H. Ju et al., DIRECT INTERACTION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE AND CAVEOLIN-1 INHIBITS SYNTHASE ACTIVITY, The Journal of biological chemistry, 272(30), 1997, pp. 18522-18525
Endothelial nitric-oxide synthase (eNOS) and caveolin-1 are associated
within endothelial plasmalemmal caveolae. It is not known, however, w
hether eNOS and caveolin-1 interact directly or indirectly or whether
the interaction affects eNOS activity. To answer these questions, we h
ave cloned the bovine caveolin-1 cDNA and have investigated the eNOS-c
aveolin-1 interaction in an in vitro binding assay system using glutat
hione S-transferase (GST)-caveolin-1 fusion proteins and baculovirus-e
xpressed 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 Nand 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 po
tently and reversibly inhibits eNOS activity by interfering with the i
nteraction of the enzyme with Ca2+/calmodulin (CaM). Regulation of eNO
S in endothelial cells, therefore, may involve not only positive allos
teric regulation by Ca2+/CaM, but also negative allosteric regulation
by caveolin-1.