(Received for publication, March 31, 1997, and in revised form, May 23, 1997)
From the Vascular Biology Center, Department of Pediatrics, and Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912
Endothelial nitric-oxide synthase (eNOS) and caveolin-1 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 Ca2+/CaM, but also negative allosteric regulation by caveolin-1.
Plasmalemmal caveolae are small membrane invaginations present in
most cells of higher eukaryotes. These membrane specializations appear
to function both as endocytotic carriers and as signal transduction
organizing centers. In the latter case, caveolae compartmentalize a
subset of signal-transducing molecules in membrane microdomains at the
cell surface (1, 2). A major structural protein of caveolae is
caveolin, a 21-24-kDa integral membrane protein that occurs in three
homologous, but distinct isoforms termed caveolins-1, -2, and -3 (3).
Full-length caveolin-1 contains three domains: a 101-residue N-terminal
domain, a 33-residue membrane-spanning region, and a 44-residue
C-terminal domain. The N- and C-terminal domains of caveolin-1 face the
cytoplasm suggesting that the membrane-spanning region forms a hairpin
loop within the membrane (4-6). A cytosolic membrane-proximal
subdomain of the N-terminal domain (residues 82-101) interacts
directly with G subunits, Ha-Ras, and Src family tyrosine kinases
(7-9). Interaction of these signaling proteins with this caveolin-1
scaffolding domain serves to sequester the proteins in caveolae and to
inhibit or suppress their catalytic activities.
Another important signaling protein known to be localized in caveolae is endothelial nitric-oxide synthase (eNOS)1 (10, 11). Production of NO by eNOS in endothelial caveolae appears to play a key role in modulating vascular tone, platelet aggregation, leukocyte adhesion, vascular smooth muscle cell proliferation, and vascular lesion formation (12, 13). eNOS has a bidomain structure consisting of an N-terminal oxygenase domain and a C-terminal reductase domain (14). Located between the oxygenase and reductase domains is a Ca2+/calmodulin (CaM)-binding region (15). Association of eNOS and caveolin-1 in cultured bovine endothelial cells has been demonstrated previously in coimmunoprecipitation experiments (16, 17). It is not known, however, whether eNOS and caveolin-1 interact directly or indirectly (i.e. through an adaptor protein). Also unidentified are the interacting domains, if any, in the two proteins. Most importantly, the functional consequences of caveolin-1 binding on eNOS catalytic activity have not been determined. Each of these questions with regard to the eNOS-caveolin-1 protein-protein interaction has been addressed in the present study.
The glutathione S-transferase
(GST)-fusion protein cloning vector, pGEX-4T-1, CaM-Sepharose 4B, and
anti-GST polyclonal antibody were obtained from Pharmacia Biotech Inc.
Sf9 insect cells were purchased from Pharmingen (San Diego, CA) and
maintained in serum-supplemented Hink's TNM-FH media from Mediatech,
Inc. (Herndon, VA). Monoclonal antibody to eNOS (clone 3) was purchased
from Transduction Laboratories (Lexington, KY).
L-[14C]Arginine and ECL reagents came from
Amersham Corp. pGBT9 (DNA binding domain hybrid cloning vector),
pGAD424 (activation domain hybrid cloning vector), and
Saccharomyces cerevisiae SFY526 were obtained from
CLONTECH (Palo Alto, CA). Oligonucleotide primers for PCR, 5-RACE kit, and TriZOL reagent were purchased from Life Technologies Inc. TA Cloning kit was obtained from Invitrogen (Carlsbad, CA). Protein assay kit was purchased from Bio-Rad. Bovine
CaM came from Sigma. Synthetic peptides were obtained from Research
Genetics, Inc. (Huntsville, AL) and were >95% pure as determined by
high performance liquid chromatography.
Total
RNA from cultured bovine aortic endothelial cells was isolated with
TriZOL reagent and subjected to reverse transcription-polymerase chain
reaction. The upstream primer for PCR was based on the first 20 nucleotides of the caveolin-1 coding sequence that are identical in
sequences previously cloned from dog, mouse, and human (18-20). The
downstream primer used was an oligo(dT)17 oligonucleotide. PCR amplification produced a 568-base pair fragment that was subcloned into the TA cloning vector and sequenced in the Molecular Biology Core
Facility of the Medical College of Georgia. Three independent PCR
reactions produced identical nucleotide sequences ruling out the
possibility of PCR-associated nucleotide incorporation errors. To
confirm that the first 20 nucleotides of the bovine coding sequence are
identical to the sequence of the upstream primer, 5-RACE (rapid
amplification of cDNA ends) was performed with a 5
-RACE kit (Life
Technologies Inc.). The sequence obtained by 5
-RACE was identical to
that obtained in the initial PCR. The nucleotide sequence has been
submitted to the GenBankTM/EMBL Data Bank with accession
number U86639.
cDNA constructs encoding GST-caveolin-1 fusion
proteins were created by subcloning into the GST-fusion protein cloning
vector, pGEX-4T-1. Caveolin-1 cDNA sequences encoding full-length
caveolin-1 (residues 1-178) and caveolin-1 residues 1-60, 1-101,
102-134, and 135-178 were generated by PCR amplification of the
full-length bovine sequence cloned into the TA cloning vector. Primers
for PCR were designed to incorporate 5 EcoRI and
SalI restriction sites for subcloning. The cDNAs
encoding the fusion proteins were sequenced to confirm the creation of
in-frame fusions devoid of PCR-associated nucleotide incorporation
errors. Fusion proteins and a GST-nonfusion protein were expressed in
Escherichia coli and purified by affinity chromatography on
glutathione-agarose as described by Frangioni and Neel (21).
Bovine eNOS was expressed in a baculovirus/Sf9 insect cell system and purified to >95% homogeneity as described previously (15, 22). eNOS was purified in buffers containing 2 mM EGTA, and purified enzyme was completely dependent on exogenous CaM for activity.
Interaction of Recombinant Baculovirus-expressed eNOS with GST-Caveolin-1 Fusion ProteinsGST or GST-caveolin-1 fusion proteins (100 pmol each, quantitated by Bio-Rad protein assay) prebound to glutathione-agarose beads were washed three times in buffer containing 50 mM Tris-HCl, pH 7.4, 20% glycerol, and the following protease inhibitors: 1% phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 5 µg/ml aprotinin. Equimolar amounts of fusion proteins in each condition were confirmed by immunoblotting with anti-GST antibody. Washed beads were incubated overnight (with shaking at 4 °C) in 1 ml of the above buffer containing 100 pmol of bovine eNOS, expressed and purified from a baculovirus system as described previously (22). Following the overnight binding reaction, beads were washed six times in 1 ml of 50 mM Hepes, pH 7.5, 120 mM NaCl, 1 mM EDTA, 0.5% CHAPS plus the protease inhibitors listed above. Bound proteins were eluted with 100 µl of 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% Triton X-100, 100 mM reduced glutathione, plus protease inhibitors. Eluted proteins were separated on SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with anti-eNOS monoclonal antibody as described previously (23). Binding experiments were also performed with GST or GST-caveolin-1 fusion proteins and purified bovine CaM. In one set of experiments the binding assay was carried out exactly as described above for eNOS. In another set of experiments, EDTA was omitted from all buffers and replaced with 2 mM CaCl2.
Interaction of eNOS and Caveolin-1 Domains in a Yeast Two-hybrid SystemConstruction of bovine eNOS oxygenase domain (residues
1-505) and reductase domain (residues 506-1205) hybrids with the GAL4 DNA binding and activation domains has been described previously (24).
Bovine caveolin-1 hybrids encoding residues 1-178, 1-101, and
135-178 were created by subcloning into the shuttle/expression vectors, pGBT9 and pGAD424 (CLONTECH). DNA
sequences for subcloning were generated by PCR amplification of the
full-length bovine caveolin-1 cDNA in the TA cloning vector. PCR
primers were designed to incorporate 5 EcoRI and
SalI restriction sites into the amplified products to
facilitate subcloning. All DNA constructs were sequenced to confirm the
creation of an in-frame hybrid devoid of PCR-associated nucleotide
incorporation errors. Transformation of yeast and colony lift filter
assay of
-galactosidase activity has been described previously (24).
All DNA-binding domain and activation domain fusion constructs were
confirmed not to reconstitute GAL4 activity by themselves, and all
activation domain fusion constructs were confirmed not to activate
transcription when combined with the unrelated pLAM 5
(human lamin
C66-230 in pGBT9).
GST or GST-caveolin-1 fusion proteins were eluted from glutathione-agarose beads with reduced glutathione, and 300 pmol of each protein (quantitated by Bio-Rad protein assay) was incubated for 5 min at 37 °C with purified, baculovirus-expressed bovine eNOS (100 pmol) in 50 mM Tris-HCl, pH 7.5, buffer. eNOS activity was then determined by monitoring the rate of formation of L-[14C]citrulline from L-[14C]arginine (50 µM) in the presence of excess cofactors including Ca2+ (2 mM), purified bovine CaM (250 pmol), NADPH (1 mM), FAD (4 µM), FMN (4 µM), and tetrahydrobiopterin (40 µM) in a 200-µl reaction volume as described previously (22). Experiments testing the effects of synthetic peptides were carried out using the same protocol and various concentrations of peptides.
CaM-Sepharose ChromatographyPurified eNOS (1 µg) was incubated with or without synthetic peptides (10 µM) in 50 mM Tris-HCl, pH 7.5, buffer for 5 min at 37 °C and then subjected to CaM-Sepharose chromatography as described previously (22). The amount of eNOS eluted from the CaM-Sepharose column was quantitated by immunoblotting with anti-eNOS antibody as described previously (23).
eNOS and caveolin-1 are known to be associated in cultured bovine
aortic and lung microvascular endothelial cells (16, 17). It is not
known, however, whether they interact directly or indirectly (i.e. through an adaptor protein). To answer this question
for purified, baculovirus-expressed bovine eNOS, we have isolated and
sequenced the cDNA encoding bovine caveolin-1. Analysis of the
deduced amino acid sequence indicates that bovine caveolin-1 shares 94, 96, 97, and 86% identity with the human, murine, canine, and chicken
sequences, respectively (18-20, 25) (Fig. 1). To determine whether eNOS interacts directly with caveolin-1, we expressed
full-length bovine caveolin-1 as a GST-fusion protein in E. coli. In addition, to determine which domains of caveolin-1 are
involved in eNOS binding, we also expressed GST-fusion proteins of
caveolin-1 residues 1-60, 1-101 (N-terminal cytoplasmic domain), 102-134 (membrane-spanning domain), and 135-178 (C-terminal
cytoplasmic domain). The fusion proteins and a GST-nonfusion protein
were purified by affinity chromatography on glutathione-agarose. The GST-caveolin-1 fusions or GST alone prebound to agarose beads were then
used in in vitro binding assays with recombinant bovine eNOS, expressed and purified from a baculovirus system (22). Beads were
incubated with eNOS at 4 °C overnight and extensively washed, and
bound proteins were eluted with reduced glutathione. Eluted proteins
were separated on SDS-polyacrylamide gels, transferred to
nitrocellulose, and immunoblotted with anti-eNOS antibody. As shown in
Fig. 2, eNOS bound specifically to the full-length GST-caveolin-1 fusion protein but not to GST alone, demonstrating that
eNOS and caveolin-1 interact directly. Furthermore, eNOS bound
specifically to GST-fusions containing only the N-terminal caveolin-1
cytoplasmic domain (residues 1-101) or only the C-terminal caveolin-1
cytoplasmic domain (residues 135-178). In contrast, GST-fusions
containing only caveolin-1 residues 1-60 or the caveolin-1 membrane-spanning domain (residues 102-134) did not bind to eNOS. The
eNOS-caveolin-1 association thus appears to involve binding of eNOS to
both cytoplasmic tails of caveolin-1. Furthermore, either cytoplasmic
domain of caveolin-1 by itself is sufficient to mediate the eNOS
binding.
To verify the conclusions reached based on the in vitro
binding assays and to determine whether caveolin-1 binds to either the
eNOS oxygenase domain or the eNOS reductase domain, or both, we have
also investigated the eNOS-caveolin-1 interaction in a yeast two-hybrid
system. Hybrid cDNA constructs were prepared that encoded
full-length bovine caveolin-1 (residues 1-178), the caveolin-1
N-terminal cytoplasmic domain (residues 1-101), the caveolin-1
C-terminal cytoplasmic domain (135-178), the bovine eNOS oxygenase
domain (1-505), and the bovine eNOS reductase domain (506-1205) fused
to either the GAL4 DNA binding domain or activation domain. Various
pairwise combinations of the plasmid constructs were used to
cotransform the yeast strain, SFY526. Interactions of hybrid proteins
were assessed by colony lift filter assay of -galactosidase reporter
gene transcription. As shown in Table I, caveolin-1
interacted with itself in the two-hybrid system through both N- and
C-terminal cytoplasmic domains, as has been demonstrated previously in
in vitro binding assays with GST-fusion proteins (26, 27).
Furthermore, both N- and C-terminal domains of caveolin-1 interacted
with eNOS in the two-hybrid system, confirming the results obtained in
the GST-fusion protein binding assays. Caveolin-1 interactions were
restricted to the eNOS oxygenase domain and did not occur with the eNOS
reductase domain.
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To determine whether interaction of eNOS with caveolin-1 alters
nitric-oxide synthase activity, we incubated equal quantities of
purified, baculovirus-expressed eNOS with equimolar quantities of the
GST alone, GST-caveolin 1-60, GST-caveolin 102-134, GST-caveolin 1-178, GST-caveolin 1-101, and GST-caveolin 135-178 fusion proteins. eNOS activity was then determined by arginine-to-citrulline conversion assay in the presence of excess cofactors, Ca2+, and CaM.
As shown in Fig. 3, the full-length caveolin-1 fusion protein inhibited eNOS activity by about 60%. Furthermore, either of
the caveolin-1 cytoplasmic domains appears to be sufficient to mediate
eNOS inhibition because the GST-caveolin 1-101 and GST-caveolin
135-178 fusion proteins also inhibited enzyme activity by about 60%.
In contrast, the GST-caveolin 1-60 and GST-caveolin 102-134 fusion
proteins were without effect on activity. To confirm that inhibition
was due to binding of the fusion proteins to eNOS rather than to CaM,
we also performed in vitro binding assays with CaM and
GST-caveolin-1 fusion proteins under the same conditions used in the
eNOS binding studies. In addition, we performed in vitro
binding assays of eNOS and CaM in which 1 mM EDTA was
omitted from all buffers and replaced by 2 mM
CaCl2. In both sets of experiments, no CaM binding to any
of the GST-caveolin-1 fusion proteins was detected.
Inhibition by GST-caveolin 1-101 but not by GST-caveolin 1-60
suggests that the inhibitory region of the N-terminal cytoplasmic domain may correspond to the caveolin-1 scaffolding domain (residues 82-101) previously shown to inhibit G subunits, Ha-Ras, and Src family tyrosine kinases (7-9). To test this hypothesis we prepared synthetic peptides corresponding to caveolin-1 residues 61-81 and
82-101. As shown in Fig. 4, the 82-101 peptide
potently inhibited eNOS activity (IC50 ~1
µM). Complete inhibition was observed at a 10 µM concentration of peptide. A 10 µM
concentration of the 61-81 peptide, on the other hand, actually
increased activity by about 30%. To determine whether inhibition was
due to an effect of the 82-101 peptide on the eNOS interaction with
Ca2+/CaM, we preincubated eNOS with and without the 61-81
and 82-101 peptides (10 µM) and then subjected the
enzyme to CaM-Sepharose chromatography. Enzyme was allowed to bind to
the column in the presence of 2 mM CaCl2 and
was eluted with 2 mM EGTA. The amount of enzyme eluted in
each condition was quantitated by immunoblotting with monoclonal
anti-eNOS antibody. As shown in Fig. 5A, the
82-101 peptide reduced binding of eNOS to CaM-Sepharose by >90% (as
determined by densitometry of immunoblots). In contrast, the 61-81
peptide had no effect on CaM binding. Furthermore, peptide inhibition of eNOS was reversible by increasing the molar excess of
Ca2+/CaM by 10-fold. eNOS was preincubated for 5 min at
37 °C with the 82-101 peptide (10 µM). Enzyme
activity was then determined in the presence of the standard molar
excess concentration of Ca2+/CaM (1.25 µM)
routinely used in the arginine-to-citrulline conversion assay.
Activity was confirmed to be completely inhibited in the presence of 10 µM peptide and 1.25 µM
Ca2+/CaM. eNOS was then incubated for an additional 5 min
at 37 °C with either 1.25 µM or 12.5 µM
Ca2+/CaM. eNOS activity was then redetermined. As shown in
Fig. 5B, increasing the Ca2+/CaM concentration
by 10-fold completely reversed the inhibitory effects of the 82-101
peptide.
In summary, the results of the present study provide several important
new insights into the eNOS-caveolin-1 interaction. First, eNOS and
caveolin-1 interact directly rather than indirectly. Second,
interaction involves both the N- and C-terminal cytoplasmic domains of
caveolin-1 and is thus fundamentally different from the interaction of
caveolin-1 with G subunits, Ha-Ras, and Src family tyrosine kinases.
Third, the caveolin-1 interaction with eNOS involves only the eNOS
oxygenase domain and not the eNOS reductase domain. Fourth, interaction
of eNOS with caveolin-1 significantly inhibits eNOS catalytic activity.
Finally, inhibition appears to be due to interference with the eNOS
interaction with Ca2+/CaM. Regulation of eNOS activity in
endothelial cells, therefore, may involve not only positive allosteric
regulation by Ca2+/CaM, but also negative allosteric
regulation by caveolin-1. It is conceivable that interaction of eNOS
with caveolin-1 provides a mechanism to deactivate the enzyme
subsequent to its activation by agonist-stimulated elevation of
intracellular Ca2+. Protein-protein interactions between
eNOS and caveolin-1, however, are probably not sufficient to mediate
membrane attachment. Fatty acylation of eNOS by myristate and palmitate
appears to also be required. This requirement has been demonstrated in
previous studies of an eNOS myristoylation-deficient mutant in which
glycine 2 has been mutated to an alanine. The mutant enzyme is neither
myristoylated nor palmitoylated and, as a result, is not
membrane-associated (22).