(Received for publication, June 27, 1995; and in revised form, August 14, 1995)
From the
Soluble guanylyl cyclase is a heterodimer consisting of an
and
subunit and stimulation occurs upon binding of NO to a
prosthetic group. Little is known about the localization of catalytic
and regulatory domains within the subunits of soluble guanylyl cyclase.
We used deletion mutagenesis to identify the regions of
and
subunits that are responsible for cGMP
production or NO-heme-mediated activation.
The amino terminus of the
subunit was necessary for NO stimulation since
deletion of the 64 NH
-terminal amino acids resulted in a
mutant with intact basal activity but complete loss of NO activation.
The amino terminus of the
subunit also appeared to be
essential for NO sensitivity since deletion of 131
NH
-terminal amino acids of
led to
markedly reduced NO activation. These results suggest that
NH
-terminal regions of
and
are involved in NO-heme-mediated signal transduction. The
NH
terminally truncated
subunit exerted a
dominant negative effect exclusively on the NO-stimulated activity of
the wild type enzyme, further underlining that the regulatory domain is
located within the NH
terminus of the enzyme. Aside from
the structural implications, the mutant represents a powerful tool to
investigate nitric oxide-sensitive signaling pathways. Coexpression of
the COOH-terminal halves of
and
were sufficient for basal cGMP production while either of the
halves expressed alone was inactive. Therefore the COOH-terminal
regions appear to contain sufficient information for dimerization and
basal enzymatic activity.
Thus, we provide the first evidence that
the regulatory and catalytic properties of soluble guanylyl cyclase can
be attributed to different regions of the subunits and that the
catalytic domain can be functionally expressed separately from the
NH-terminal regulatory domain. Taken together with findings
on the membrane bound enzyme form, guanylyl cyclases, appear to
resemble fusion proteins where different regulatory domains have been
joined with a common cGMP-forming segment.
Guanylyl cyclases (GTP pyrophosphate-lyase (cyclizing); EC
4.6.1.2), the enzymes catalyzing the formation of cGMP, exist in
membrane-bound and soluble forms. The membrane-bound enzymes are
stimulated by different peptide hormones and belong to the group of
receptor-linked enzymes with one membrane spanning region(1) .
Accordingly, their structure can be divided into three domains: an
amino-terminal extracellular ligand-binding domain, an intracellular
protein kinase-like domain, which has been proposed to function as a
negative regulatory element(2) , and the COOH-terminal
catalytic region comprising about 250 amino acids responsible for the
synthesis of cGMP, as shown by a deletion mutant of the membrane-bound
guanylyl cyclase GC-A(3, 4) . In contrast to the
membrane-bound enzymes which exist as homodimers or higher ordered
structures, soluble guanylyl cyclase consists of two different subunits
designated and
, which are both required for catalytic
activity(5) . Four subunits have been reported up to date
(
,
,
, and
). The
heterodimer
corresponds to the enzyme purified from bovine lung, whereas the
and
subunits have been identified
by homology screening but have not yet been detected on the protein
level. All subunits show some homologies over the whole length of the
polypeptide chains; the strongest homologies are found in the
COOH-terminal regions which are also shared with the membrane-bound
guanylyl cyclases and adenylyl cyclases(6) .
Soluble
guanylyl cyclase is a heme protein with spectral properties indicative
of a 5-coordinate ferrous heme with histidine as the axial
ligand(7) , and it is the heme moiety which serves as the
receptor for nitric oxide (NO), ()the activator of the
soluble enzyme(8) . Recently, histidine 105 of the
subunit has been shown to be essential for the stimulation by NO
since substitution by phenylalanine yielded an enzyme that was
catalytically active but insensitive to NO(9) .
It is
unknown whether heme binding and catalytic activity can be attributed
to distinct domains of soluble guanylyl cyclase. Here, we show that the
COOH-terminal halves of the and
subunits comprising the putative catalytic domain are sufficient
for cGMP formation. Moreover, deletions of the poorly conserved
NH
termini of the
(
-
N
) and
subunits
(
-
N
) led to severe impairment of NO
stimulation, thus underlining the importance of the
NH
-terminal regions of both subunits for heme binding
and/or transduction of the stimulatory binding signal to the catalytic
domain. The NH
-terminally truncated
subunit specifically blocks only the stimulated activity of
wild-type enzyme while leaving the
non-stimulated activity unchanged. Therefore, this mutant offers a new
and potentially powerful means by which to inhibit the NO stimulated
but not basal catalytic activity of the soluble form of guanylyl
cyclase.
NH-terminal truncated mutants of
and
subunits were constructed by polymerase
chain reaction (PCR). Fragments coding for shortened NH
termini were amplified and used to substitute the respective
fragments in the original cDNA clones. Primers used in the PCR had an
average length of 22 bases matching the sequences used for
amplification. In the sense oligonucleotides, the beginning of the
coding region was preceded by the ribosomal binding site derived from
the sequence of the
subunit, and a HindIII
site was added to the 5`-end to aid subcloning of the amplified
products. The positions of the antisense oligonucleotides were selected
to cover an internal restriction site allowing convenient substitution
in the original cDNA. PCR was carried out in 30 cycles (94 °C for
60 s; 55 °C for 150 s; 72 °C for 150 s), using 1 µg of cDNA
as template, 80 pmol of each primer, and 5 units of Taq-Polymerase (Promega) under the conditions suggested by the
manufacturer. After subcloning the PCR products, the truncated mutants
were constructed as follows.
For construction of the
-
N
mutant, a HindIII/SphI fragment (151 base pairs) of the
subcloned PCR product was used to substitute for the respective
fragment in the cDNA of the
subunit subcloned in the HindIII/EcoRI sites of pUC BM20. The mutant was
subcloned into the HindIII/SmaI sites of pCMV by
cutting with EcoRI, filling the recessed 3` termini, and
subsequently cutting with HindIII. For expression in Sf9
cells, the pCMV clone was cut with EcoRI/SspI and
subcloned into the EcoRI/SmaI sites of pVL1392.
For construction of the -
N
mutant, the cDNA of
subunit was cut with BamHI/EcoRI, and the resulting fragment coding for
the COOH-terminal part of the subunit was ligated to the BamHI/EcoRI-cut PCR product subcloned in pUC BM20.
For expression in Sf9 cells, the pUC BM20 clone was cut with HindIII, the recessed 3` termini were refilled and, after a EcoRI cut, the mutant was subcloned into the SmaI/EcoRI sites of pVL1393.
For construction of
the -
N
mutant, the cDNA of
subunit cloned into the EcoRI site of pBR322
was cut with SacI/EcoRI, and the resulting fragment
coding for the COOH-terminal part of the subunit was ligated to the SacI/EcoRI-cut PCR product coding for the shortened
NH
terminus subcloned in pUC BM20. The resulting mutant was
subcloned into the HindIII/SmaI sites of pCMV after
cutting with EcoRI, filling of the recessed 3` termini and a HindIII cut. For expression in Sf9 cells, the pCMV clone was
cut with EcoRI and SspI and subcloned into the EcoRI/SmaI sites of pVL1392.
For construction of
the -
N
mutant, the cDNA of
subunit cloned into the EcoRI site of pBR322
was cut with ApaI/EcoRI, and the resulting fragment
coding for the COOH-terminal part of the subunit was ligated to the ApaI/EcoRI-cut PCR product coding for the shortened
NH
terminus subcloned in pUC BM20. The mutant was subcloned
into the HindIII/SmaI sites of pCMV using the EcoRI and HindIII sites of the mutant cDNA clone in
pUC BM20. For expression in Sf9 cells, the NH
-terminal
fragment of the mutant (BglII/NcoI) was used to
substitute for the respective region (BamHI/NcoI) of
the wild type cDNA of
in pVL1393. Construction of the
wild type
and
subunits, generation
of recombinant viruses, and cell culture of Sf9 cells were performed as
described(9) . To obtain cytosolic fractions containing the
recombinant protein, spinner cultures of Sf9 cells grown to 1.5
10
cells/ml were coinfected with combinations of the
respective baculoviruses as the indicated apparent multiplicity of
infection (m.o.i.). Cells were collected by centrifugation 42 h after
infection and resuspended in 40 µl (1 mM EDTA, 2
mM dithiothreitol, 0.2 µM benzamidine, 50 mM triethanolamine hydrochloride, pH 7.4) per ml of the original cell
suspension. After passing the cells 10 times through a 22-gauge needle,
the homogenates were centrifuged for 30 min at 30,000
g. The resulting supernatants (50-100 µg) were
assayed for cyclase activity as described previously (9) in the
presence of 0.1 mM GTP, 3 mM Mg
or
Mn
with or without 100 µMS-nitrosoglutathione (GSNO) unless stated otherwise.
Endogenous cGMP formation of Sf9 cells infected with only the
virus (up to 10 pmol of cGMP
min
mg
) was substracted as control from
recombinant heterodimer activity. Data are shown for typical
experiments performed in duplicate which were repeated at least once.
Western blots were performed as described(10) .
In order to be able to encircle functional domains of soluble
guanylyl cyclase, we analyzed the homology of different regions of the
subunits. As outlined above, each subunit contains a region homologous
to the catalytic domain of the membrane-bound enzyme which is also
conserved in the adenylyl cyclases. This COOH-terminal region is
preceded by a central part of the polypeptide chains with less but
still pronounced homology between guanylyl cyclase subunits. In
contrast, the overall homology of the NH-terminal parts of
the subunits is low. Notable is a stretch of about 100 amino acids
within this NH
-terminal region revealing a significantly
higher degree of identical amino acids between
and
or
and
than
between
and
subunits. In one set of deletions, we omitted
the amino acids preceding this stretch (131 and 64 amino acids of the
and
subunits, respectively) to
investigate the potential function of these very low conserved NH
termini. In another set of mutations, the entire
NH
-terminal half of either subunit was deleted so that the
resulting truncated subunits (
-
N
and
-
N
) contained the
putative catalytic regions. All deletion mutants were coexpressed with
their corresponding wild type subunit in Sf9 cells. In order to obtain
comparable amounts of expressed proteins, the m.o.i. used for the
different viruses was varied, and the expression of the recombinant
proteins was monitored in cytosolic fractions of infected cells in
Western blots using antibodies directed against COOH-terminal peptides
of the
and
subunit. Fig. 1shows the results of a Western blot in which the m.o.i.
used for the respective heterodimers were adjusted. Accordingly, the
expression of the recombinant truncated mutants and the wild type
subunits was quite similar. Like the wild type
subunit (calculated molecular mass of 78 kDa), all deletional
mutants of the
subunit showed a slightly faster
migration on SDS gels than expected from the predicted molecular
masses.
Figure 1:
Immunoblots demonstrating the
expression of the deletional mutants in the cytosol of the infected
cells. Sf9 cells were coinfected with the viruses coding for the
indicated mutants and the respective corresponding wild type subunit. A
m.o.i. of 0.5 was used for the subunit, 1 m.o.i. for
the
subunit, 0.5 m.o.i. for the
-
N
, 0.1 m.o.i. for the
-
N
, 0.5 m.o.i. for
-
N
, and 0.1 m.o.i. for the
-
N
. Cytosolic proteins (25 µg)
were separated on SDS-polyacrylamide gel electrophoresis gels and
blotted on nitrocellulose, peptide antibodies against the COOH termini
of the
and
subunits were used for
detection in all samples except in the sample with the
-
N
mutant where the antibody
against the
subunit was omitted to allow detection of
a single band. The arrows indicate the position of the
respective deletion mutant with calculated molecular masses of 63 kDa
for
-
N
, 36 kDa for
-
N
, 63 kDa for
-
N
, and 36 kDa for
-
N
. The other protein bands
recognized by the antibodies represent the wild type
subunits.
Subsequently, enzyme activity of the modified heterodimers
was determined under basal and NO-stimulated conditions in the presence
of 3 mM Mg or 3 mM Mn
as divalent cations (Table 1). cGMP production by the
wild type in the presence of
Mg
was 50 pmol of cGMP
min
mg
; addition of GSNO resulted in a
90-fold increase in catalytic activity. Among the modified guanylyl
cyclases, only
-
N
revealed
measurable basal activity (40 pmol of cGMP
min
mg
) in the presence of
Mg
. Whereas basal activity was comparable to that of
the wild type enzyme, GSNO did not alter enzyme activity of this
mutant. In contrast,
-
N
exhibited
some sensitivity to NO in the presence of Mg
as the
enzyme showed cGMP formation (10 pmol of cGMP
min
mg
) under stimulated conditions
whereas basal activity was below the detection limit.
The use of
Mn instead of Mg
as divalent cation
led to an increased basal activity of the wild type (400 pmol of cGMP
min
mg
), with the
activator causing only a 10-fold stimulation. These findings are in
accordance with earlier results(11) . Under these conditions,
all modified heterodimers were catalytically active but insensitive to
NO. Basal activity of
-
N
(503 pmol
of cGMP
min
mg
)
was again in the range of the wild type enzyme, whereas cGMP formation
of the other mutant enzymes was reduced to 9-22% of wild type
activity (see Table 1). After establishing intact basal activity
of
-
N
but insensitivity to NO, this
mutant was further characterized. To compare the kinetic properties of
this mutant with the wild type enzyme, cGMP formation was determined in
the presence of increasing GTP concentrations. A Lineweaver-Burk plot
of the data revealed similar apparent K
values of 106 and 110 µM GTP for
and
-
N
, respectively (Fig. 2), suggesting proper folding of the catalytic domain.
Figure 2:
Comparison of kinetic properties of the
wild type and the -
N
heterodimers.
Shown are double reciprocal plots of cGMP formation as a function of
MnGTP concentrations in the cytosol of Sf9 cells coinfected with
viruses coding for the
(0.5 m.o.i.) plus
subunits (1 m.o.i.) and
(0.5 m.o.i.) plus
-
N
(0.25 m.o.i.). Preparation of
cytosol and determination of cyclase activity were as described under
``Experimental Procedures.''
Since the -
N
heterodimer is not
activated by the NO-releasing compound GSNO nor by protoporphyrin IX
(data not shown), we tested whether the mutant could function as a
dominant-negative protein inhibiting only the stimulated wild type
enzyme while leaving the basal activity unchanged. For this purpose,
Sf9 cells were infected with the viruses coding for wild type subunits
as well as with increasing amounts of the virus coding for the
-
N
mutant. Parallel to the
determination of the catalytic activity, we performed Western blots of
the different samples to ensure that the loss of stimulated activity
was not due to a decrease in expression of the wild type subunits (Fig. 3B). Catalytic activity of the coexpressed
subunits in cytosolic fractions was determined under basal and
stimulated conditions (100 µM GSNO) in the presence of 3
mM Mn
. As shown in Fig. 3A,
increasing amounts of
-
N
virus led
to a decline of the stimulated activity to 60, 37, 29, 23, and 20% of
the original activity (5200 pmol of cGMP
min
mg
) of the wild type enzyme, whereas
basal cGMP formation remained constant over a range of 10 to 30 pmol of
cGMP
min
mg
.
Figure 3:
Dominant-negative effect of the
-
N
mutant on the stimulated activity
of the wild type. Sf9 cells were coinfected with viruses coding for the
(0.1 m.o.i.) and
subunits (1
m.o.i.) alone (WT) or in the presence of increasing amounts of the
virus coding for the
-
N
mutant
(0.02, 0.04, 0.06, 0.08, and 0.10 m.o.i.) or with the viruses coding
for the the
(0.1 m.o.i.) and the
-
N
mutant (0.05 m.o.i.). A,
cyclase activity was determined in the presence of 3 mM Mn
as described under ``Experimental
Procedures.'' B, shown are immunoblots of the same
cytosolic protein preparations that were used for the determination of
enzymatic activity. Cytosolic proteins (30 µg) were separated on
SDS-polyacrylamide electrophoresis gels and blotted onto
nitrocellulose, peptide antibodies against the COOH termini of the
and
subunits were used for
detection of the wild type subunits and the
-
N
mutant.
In the present study, we demonstrate that coexpression of the
COOH-terminal halves of the and
subunits of soluble guanylyl cylcase, comprising the region
conserved in all cyclases, yields an enzyme sufficient for the
formation of cGMP but insensitive to NO. Preliminary results indicate
that COOH-terminally truncated mutants are catalytically inactive, thus
further underlining the importance of the catalytic consensus domain.
Our results are in good agreement with reports on an
NH
-terminally truncated GC-A receptor mutant exhibiting
ligand-independent cGMP production. In the deletional mutant of the
GC-A receptor, the cyclase catalytic domain is preceded by the
so-called hinge region (47 amino acids) and a small portion (15 amino
acids) of the carboxyl segment of the consensus protein kinase-like
domain. These additional amino acids were shown to be essential for a
properly folded catalytically active site, and it was suggested that
these sequences may be required for dimerization (12) . In a
recent report, the 43 amino acids in front of the catalytic domain have
indeed been shown to be required for dimerization(13) . In
analogy, the 80 additional amino acids NH
-terminal of the
putative catalytic region present in the COOH-terminal halves of the
and
subunits may be required for
proper folding or dimerization of the subunits. Dimerization of the
subunits is a prerequisite for enzyme activity since expression of one
subunit or a truncated mutant did not yield any cGMP forming activity.
The various adenylyl cyclases also contain two cyclase homology
domains which in contrast to soluble guanylyl cyclase are localized on
one polypeptide chain. Similarily as in soluble guanylyl cyclase, both
domains present in adenylyl cyclases are required for catalysis as the
separate expression of either region results in a loss of enzyme
activity(14) . Moreover, the ability of the
NH-terminally truncated GC-A receptor mutant to dimerize
coincided with intact catalytic activity, further underlining the
necessity of two catalytic consensus domains. The significance of the
association of two different catalytic domains in soluble guanylyl
cyclases and adenylyl cyclases as opposed to the existence of two
identical catalytic domains in the membrane bound guanylyl cyclases,
however, is unknown.
Besides the identification of the catalytic
domain, we show that even the very low conserved
NH-terminal part of the
subunit (64 amino
acids) is required for the stimulation by NO. Recently, we identified
histidine 105 of the
subunit as a likely candidate to
be the residue forming a linkage to the central iron atom of the
prosthetic heme group, since substitution of this histidine 105 by
phenylalanine yielded an NO-insensitive enzyme lacking the prosthetic
heme group(9) . Although histidine 105 of the
subunit is present in the mutant
-
N
, the loss of NO sensitivity
suggests disturbance of either a configuration required for heme
binding or lack of structures involved in the transduction of the
stimulatory effect to the catalytic center. Deletion of the
NH
-terminal part of the
subunit (131
amino acids) yielded an enzyme still activated by NO, although the
non-responsiveness in the presence of Mn
suggests a
severe impairment of NO stimulation. As further deletion of the
subunit also destroys NO activation of the enzyme, we
conclude that the NH
-terminal regions of the
and
subunits are responsible for the regulation
of the enzyme. Characterization of the purified mutants will reveal
whether those are still able to bind heme or whether the mediation of
the stimulatory signal is impaired. When coexpressed with the wild type
enzyme, the NH
-terminally truncated
subunit inhibits only the stimulated activity but leaves the
basal activity unchanged. The ability of the mutant to compete with the
wild type
subunit for dimerizing with the
subunit further emphasizes the NH
-terminal location
of the regulatory domain. The dominant-negative effect of the mutant on
the stimulated activity will allow the selective inhibition of nitric
oxide-dependent cGMP formation in intact cells. Hence, the mutant will
be a helpful tool in forthcoming studies of the NO-cGMP signaling
system and, in this regard, the retained ability of basal cGMP
formation is probably beneficial when compared to the recently
identified dominant-negative mutants that knock out guanylyl cyclase
activity completely(16) .
Our results show that the two
defining features of soluble guanylyl cyclase, namely the catalytic
activity and the regulation by NO, can be attributed to different
regions on the subunits and that the catalytic domain can be expressed
separately. Similar results exist for the bacterial protein FixL, a
dimeric hemoprotein kinase whose enzymatic activity is reversibly
blocked by oxygen binding to the heme. There the heme binding domain
and the phosphotransferase activity were also attributed to the
NH-terminal and COOH-terminal regions, respectively; they
still contained their heme-dependent oxygen-binding and catalytic
properties when expressed separately(15) . These findings
suggest that during evolution, regulatory segments have been connected
to distinct catalytic domains. In the case of guanylyl cyclases, a
common cGMP-forming catalytic unit occurs under the control of
different regulatory domains; in that respect, the regulatory
heme-containing domain of the soluble enzyme appears to represent the
equivalent to the ligand-binding and kinase-like domains of the
membrane bound guanylyl cyclases.