Direct interaction of endothelial nitric oxide synthase and caveolin-1 inhibits synthase activity

Richard C Venema, H. Ju, R. Zou, V. J. Venema

Research output: Contribution to journalArticle

Abstract

Endothelial nitric oxide synthase (eNOS) generates NO from L-arginine fol: lowing activation of the enzyme by Ca2+/calmodulin. NO synthesized by eNOS plays a key role in regulation of vascular tone and platelet aggregation. Association of eNOS with caveolin-1 in plasmalemmal caveolae has been shown previously in coimmunoprecipitation experiments. It is not known, however, whether eNOS and caveolin-1 interact directly or indirectly or whether interaction affects eNOS activity, eNOS contains an N-terminal oxygenase domain and a C-terminal reductase domain. Caveolin-1 Contains three domains consisting of N- and C-terminal cytosolic domains separated in the polypeptide sequence by a membrane spanning domain. To determine whether eNOS and caveolin-1 interact directly and to map the interacting domains in the two proteins, we have investigated the eNOS-caveolin-1 interaction using an in vitro binding assay with glutathione S-transferase fusion proteins. We have also mapped the domains involved in the interaction using a yeast two-hybrid system. Results obtained using both approaches show that both N- and Cterminal cytosolic domains of caveolin-1 interact directly with the eNOS oxygenase domain. Furthermore, interaction of eNOS with caveolin-1 significantly inhibits eNOS catalytic activity. Negative allosteric regulation of eNOS by caveolin-1 may thus function in opposition to the well-known positive allosteric regulation of eNOS by Ca2+/calmodulin.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - Dec 1 1997

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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