Acylation targets endothelial nitric oxide synthase to plasmalemmal caveolae

P. W. Shaul, E. J. Smart, L. J. Robinson, Z. German, I. Yuhanna, Y. Ying, R. G W Anderson, T. Michel

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

The endothelial isoform of nitric oxide synthase (ecNOS) is a key determinant of vascular tone and platelet aggregation. We showed previously that ecNOS is targeted to the endothelial paniculate subcellular fraction by two distinct acylations, N-terminal myristoylation and reversible thiopalmitoylation. We now explore the specific paniculate fraction to which ecNOS is targeted. Some palmitoylated proteins are localized to plasmalemmal caveolae. Caveolae are specialized plasma membrane invaginations that may serve to localize cell surface signaling domains, and may contain Gproteins, receptors and calcium channels. Using a newly-developed detergent-free method, we isolated caveolae from endothelial cells and found that ecNOS activity (assayed by 3H-citrulline formation) and protein expression (analyzed by immunoblot) is >9-fold enriched in caveolae relative to plasma membrane fractions. Immunoelectron microscopy also specifically localized ecNOS to endothelial caveolae. In COS-7 cells transfected with cDNA encoding wild-type ecNOS, the enzyme is targeted to caveolae. By contrast, neither palmitoylation- nor myristoylation-deficient ecNOS mutants are localized to caveolae in transfected COS-7 cells. Acylation of ecNOS is therefore required for its caveolar targeting. The localization of ecNOS to plasmalemmal caveolae may play a key role both in enzyme activation and in extracellular NO generation; agonist-induced depalmitoylation of caveolar ecNOS may serve as an important feedback mechanism for NO signaling.

Original languageEnglish (US)
JournalJournal of Investigative Medicine
Volume44
Issue number3
StatePublished - 1996

Fingerprint

Caveolae
Acylation
Nitric Oxide Synthase Type III
Cell membranes
Citrulline
Endothelial cells
Enzymes
Calcium Channels
Platelets
Nitric Oxide Synthase
Detergents
Microscopic examination
Protein Isoforms
Proteins
Agglomeration
Complementary DNA
Chemical activation
Feedback
COS Cells
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Shaul, P. W., Smart, E. J., Robinson, L. J., German, Z., Yuhanna, I., Ying, Y., ... Michel, T. (1996). Acylation targets endothelial nitric oxide synthase to plasmalemmal caveolae. Journal of Investigative Medicine, 44(3).

Acylation targets endothelial nitric oxide synthase to plasmalemmal caveolae. / Shaul, P. W.; Smart, E. J.; Robinson, L. J.; German, Z.; Yuhanna, I.; Ying, Y.; Anderson, R. G W; Michel, T.

In: Journal of Investigative Medicine, Vol. 44, No. 3, 1996.

Research output: Contribution to journalArticle

Shaul, PW, Smart, EJ, Robinson, LJ, German, Z, Yuhanna, I, Ying, Y, Anderson, RGW & Michel, T 1996, 'Acylation targets endothelial nitric oxide synthase to plasmalemmal caveolae', Journal of Investigative Medicine, vol. 44, no. 3.
Shaul, P. W. ; Smart, E. J. ; Robinson, L. J. ; German, Z. ; Yuhanna, I. ; Ying, Y. ; Anderson, R. G W ; Michel, T. / Acylation targets endothelial nitric oxide synthase to plasmalemmal caveolae. In: Journal of Investigative Medicine. 1996 ; Vol. 44, No. 3.
@article{27dbed0f693649c59531bff746577c03,
title = "Acylation targets endothelial nitric oxide synthase to plasmalemmal caveolae",
abstract = "The endothelial isoform of nitric oxide synthase (ecNOS) is a key determinant of vascular tone and platelet aggregation. We showed previously that ecNOS is targeted to the endothelial paniculate subcellular fraction by two distinct acylations, N-terminal myristoylation and reversible thiopalmitoylation. We now explore the specific paniculate fraction to which ecNOS is targeted. Some palmitoylated proteins are localized to plasmalemmal caveolae. Caveolae are specialized plasma membrane invaginations that may serve to localize cell surface signaling domains, and may contain Gproteins, receptors and calcium channels. Using a newly-developed detergent-free method, we isolated caveolae from endothelial cells and found that ecNOS activity (assayed by 3H-citrulline formation) and protein expression (analyzed by immunoblot) is >9-fold enriched in caveolae relative to plasma membrane fractions. Immunoelectron microscopy also specifically localized ecNOS to endothelial caveolae. In COS-7 cells transfected with cDNA encoding wild-type ecNOS, the enzyme is targeted to caveolae. By contrast, neither palmitoylation- nor myristoylation-deficient ecNOS mutants are localized to caveolae in transfected COS-7 cells. Acylation of ecNOS is therefore required for its caveolar targeting. The localization of ecNOS to plasmalemmal caveolae may play a key role both in enzyme activation and in extracellular NO generation; agonist-induced depalmitoylation of caveolar ecNOS may serve as an important feedback mechanism for NO signaling.",
author = "Shaul, {P. W.} and Smart, {E. J.} and Robinson, {L. J.} and Z. German and I. Yuhanna and Y. Ying and Anderson, {R. G W} and T. Michel",
year = "1996",
language = "English (US)",
volume = "44",
journal = "Journal of Investigative Medicine",
issn = "1081-5589",
publisher = "Lippincott Williams and Wilkins",
number = "3",

}

TY - JOUR

T1 - Acylation targets endothelial nitric oxide synthase to plasmalemmal caveolae

AU - Shaul, P. W.

AU - Smart, E. J.

AU - Robinson, L. J.

AU - German, Z.

AU - Yuhanna, I.

AU - Ying, Y.

AU - Anderson, R. G W

AU - Michel, T.

PY - 1996

Y1 - 1996

N2 - The endothelial isoform of nitric oxide synthase (ecNOS) is a key determinant of vascular tone and platelet aggregation. We showed previously that ecNOS is targeted to the endothelial paniculate subcellular fraction by two distinct acylations, N-terminal myristoylation and reversible thiopalmitoylation. We now explore the specific paniculate fraction to which ecNOS is targeted. Some palmitoylated proteins are localized to plasmalemmal caveolae. Caveolae are specialized plasma membrane invaginations that may serve to localize cell surface signaling domains, and may contain Gproteins, receptors and calcium channels. Using a newly-developed detergent-free method, we isolated caveolae from endothelial cells and found that ecNOS activity (assayed by 3H-citrulline formation) and protein expression (analyzed by immunoblot) is >9-fold enriched in caveolae relative to plasma membrane fractions. Immunoelectron microscopy also specifically localized ecNOS to endothelial caveolae. In COS-7 cells transfected with cDNA encoding wild-type ecNOS, the enzyme is targeted to caveolae. By contrast, neither palmitoylation- nor myristoylation-deficient ecNOS mutants are localized to caveolae in transfected COS-7 cells. Acylation of ecNOS is therefore required for its caveolar targeting. The localization of ecNOS to plasmalemmal caveolae may play a key role both in enzyme activation and in extracellular NO generation; agonist-induced depalmitoylation of caveolar ecNOS may serve as an important feedback mechanism for NO signaling.

AB - The endothelial isoform of nitric oxide synthase (ecNOS) is a key determinant of vascular tone and platelet aggregation. We showed previously that ecNOS is targeted to the endothelial paniculate subcellular fraction by two distinct acylations, N-terminal myristoylation and reversible thiopalmitoylation. We now explore the specific paniculate fraction to which ecNOS is targeted. Some palmitoylated proteins are localized to plasmalemmal caveolae. Caveolae are specialized plasma membrane invaginations that may serve to localize cell surface signaling domains, and may contain Gproteins, receptors and calcium channels. Using a newly-developed detergent-free method, we isolated caveolae from endothelial cells and found that ecNOS activity (assayed by 3H-citrulline formation) and protein expression (analyzed by immunoblot) is >9-fold enriched in caveolae relative to plasma membrane fractions. Immunoelectron microscopy also specifically localized ecNOS to endothelial caveolae. In COS-7 cells transfected with cDNA encoding wild-type ecNOS, the enzyme is targeted to caveolae. By contrast, neither palmitoylation- nor myristoylation-deficient ecNOS mutants are localized to caveolae in transfected COS-7 cells. Acylation of ecNOS is therefore required for its caveolar targeting. The localization of ecNOS to plasmalemmal caveolae may play a key role both in enzyme activation and in extracellular NO generation; agonist-induced depalmitoylation of caveolar ecNOS may serve as an important feedback mechanism for NO signaling.

UR - http://www.scopus.com/inward/record.url?scp=33749427210&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33749427210&partnerID=8YFLogxK

M3 - Article

VL - 44

JO - Journal of Investigative Medicine

JF - Journal of Investigative Medicine

SN - 1081-5589

IS - 3

ER -