Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo

Guillermo García-Cardeña, Pavel Martasek, Bettie Sue Siler Masters, Phillip M. Skidd, Jacques Couet, Shengwen Li, Michael P. Lisanti, William C. Sessa

Research output: Contribution to journalArticle

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Abstract

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin- 1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathione S-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GST-caveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61- 101) and to a lesser extent the C-terminal tail (amino acids 135-178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.

Original languageEnglish (US)
Pages (from-to)25437-25440
Number of pages4
JournalJournal of Biological Chemistry
Volume272
Issue number41
DOIs
StatePublished - Oct 10 1997

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Caveolins
Nitric Oxide Synthase Type III
Nitric Oxide Synthase
Caveolin 1
Caveolin 3
Caveolae
Endothelial cells
Glutathione Transferase
Endothelial Cells
Amino Acids
Muscle
Caveolin 2
Mutagenesis
Molecular Chaperones
Capsid Proteins
Nitric Oxide Synthase Type II
Site-Directed Mutagenesis
Cardiac Myocytes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo. / García-Cardeña, Guillermo; Martasek, Pavel; Masters, Bettie Sue Siler; Skidd, Phillip M.; Couet, Jacques; Li, Shengwen; Lisanti, Michael P.; Sessa, William C.

In: Journal of Biological Chemistry, Vol. 272, No. 41, 10.10.1997, p. 25437-25440.

Research output: Contribution to journalArticle

García-Cardeña, G, Martasek, P, Masters, BSS, Skidd, PM, Couet, J, Li, S, Lisanti, MP & Sessa, WC 1997, 'Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo', Journal of Biological Chemistry, vol. 272, no. 41, pp. 25437-25440. https://doi.org/10.1074/jbc.272.41.25437
García-Cardeña, Guillermo ; Martasek, Pavel ; Masters, Bettie Sue Siler ; Skidd, Phillip M. ; Couet, Jacques ; Li, Shengwen ; Lisanti, Michael P. ; Sessa, William C. / Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 41. pp. 25437-25440.
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