(Received for publication, July 20, 1995; and in revised form, August 28, 1995)
From the
Nitric oxide produced by the endothelial isoform of nitric oxide
synthase (ecNOS) is a key determinant of vascular tone. In contrast to
other nitric oxide synthase (NOS) isoforms, which have been
characterized as soluble homodimeric enzymes, ecNOS is predominantly
membrane-associated, a feature that has hindered direct biochemical
analyses of its oligomeric structure. We investigated ecNOS
oligomerization using co-immunoprecipitation experiments in transiently
transfected COS-7 cells. When COS-7 cells co-transfected with
constructs encoding wild-type ecNOS and an epitope-tagged
myristoylation-deficient mutant were biosynthetically labeled with
[H]myristate, the antibody to the epitope tag
specifically immunoprecipitated
H-labeled ecNOS, reflecting
enzyme oligomerization. In COS-7 cells transfected with cDNAs encoding
epitope-tagged truncation mutants and untagged full-length ecNOS, the
wild-type enzyme could be immunoprecipitated by the antibody to the
epitope tag. Co-immunoprecipitation of ecNOS with truncation mutants
documented that both N- and C-terminal domains are involved in ecNOS
oligomerization. When these truncation mutants are co-expressed with
wild-type ecNOS, they exert a marked dominant negative effect on enzyme
activity. Since NOS oligomerization itself may be subject to dynamic
modulation, the regulation of ecNOS assembly may have implications for
NO signaling in the vascular wall.
Nitric oxide, a ubiquitous messenger molecule, is synthesized by
a family of nitric oxide synthases (NOS) ()and participates
in diverse cellular processes, including neurotransmission, immune
regulation, and vascular homeostasis(1) . Three distinct NOS
isoforms have been identified: neuronal (nNOS), inducible (iNOS), and
endothelial NOS (ecNOS). These NOS isoforms subserve disparate
physiological roles and differ markedly in their tissue distribution
and mechanisms of regulation (see (2, 3, 4) for review). Nevertheless, the NOS
isoforms appear to share many biochemical features, including common
cofactor and substrate requirements. All known NOS isoforms catalyze
the calmodulin-dependent oxidation of L-arginine to form
nitric oxide plus L-citrulline. Recently, it was shown that
dimerization of iNOS is required for catalytic activity(5) ;
the active nNOS is also a homodimer(6) . The oligomeric
structure of ecNOS is unknown.
Oligomerization may serve as an important mechanism both for enzyme regulation and for signal transduction(7, 8) . Dimerization of iNOS appears to depend on the presence of the enzyme cofactors tetrahydrobiopterin and heme, as well as the substrate L-arginine(5) . The nNOS homodimer is also stabilized by tetrahydrobiopterin and L-arginine (6) . Since cellular levels of cofactors and substrate may themselves be regulated, enzyme oligomerization may provide a mechanism for modulation of NOS activity.
In contrast to the other NOS isoforms, which can be studied as soluble dimeric enzymes, ecNOS is predominantly membrane-associated(9) , a feature that has hampered direct hydrodynamic analyses of its oligomeric structure. In the present study, we use co-immunoprecipitation experiments to show that ecNOS is an oligomeric protein and that both N- and C-terminal domains of the enzyme appear to be involved in its assembly. Furthermore, these studies document that co-expression of ecNOS truncation mutants with the wild-type enzyme exerts a dominant negative effect on enzyme activity.
Figure 1:
Schematic of ecNOS constructs
used in co-transfection experiments. cDNA encoding the full-length
wild-type ecNOS (135 kDa) was cleaved at propitious restriction enzyme
sites to yield two truncation mutants, each cloned such that the 3`-end
is joined to the sequence for the HA epitope tag followed by a stop
codon. The protein encoded by the mutant cDNA pKpn contains
residues 1-734 of ecNOS. The pKCT truncation mutant encodes
residues 512-1204 of ecNOS. The site for mutagenesis (G2A) in
the myristoylation consensus sequence is shown (myr
);
this mutation results in formation of acylation-deficient ecNOS, which
was epitope-tagged at the 3`-end of the construct. Also noted are the
putative sites for heme, calmodulin (CAM), FMN, FAD, and NADPH
binding to ecNOS, as well as the location (P1) of the ecNOS
peptide sequence (residues 597-612) used as an epitope for
generation of ecNOS antiserum. aa, amino
acids.
Figure 2:
Co-immunoprecipitation of wild-type ecNOS
with epitope-tagged myr ecNOS in
[
H]myristate-labeled COS-7 cells. Shown in this
figure are the results of SDS-PAGE and fluorography of wild-type and
mutant ecNOS immunoprecipitated from cells transfected with cDNAs
encoding the epitope-tagged, myristoylation-deficient mutant (myr
) or the wild-type untagged enzyme (ecNOS) or co-transfected with both cDNAs, as indicated below
the fluorogram. The transfected cells were biosynthetically labeled
with [
H]myristic acid; duplicate aliquots from
each cell lysate were immunoprecipitated either with the 12CA5
monoclonal antibody directed against the HA epitope (
HA)
or antiserum directed against ecNOS (
ecNOS) as indicated.
The immunoprecipitated proteins were resolved by SDS-PAGE and subjected
to fluorography on x-ray film for 3 weeks. The molecular mass of
standards is shown in kDa; the site for migration of ecNOS is noted.
The results shown are representative of three independent
experiments.
Figure 3:
NADPH diaphorase staining of wild-type and
truncation mutant ecNOS transfected into COS-7 cells. Shown are
photomicrographs of COS-7 cells transfected with the plasmid vector
alone (sham), with cDNAs encoding wild-type ecNOS (wild-type) or truncation mutants expressing either N-terminal
(Kpn) or C-terminal (pKCT) domains of ecNOS (see Fig. 1). Seventy-two hours after transfection, cultures were
fixed and stained with nitroblue tetrazolium to detect NADPH diaphorase
activity.
We investigated which domains are involved in
ecNOS oligomerization by co-expression and immunoprecipitation of
wild-type ecNOS with these epitope-tagged truncation mutants. As shown
in Fig. 4, the protein encoded by the pKpn truncation
mutant can be expressed in COS cells and is immunoprecipitated with
either the ecNOS antibody or the antibody to the epitope tag. Wild-type
ecNOS, when expressed alone, can be immunoprecipitated only by the
ecNOS antibody. However, when wild-type ecNOS is co-expressed with the
epitope-tagged mutant, the full-length enzyme is now immunoprecipitated
by the HA antibody (Fig. 4). This suggests that the full-length
protein associates with the tagged truncation mutant in a
hetero-oligomeric complex. When lysates of cells transfected separately
with ecNOS or p
Kpn were combined in vitro, ecNOS did not
co-immunoprecipitate with the truncation mutant (Fig. 4). This
result implies that the observed association of co-expressed
full-length and truncated ecNOS primarily occurs intracellularly and
not following cell lysis.
Figure 4:
Co-immunoprecipitation of full-length
ecNOS and C-terminal truncation mutant. Shown is a fluorogram of the
SDS-PAGE analysis of proteins immunoprecipitated from COS-7 cells
transfected with cDNAs for untagged full-length ecNOS and/or an
epitope-tagged truncation mutant lacking the protein's C terminus
(Kpn-HA). Transfected cells were biosynthetically labeled with
[
S]methionine, and duplicate samples of cell
lysates were immunoprecipitated using the HA antibody against the
epitope tag or anti-ecNOS antiserum, as noted above the fluorogram.
Minor bands reflect nonspecific immunoprecipitation of cellular
proteins by the monoclonal HA antibody, as previously
reported(11) . In the first two lanes are the results
of immunoprecipitations from cultures co-transfected with both the
p
Kpn and wild-type ecNOS cDNAs. In the following lanes are
proteins immunoprecipitated from cultures transfected with only one of
these constructs (indicated below the fluorogram); the last two
lanes show the pattern of immunoprecipitation seen when such
lysates (containing either p
Kpn or wild-type ecNOS) are mixed
together immediately prior to immunoprecipitation (post-hoc).
Immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography;
this film was exposed for 3 days. On the right, arrows indicate bands corresponding to the full-length ecNOS (135 kDa)
and the
Kpn-HA truncation mutant protein (82 kDa). The molecular
mass standards are shown in kDa on the left. The results shown
are representative of three independent
experiments.
A similar approach was employed to examine the role of the C-terminal domain in enzyme oligomerization. COS-7 cells were transfected with constructs for wild-type ecNOS or the HA-tagged truncation mutant, pKCT (Fig. 5); as before, when expressed alone, the untagged wild-type enzyme was immunoprecipitated by the ecNOS antibody but not by the antibody to the epitope tag. Epitope-tagged pKCT could be immunoprecipitated by either antibody. When the wild-type enzyme was co-expressed with pKCT, full-length ecNOS could now be immunoprecipitated by the HA antibody, consistent with the formation of a hetero-oligomeric complex. Together these results suggest that both the N- and C-terminal domains of ecNOS may be involved in oligomerization of the enzyme. These findings may be contrasted with a recent study of iNOS oligomerization by Ghosh and Stuehr(19) . In this report, limited tryptic digestion of purified iNOS (which cleaves in midmolecule to separate N- and C-terminal domains) led to the formation of dimeric N-terminal and monomeric C-terminal peptides. Therefore, it was proposed for iNOS that only the N-terminal domain, and not the C-terminal domain, participates in dimer formation. However, our results document specific interactions between the full-length enzyme and both the N- and C-terminal domains, since the N- and C-terminal truncation mutants are each able to co-immunoprecipitate ecNOS. These divergent results may represent intrinsic differences between iNOS and ecNOS oligomerization or may reflect differences in experimental approach.
Figure 5:
Co-immunoprecipitation of full-length
ecNOS and N-terminal truncation mutant. This figure shows a fluorogram
of the SDS-PAGE analysis of proteins immunoprecipitated from COS-7
cells biosynthetically labeled with
[S]methionine following transfection with cDNAs
(indicated below the fluorogram) encoding the untagged full-length
ecNOS and/or an epitope-tagged N-terminal truncation mutant of ecNOS (pKCT). Proteins were immunoprecipitated from duplicate
samples of cell lysates using either the HA or ecNOS antibody, as noted
above the fluorogram. The immunoprecipitated proteins were analyzed by
SDS-PAGE and fluorography; this film was exposed for 3 days. On the right, arrows indicate bands corresponding to the
full-length ecNOS (135 kDa) and the pKCT truncation mutant protein (78
kDa). The molecular weight standards are shown in kDa on the left. Results are representative of three independent
experiments.
Both the N- and
C-terminal ecNOS truncation mutants thus appear to form
hetero-oligomeric complexes with the wild-type enzyme. However, only a
fraction of the wild-type ecNOS is co-immunoprecipitated by these
truncation mutants ( Fig. 4and Fig. 5). It is possible
that stronger associations between full-length ecNOS monomers result in
preferential formation of homo-oligomers of the wild-type enzyme.
Indeed, when ecNOS is expressed with the full-length HA-tagged
myr mutant, a larger fraction of the wild-type enzyme
is co-immunoprecipitated by the HA antibody (Fig. 2). Weaker
interactions between truncation mutants and full-length ecNOS might
also lead to a substantial dissociation of hetero-oligomeric complexes
during immunoprecipitation, with consequent loss of the full-length
enzyme.
Figure 6:
Dominant negative effect of truncation
mutants on wild-type ecNOS enzyme activity. NOS activity was assayed in
lysates of transfected cells by measuring the conversion of
[H]arginine to
[
H]citrulline. The effects of co-expression of
the truncation mutants p
Kpn (panel A) and pKCT (panel
B) with wild-type ecNOS were analyzed in separate experiments. The
activity data shown (mean ± S.D.) are expressed as a percentage
of the NOS activity of the wild-type enzyme alone; for the experiment
shown in panel A, wild-type NOS activity was 1.4 pmol of
[
H]citrulline formed/min
mg protein and for panel B was 1.2 pmol/min
mg protein. For each
transfection, the total amount of DNA transfected is kept constant by
the addition of vector DNA. Duplicate aliquots of the transfected cell
lysates were also analyzed by protein immunoblot (probed with ecNOS
antiserum); the transfection of equal quantities of wild-type ecNOS
cDNA yielded similar levels of protein expression (data not shown). In
both panels A and B, the first column represents NOS activity in COS cells transfected with vector alone
(sham) and the next column the activity of the wild-type enzyme (5
µg of cDNA/culture) expressed alone. The following three
columns show the results of co-expressing the same quantity of
wild-type cDNA with 2, 5, or 10 µg of truncation mutant cDNA, as
noted below the histogram; the next column represents activity
in COS cells transfected with only the truncation mutant (5 µg of
DNA/culture). The last column represents activity measured when cells
are transfected separately with either wild-type or truncated ecNOS
cDNAs, and the lysates, containing either wild-type or mutant ecNOS,
are subsequently combined in vitro just prior to the activity
assay (post-hoc). The experiments shown are representative of
three similar experiments, each conducted in
triplicate.
Dimerization has been shown to be necessary for the catalytic activity of other isoforms of NOS and may be modulated by levels of substrates and cofactors(5, 6) . It seems likely that ecNOS oligomerization is also required for catalysis, most plausibly involving formation of an intact ecNOS homodimer. The dominant negative effects of ecNOS truncation mutants may reflect their competition with full-length monomers for the same sites of association. These results also suggest that intersubunit interactions are involved in the complex redox pathway from arginine to citrulline plus NO. These speculations will be refined by studies of the structural features of hetero-oligomeric ecNOS complexes; such studies are likely to provide important insights into the mechanisms underlying the dominant negative effect of the truncation mutants. Characterization of the regions involved in NOS dimerization may lead to the identification of a new class of enzyme inhibitors that exert a dominant negative effect on NOS activity by inhibiting enzyme oligomerization.