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

Hong Ju, Rong Zou, Virginia J. Venema, Richard C. Venema

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Abstract

Endothelial nitric-oxide synthase (eNOS) and caveolind are associated within endothelial plasmalemmal caveolae. It is not known, however, whether eNOS and caveolin-1 interact directly or indirectly or whether the interaction affects eNOS activity. To answer these questions, we have cloned the bovine caveolin-1 cDNA and have investigated the eNOS-caveolin-1 interaction in an in vitro binding assay system using glutathione S- transferase (GST)-caveolin-1 fusion proteins and baculovirus-expressed bovine eNOS. We have also mapped the domains involved in the interaction using an in vivo yeast two-hybrid system. Results obtained using both in vitro and in vivo protein interaction assays show that both N- and C-terminal cytosolic domains of caveolin-1 interact directly with the eNOS oxygenase domain. Interaction of eNOS with GST-caveolin-1 fusion proteins significantly inhibits enzyme catalytic activity. A synthetic peptide corresponding to caveolin-1 residues 82-101 also potently and reversibly inhibits eNOS activity by interfering with the interaction of the enzyme with Ca2+/calmodulin (CaM). Regulation of eNOS in endothelial cells, therefore, may involve not only positive allosteric regulation by Ca/2+/CaM, but also negative allosteric regulation by caveolin-1.

Original languageEnglish (US)
Pages (from-to)18522-18525
Number of pages4
JournalJournal of Biological Chemistry
Volume272
Issue number30
DOIs
StatePublished - Aug 12 1997

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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