions: The potential physiological relevance of NOS-I regulation by PMCA 4b was tested in neuro-2a neuroblastoma cells, a commonly used model system in neuronal biology (Olmsted et al., 1970). Similarly to HEK293 cells, tight functional coupling of PMCA4b and NOS-I was observed: NOS-I activity was strongly downregulated by the PMCA4b (Fig. 3 A). This effect was reversed by the NO donor NOC-18 (2,2′-[hydroxynitrosohydrazino]bis-ethanamine), suggesting that interaction of these proteins not only occurs in HEK293 cells, but also in a neuroblastoma-derived cell line.

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