(Received for publication, April 14, 1997, and in revised form, April 29, 1997)
From the The endothelial nitric-oxide synthase
(eNOS) is a key determinant of vascular homeostasis. Like all known
nitric-oxide synthases, eNOS enzyme activity is dependent on
Ca2+-calmodulin. eNOS is dynamically targeted to
specialized cell surface signal-transducing domains termed plasmalemmal
caveolae and interacts with caveolin, an integral membrane protein that comprises a key structural component of caveolae. We have previously reported that the association between eNOS and caveolin is quantitative and tissue-specific (Feron, O., Belhassen, L., Kobzick, L., Smith, T. W., Kelly, R. A., and Michel, T. (1996) J. Biol. Chem.
271, 22810-22814). We now report that in endothelial cells the
interaction between eNOS and caveolin is importantly regulated by
Ca2+-calmodulin. Addition of calmodulin disrupts the
heteromeric complex formed between eNOS and caveolin in a
Ca2+-dependent fashion. In addition,
overexpression of caveolin markedly attenuates eNOS enzyme activity,
but this inhibition is reversed by purified calmodulin. Caveolin
overexpression does not affect the activity of the other NOS isoforms,
suggesting eNOS-specific inhibition of NO synthase by caveolin. We
propose a model of reciprocal regulation of eNOS in endothelial cells
wherein the inhibitory eNOS-caveolin complex is disrupted by binding of
Ca2+-calmodulin to eNOS, leading to enzyme activation.
These findings may have broad implications for the regulation of
Ca2+-dependent signal transduction in
plasmalemmal caveolae.
Nitric oxide is a ubiquitous molecule implicated in diverse
biological processes and is synthesized in mammalian cells by a family
of Ca2+-calmodulin-dependent nitric-oxide
synthase (NOS)1 enzymes (1-3).
Endothelium-derived nitric oxide (NO), formed by the endothelial
isoform of nitric-oxide synthase (eNOS), serves as an important
determinant of blood pressure and platelet aggregation. In endothelial
cells, increases in intracellular Ca2+ elicited by diverse
extracellular signals lead to activation of eNOS. The three known
mammalian nitric-oxide synthases share similar overall
Ca2+-calmodulin-dependent catalytic pathways.
However, the eNOS enzyme is unique among the three known NOS isoforms
in being localized to the specialized cell surface signal-transducing
domains termed plasmalemmal caveolae (4, 5).
Plasmalemmal caveolae are small invaginations in the plasma membrane
that may serve as sites for the sequestration of signaling proteins (6,
7) including receptors, G proteins, and protein kinases, as well as
eNOS. The principal protein in caveolae is the integral membrane
protein caveolin, an oligomeric protein that serves as a structural
"scaffold" within caveolae (8). eNOS can be quantitatively
immunoprecipitated by antibodies directed against caveolin; conversely,
eNOS antiserum also immunoprecipitates caveolin (5), although it has
not yet been established whether the interaction between these two
proteins is direct. Moreover, a functional role of the eNOS-caveolin
association, beyond its postulated role in subcellular targeting of the
enzyme, remains to be determined. We document in this report that the
interaction between eNOS and caveolin may be regulated by
Ca2+-calmodulin, and we show that caveolin can specifically
inhibit eNOS enzyme activity.
A plasmid construct encoding eNOS has
been described previously (9, 10). cDNA clones encoding iNOS (11)
and nNOS (12) were kindly provided by Carl Nathan (Cornell University
Medical College) and Solomon Snyder (Johns Hopkins University),
respectively. Caveolin-1 cDNA (13) in the eukaryotic expression
vector pCB-7 was obtained from Michael Lisanti (Whitehead Institute).
An irrelevant plasmid encoding Cultures of bovine aortic
endothelial cells (studied between passages 4 and 10) and COS-7 cells
were performed as described previously (9, 14). COS-7 cells were
transfected with 10 µg of total plasmid DNA in 100-mm cell culture
plates using LipofectAMINETM (Life Technologies, Inc.) according to
the manufacturer's protocol.
Endothelial cells were lysed and solubilized
either with: 1) a previously described Ca2+-free CHAPS
buffer (5, 14) supplemented with 1 mM EGTA/1 mM
EDTA or 2) an otherwise identical CHAPS buffer containing 1 mM CaCl2 and no EDTA/EGTA. CHAPS-solubilized
bovine aortic endothelial cell lysates were incubated either with a
polyclonal caveolin antibody (Transduction Laboratories) at a final
concentration of 4 µg/ml or with a previously characterized
polyclonal antiserum directed against eNOS (9) used at a final dilution
of 1:100. Immunoprecipitated complexes were then recovered, denatured
in Laemmli sample buffer, separated on 12% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (5). Monoclonal antibodies directed against eNOS or caveolin-1 (Transduction Laboratories) were
then used to detect eNOS and caveolin-1 using chemiluminescence, as
described previously (5). Calmodulin was detected using a previously
characterized monoclonal calmodulin antibody (15). Expression of
immunoblotted proteins was quantitated by laser densitometric analysis
of x-ray films following chemiluminescence detection.
NO synthase activity in lysates
prepared from transfected COS-7 cell was determined by measuring
conversion of [3H]L-arginine to
[3H]L-citrulline as described previously (9,
14). In some experiments, this NO synthase activity assay was performed
in washed membrane fractions prepared from endothelial cell lysates as
described previously (9), except that calmodulin concentrations were varied by the addition of purified bovine brain calmodulin (Sigma) as
indicated.
In exploring the factors governing eNOS-caveolin association, we
discovered that the co-immunoprecipitation of eNOS and caveolin was
markedly attenuated when endothelial cell lysates were prepared in
buffers containing excess free Ca2+ (Fig.
1). Under no conditions did the presence of
Ca2+ affect the recovery of caveolin or eNOS from
endothelial cell lysates when the proteins were directly
immunoprecipitated by their cognate antibody. Only
co-immunoprecipitation was abrogated by Ca2+, as
shown in Fig. 1.
We next investigated the role of Ca2+ in modulating the
association of eNOS and calmodulin. This is particularly important
because calmodulin, a ubiquitous Ca2+-binding protein,
plays a central role in nitric-oxide synthase catalysis (16).
Co-immunoprecipitation experiments using eNOS antiserum to explore
eNOS-calmodulin interactions in endothelial cell lysates are shown in
Fig. 2. We could detect co-immunoprecipitation of eNOS
and calmodulin only when free Ca2+ was present. This result
is in striking contrast to the loss of eNOS-caveolin
co-immunoprecipitation observed in the presence of Ca2+
(Fig. 1). Taken together, these data suggest that Ca2+
differentially modulates the association of eNOS with caveolin versus calmodulin in endothelial cell lysates.
We next used antibodies against caveolin to co-immunoprecipitate eNOS
in Ca2+-free buffers and then washed the immune complexes
extensively to remove residual Ca2+ and calmodulin.
Subsequent addition of either Ca2+ or calmodulin alone had
no effect on the subsequent recovery of co-immunoprecipitated eNOS from
the eNOS-caveolin complex (Fig. 3, upper
panel). However, when Ca2+ and calmodulin were added
together, eNOS was entirely lost from the caveolin immune complex. This
"lost" eNOS could be completely recovered in the supernatant of the
immune complex (Fig. 3, middle panel), indicating that the
eNOS molecule had been released and not degraded following treatment of
the caveolin-eNOS complex with Ca2+ plus calmodulin. None
of these treatments affected the recovery of caveolin itself from the
immune complex (Fig. 3, lower panel). The extensive washing
of these immune complexes, required to remove the endogenous
calmodulin, likely led to the loss of some eNOS (because the affinity
of the eNOS-caveolin interaction is undoubtedly less than the affinity
of the antibody for caveolin), leading to the detection of a relatively
faint but highly reproducible (n = 4) signal for eNOS
released from these complexes by the combination of Ca2+
plus calmodulin.
Diverse experimental approaches have shown that agonist activation of
eNOS in endothelial cells is dependent on Ca2+-calmodulin
(1, 2). The striking effects of Ca2+-calmodulin on the
interactions of eNOS and caveolin therefore suggested to us that
caveolin may influence eNOS enzyme activity. Indeed, caveolin has
recently been shown in vitro to interact with other
signaling proteins, including H-ras and G protein
Taken together, these studies suggest that the interaction between eNOS
and caveolin is dynamically and specifically regulated by
Ca2+-calmodulin and may serve as an important point of
control in NO-dependent signaling. A direct interaction of
caveolin with calmodulin appears unlikely to us because there was no
influence of caveolin on the activity of other calmodulin-binding
proteins (iNOS and nNOS) closely related to eNOS. This hypothesis is
consistent with our failure to detect co-immunoprecipitation of
calmodulin with caveolin (Fig. 2), under conditions in which eNOS was
shown to associate with either one or the other protein. Furthermore, the amino acid sequence of caveolin isoforms show no obvious sequence or structural homologies to the known NOS isoforms (10-12) nor to any
known calmodulin-binding protein sequences. Caveolin can attenuate the
tyrosine kinase activity of c-src, an enzyme that bears no
structural homology to eNOS, and is not known to be regulated by
calmodulin (13). We speculate that there is a common higher order
structure assumed by the inactive conformation of diverse caveolae-targeted signaling proteins that forms the basis for their
common interaction with caveolin.
The targeting of eNOS to caveolae is likely to facilitate the
interactions of eNOS with other co-localized signaling and regulatory molecules (3). Formation of an inhibitory eNOS-caveolin heteromeric complex may serve to ensure the latency of the NO signal until calcium-mobilizing extracellular stimuli destabilize this complex and
activate the enzyme. This close control of enzyme activity may be
particularly important for eNOS in caveolae, where calmodulin, the
enzyme's key allosteric activator, also is localized (4) and where
even subtle increases in intracellular Ca2+ could thus lead
to enzyme activation if the interactions of caveolin with eNOS were not
keeping the system in check. Because nitric oxide has cytotoxic as well
as signaling functions (1, 2), attenuation of basal enzyme
"leakiness" by caveolin may be of particular importance. The
reciprocal regulation of eNOS by caveolin and calmodulin may represent
a novel mechanism for the concerted control of NO production in the
vascular wall.
This report is dedicated to the memory of Professor Thomas W. Smith. We are grateful to John Joyal for helpful
discussions and for assistance performing calmodulin immunoblots.
Cardiovascular Division,
Department of Pathology, Brigham and Women's
Hospital, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Plasmid Constructs
-galactosidase was used as a control
to maintain identical amounts of DNA in each transfection.
Fig. 1.
Co-immunoprecipitation of eNOS with caveolin
is blocked by Ca2+. Immunoprecipitation
(IP) of eNOS and caveolin was performed in endothelial cell
lysates prepared in the presence (1 mM CaCl2) or the absence (1 mM EDTA, 1 mM EGTA) of excess
calcium, as described in the text. The immunoblots in the upper
panels were probed with eNOS antibody, and those in the
lower panels were probed with caveolin antibody. In the
left panels are shown the results of immunoblots performed
directly on endothelial cell lysates prepared in the presence and the
absence of added Ca2+, documenting no change in the
abundance of these proteins in cell lysates. In the middle panels
are shown the results of immunoblot analyses following
immunoprecipitations with eNOS antiserum, and in the right
panels are shown the immunoblots probed following immunoprecipitation with the caveolin antibody. This experiment was
repeated three times with equivalent results.
[View Larger Version of this Image (49K GIF file)]
Fig. 2.
Co-immunoprecipitation of eNOS with
calmodulin requires Ca2+. Shown are the results of
SDS-PAGE and immunoblots probed with monoclonal calmodulin antibody.
Endothelial cell lysates were solubilized with CHAPS buffer containing
either EDTA/EGTA or CaCl2 as noted above each
lane. Immunoprecipitation (IP) of calmodulin by
the eNOS antiserum (middle lanes) required Ca2+
and was not seen in the presence of EGTA/EDTA. Co-immunoprecipitation of calmodulin by caveolin (right lanes) was not detected
under any conditions.
[View Larger Version of this Image (28K GIF file)]
Fig. 3.
Release of eNOS from caveolin immune
complexes by Ca2+-calmodulin and its recovery.
Endothelial cell lysates were solubilized in Ca2+-free (1 mM EDTA, 1 mM EGTA) CHAPS buffer, and caveolin
was immunoprecipitated as described in the text. Immune complexes were
precipitated by the addition of protein G-Sepharose (16). The immune
complexes bound to protein G-Sepharose beads were washed ten times in
CHAPS buffer, and the beads were then equally distributed in four
separate aliquots and incubated for 1 h at 4 °C as described
below and then processed for SDS-PAGE and immunoblot analysis. The
first lane shows results following incubation with CHAPS
buffer alone; the second lane shows the results when 1 mM CaCl2 is added; calmodulin (1 µg/ml) was
added for the third lane; in the last lane both CaCl2 and calmodulin are added to the incubation. Following
the incubation, the beads are pelleted and the supernatant
(supe) is saved (and analyzed in the middle panel
of the figure). After a final wash, the immune complexes are eluted
from beads with SDS-PAGE sample buffer and then analyzed by SDS-PAGE;
immunoblots were then probed with eNOS antibody (top panel)
or caveolin antibody (bottom panel). These experiments were
performed four times with similar results.
[View Larger Version of this Image (36K GIF file)]
subunits,
preferentially associating with the "inactive" forms of these
proteins (17), but the cellular regulation of these interactions is
less well understood. We explored the functional consequences of the
interaction between eNOS and caveolin using transient transfection
experiments in COS-7 cells and analyzed eNOS enzyme activity in
transfected cells by assaying the conversion of
[3H]L-arginine to
[3H]L-citrulline in cell lysates, as shown in
Fig. 4. In three separate experiments, each conducted in
triplicate, we found that the co-transfection of a plasmid cDNA
construct encoding caveolin with eNOS cDNA led to a marked
attenuation of NOS activity (3.4 ± 0.3 versus 1.6 ± 0.1 pmol citrulline/min·mg protein in the absence or the presence of caveolin co-expression, respectively; see Fig. 4A). There
was no change in the abundance of eNOS protein associated with caveolin co-transfection, as assessed in immunoblots of these cellular lysates
analyzed in each experiment (data not shown). Importantly, caveolin
co-transfection failed to attenuate the enzyme activity expressed by
transfected iNOS or nNOS cDNA, shown in Fig. 4B. As for
eNOS, the enzyme activity of iNOS and nNOS is
calmodulin-dependent (although important differences in the
Ca2+ dependence of the different NOS isoforms have been
noted). In further contrast to eNOS, the other NOS isoforms are not
targeted to caveolae. To explore the specificity of the inhibitory
effect of caveolin co-expression on eNOS enzyme activity, we performed activity assays in the presence of varying concentrations of purified calmodulin added to washed membrane fractions prepared from transfected COS-7 cells. As shown in Fig. 4C, in cells transfected with
eNOS cDNA alone, there is a robust NOS activity even in the absence of added calmodulin (presumably due the presence of endogenous calmodulin in these membranes); enzyme activity increases only slightly
with the addition of exogenous calmodulin. By contrast, caveolin
co-expression markedly inhibits eNOS activity (by >90%) in the
absence of added calmodulin; addition of increasing concentrations of
exogenous calmodulin relieves this enzyme inhibition in a
dose-dependent fashion, documenting that the caveolin
inhibitory effect may be specifically overcome by purified
calmodulin.
Fig. 4.
Attenuation of eNOS enzyme activity by
co-expression of caveolin in transfected cells (A); no
effects on iNOS or nNOS activity (B); and reversal of
inhibition by calmodulin (C). Shown are the results of
a [3H]L-arginine [3H]L-citrulline nitric-oxide synthase
activity assay performed in lysates of transfected COS-7 cells as
described above. A, COS-7 cells were transfected with an
expression plasmid encoding eNOS, in a co-transfection either with
caveolin (cav) cDNA (as noted) or with an equivalent
quantity of plasmid DNA encoding an irrelevant protein
(
-galactosidase). This experiment was conducted in triplicate three
times with equivalent results. B, transfection of COS-7 cells with eNOS, iNOS, or nNOS cDNAs without (black
bars) or with (shaded bars) caveolin cDNA were
performed as described for A, with NOS enzyme activity
measured in cell lysates and normalized to the activity seen in the
absence of caveolin. Maximal NOS enzyme activities (in the absence of
caveolin) in different experiments averaged 4.2 ± 2.1 pmol
citrulline formed/min·mg protein. The experiment shown was repeated
three times in triplicate with identical results. C, NOS
enzyme activity was measured in washed membranes prepared from COS-7
cells transfected with eNOS cDNA either without (black
bars) or with (hatched bars) caveolin cDNA. NOS
activity was assayed under conditions identical to those described
above except that varying concentrations of purified calmodulin were added as noted on the abscissa. This experiment was performed twice in
duplicate, yielding equivalent results.
[View Larger Version of this Image (28K GIF file)]
*
This work was supported by awards (to T. M.) from the
National Institutes of Health, the American Heart Association, and the Burroughs Wellcome Fund.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Bugher-American Heart Association Fellow in Cardiovascular
Molecular Biology.
¶
Supported by a fellowship from the Belgian American
Educational Foundation and by a grant from the Patrimoine Facultaire de l'Université Catholique de Louvain (Belgium).
**
Established Investigator of the American Heart Association and a
Burroughs Wellcome Scholar in Experimental Therapeutics. To whom
correspondence should be addressed: Brigham and Women's Hospital,
Harvard Medical School, 75 Francis St., Boston, MA 02115. Tel.:
617-732-7376; Fax: 617-732-5132; E-mail:
Michel{at}Calvin.BWH.Harvard.Edu.
1
The abbreviations used are: NOS, nitric-oxide
synthase; NO, nitric oxide; eNOS, endothelial isoform of NOS; iNOS,
inducible isoform of NOS; nNOS, neuronal isoform of NOS; PAGE,
polyacrylamide gel electrophoresis; CHAPS,
3-[(3-holamidopropyl)dimethylammonio]-1-propanesulfonic acid.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.