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J Biol Chem, Vol. 274, Issue 26, 18149-18152, June 25, 1999
From the Department of Pharmacology and Toxicology,
Julius-Maximilians-University, D-97078 Wuerzburg, Germany
Soluble guanylyl cyclase (sGC) is an
Nitric oxide plays an important role as an intercellular messenger
in a great variety of physiological processes (1, 2). To mediate these
effects, NO binds to and regulates several proteinaceous and
nonproteinaceous cellular targets. The presently best characterized signal-transducing receptor for NO is the heme-containing enzyme soluble guanylyl cyclase (sGC)1
(3-5). Upon binding of NO to the
prosthetic heme group, sGC catalyzes the conversion of GTP to cGMP,
which in turn regulates various effector proteins, such as protein
kinases, phosphodiesterases, and ion channels (6, 7).
Native sGC purifies as a heterodimeric complex composed of a larger Whereas active pGCs are formed by homodimerization, i.e.
association of identical CHDs (14), sGC activity depends on
heterodimerization. Only the coexpression of The central parts of sGC Using glutathione S-transferase (GST)-tagged recombinant
human sGC subunits expressed in a baculovirus expression system, we
here present a single-step purification method for recombinant human
sGC (rhsGC) and its subunits, which enabled us to analyze the
oligomerization behavior of the sGC subunits. Here we demonstrate for
the first time the formation of homodimeric yet inactive sGC complexes.
The possible physiological implications for regulation of sGC activity
in intact cells are discussed.
Materials--
GSH was purchased from Roche Molecular
Biochemicals; cell culture materials were from Life Technologies, Inc.
(Eggenstein, Germany). All other chemicals were of the highest purity
grade available and obtained from either Sigma Chemicals (Deisenhofen, Germany) or Merck AG (Darmstadt, Germany). Water was deionized to 18 M Baculovirus Construction--
cDNAs comprising the complete
coding sequences for hsGC Sf9 Cell Culture and Production of rhsGC--
Sf9
cells were cultured as described (17). For expression of nontagged
rhsGC subunits, spinner cultures (2 × 106 cells
ml GSH-Sepharose Affinity Chromatography--
Glutathione-Sepharose
4B (Amersham Pharmacia Biotech, Freiburg, Germany) was equilibrated
with lysis buffer (see above) containing 75 mM NaCl,
incubated with crude supernatant fractions of rhsGC-containing Sf9 cells for 1 h at 25 °C in a rotation mixer, and
washed two or three times with lysis buffer containing 75 mM NaCl. For GSH elution, GSH-Sepharose was incubated with
5 mM GSH in 50 mM Tris-HCl, pH 8.0, for 5 min
at 25 °C. Fractions were brought to a final concentration of 10%
(v/v) glycerol and kept at Size Exclusion Chromatography--
Crude rhsGC-containing
Sf9 supernatant fractions were subjected to fast protein liquid
chromatography on a Superose 6 column (Amersham Pharmacia Biotech) at a
flow rate of 0.2 ml min sGC Activity Assay--
sGC activity was measured as the
formation of cGMP at 37 °C for 10 min in a total volume of 100 µl,
containing 50 mM triethanolamine HCl, pH 7.4, 3 mM GSH, 1 mM 3-isobutyl-1-methylxanthine, 5 mM creatine phosphate, 0.25 mg ml Western Blot--
Immunodetection of nontagged and GST-tagged
rhsGC subunits was performed as described previously (17), using
polyclonal antibodies raised against peptide sequences that correspond
to hsGC To examine sGC subunit association, we established a rapid and
efficient method to purify rhsGC and its subunits. Fusion proteins composed of a GST affinity tag (25 kDa), and the recombinant human sGC As shown in Fig. 1A, the
recombinant GST-rhsGC As shown in Fig. 1B, the GST-tagged sGC subunits retained
their ability to heterodimerize with the respective complementary nontagged subunit. When coexpressed in Sf9 cells, nontagged
COMMUNICATION
Homodimerization of Soluble Guanylyl Cyclase Subunits
DIMERIZATION ANALYSIS USING A GLUTATHIONE
S-TRANSFERASE AFFINITY TAG*
,
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/
-heterodimeric hemoprotein that, upon interaction with the
intercellular messenger molecule NO, generates cGMP. Although the
related family of particulate guanylyl cyclases (pGCs) forms active
homodimeric complexes, it is not known whether homodimerization of sGC
subunits occurs. We report here the expression in Sf9 cells of
glutathione S-transferase-tagged recombinant human sGC1
and 1 subunits, applying a novel and rapid purification method based
on GSH-Sepharose affinity chromatography. Surprisingly, in intact
Sf9 cells, both homodimeric GST/ and GST/ complexes
were formed that were catalytically inactive. Upon coexpression of the
respective complementary subunits, GST/ or GST/
heterodimers were preferentially formed, whereas homodimers were still
detectable. When subunits were mixed after expression, e.g.
GST and or GST and , no dimerization was observed. In conclusion, our data suggest the previously unrecognized possibility of
a physiological equilibrium between homo- and heterodimeric sGC complexes.
INTRODUCTION
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(~80 kDa) and a smaller subunit (~70 kDa) (3, 8). The
N-terminal domains of both subunits are essential for the stimulation
of the enzyme by NO (9, 10), whereas heme-binding occurs solely in the
subunit (11-13). Both the and subunit contain a C-terminal
cyclase homology domain (CHD), which, in analogy to adenylyl cyclases
and pGCs, constitutes a bipartite catalytic center by the association
of the and C-terminal domains (4, 14).
and subunit
cDNAs in heterologous expression systems constitutes active sGC
(15-17), whereas separate expression of or subunits yields
neither NO-sensitive nor basally active enzyme. Moreover, sGC activity
could not be restored by mixing of the expressed subunits (15).
and (9) share extensive homologies with
each other and with a 43-amino acid sequence in pGC that is essential
for homodimerization (18). This prompted us to speculate whether
homodimer formation of sGC or sGC may also occur. It is, however,
unknown whether / and/or / homodimers can assemble
intracellularly or whether separately expressed sGC subunits stay
monomeric in the absence of a complementary subunit. This would prevent
the formation of a "two-CHD" catalytic center and thereby explain
the lack of cGMP formation.
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cm (Milli-Q; Millipore, Eschborn, Germany).
and subunits (17, 19) were cloned into
the pAcG2T baculovirus transfer vector (Pharmingen, San Diego, CA),
which allows the expression of GST-sGC fusion proteins and provides a
thrombin cleavage site for proteolytic removal of the GST tag. For
in-frame cloning, BamHI sites (underlined below) were
introduced by polymerase chain reaction immediately upstream of
the translational start sites. Fragments were amplified with the primer
pairs 5'-AAAAGGATCCATGTTCTGCACGAAGCTC-3' (bp 524-541) and
5'-ATTATGGAAGCAGGGAGG-3' (bp 1249-1232) for 1 and
5'-AAAAGGATCCATGTACGGATTTGTGAAT-3' (bp 89-106) and
5'-ATGCGTGATTCCTGGGTACC-3' (bp 711-692) for 1. Products were
cut with BamHI/BsaAI (1) or BamHI/KpnI (1) and ligated with
BsaAI/EcoRI (bp 1193-3015, 1) or
KpnI/EcoRI (bp 692-2444, 1) cDNA
fragments to the BamHI/EcoRI-cleaved vector.
Recombinant GST-hsGC1 and GST-hsGC1 baculoviruses were isolated
as described for hsGC1 and hsGC1 baculoviruses (17).
1) were infected with recombinant baculoviruses coding
for hsGC1 or hsGC1 (17) at a multiplicity of infection (m.o.i.)
of 5 plaque-forming units/cell. This high m.o.i. ensured that nearly all cells (>99%) were infected with the viruses encoding the
nontagged subunits, which is necessary in the triple expression
experiment shown in Fig. 2D (see "Results"). Infections
with GST-hsGC and GST-hsGC viruses (see above) were performed at
an m.o.i. of 0.5 plaque-forming units/cell, because expression levels
of the GST-tagged rhsGC subunits were substantially higher (data not
shown). Infection of cells with a baculovirus coding for GST (kind gift
from C. Weber, Institut für Medizinische Strahlenkunde und
Zellforschung, Würzburg) were performed at a m.o.i. of 2.5 plaque-forming units/cell. Cells were harvested 72 h post
infection, and all subsequent procedures were performed at 4 °C.
Cells were lysed for 15 min on ice in hypotonic lysis buffer (25 mM triethanolamine, pH 7.8, 1 mM EDTA, 5 mM dithiothreitol, 1 µM leupeptin, 0.5 µg
ml1 soy bean trypsin inhibitor), and crude supernatant
and particulate fractions were separated by centrifugation (20,000 × g) for 15 min at 4 °C. Supernatant fractions were
brought to a final concentration of 75 mM NaCl; for storage
at 
20 °C, a final concentration of 10% (v/v) glycerol was used.
Protein concentrations were determined according to Bradford (20),
using bovine serum albumin as a standard.
20 °C.
1 in 50 mM Tris-HCl,
pH 6.7, 300 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol. Aliquots of each fraction were assayed
for rhsGC-immunoreactive protein by Western blot. Signals were
quantitated by flatbed scanning and densitometry using the NIH Image
software (Division of Computer Research and Technology, National
Institutes of Health, Bethesda, MD). The column was calibrated with
standard proteins (Sigma) of known Stoke's radii: thyroglobulin (8.5 nm), apoferritin (6.1 nm), alcohol dehydrogenase (4.55 nm), bovine
serum albumin (3.55 nm), and carbonic anhydrase (2.01 nm).
1 creatine
kinase, 500 µM GTP, and either 3 mM
MgCl2 or 3 mM MnCl2. Reactions were
started by adding the enzyme-containing fraction and immediately
thereafter the sGC activator sodium nitroprusside (100 µM). The cGMP content was determined by an enzyme-linked immunoassay (Biotrend, Cologne, Germany). Results are expressed as the
means ± S.E. of at least three experiments.
1 (amino acids 634-647) and hsGC1 (amino acids 593-614), which were affinity-purified against the respective peptides. Blots
were developed using the ECL detection system (Amersham Pharmacia
Biotech) according to the manufacturer's protocol.
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1 and 1 subunits (rhsGC1, 79.5 kDa, and rhsGC1, 68, 5 kDa (17)) were constructed and expressed in the baculovirus/Sf9 cell system.
1 and GST-rhsGC1 (GST, GST) fusion
proteins migrated with the expected apparent molecular masses of 105 and 94 kDa, bound to GSH-Sepharose, and were specifically eluted with
GSH (lanes 2-5). In contrast, the nontagged rhsGC1 ()
and rhsGC1 () subunits did not bind to the GSH beads (lanes
6-9). In addition to the full-length recombinant proteins, some
sGC-immunoreactive degradation products were recognized in crude
Sf9 lysates (lanes 2, 4, and
6), which did not appear in lysates from noninfected
Sf9 cells (lane 1).
View larger version (13K):
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Fig. 1.
Expression and heterodimerization of
GST-tagged and nontagged rhsGC 1 and
1 subunits. Crude supernatant fractions of
Sf9 cells expressing GST-tagged rhsGC1 (GST),
GST-tagged rhsGC1 (GST), nontagged rhsGC1
(), and/or 1 () were subjected to
GSH-Sepharose affinity chromatography (see "Experimental
Procedures"). Load and eluate fractions were analyzed by Western
blot, which was developed simultaneously with 1- and 1-specific
antibodies (see "Experimental Procedures"). A, GST
and GST specifically bind to GSH-Sepharose. GST, GST, , and
were expressed separately. Sf9, crude supernatant
fraction of noninfected Sf9 cells. B,
heterodimerization of GST-tagged rhsGC. GST and (GST and )
were coexpressed (lanes 1-4) or mixed after separate
expression and incubated for 15 min at room temperature prior to
GSH-Sepharose binding (lanes 5-8). L, load;
E, GSH eluate.
co-eluted with GST and vice versa (lanes
1-4), indicating a direct physical interaction and demonstrating
that addition of the GST tag did apparently not interfere with the
dimerization function. Whereas coexpression of GST and subunits
yielded active and NO-sensitive sGC in crude Sf9 cell
supernatant fractions, only basal sGC activity was obtained upon
coexpression of GST and (Table I).
Apparently, GST tagging of the sGC N terminus interfered with NO
stimulation, probably because of the close proximity of the GST to the
heme-binding site (11-13). GST/ activity in the presence of
Mg2+ was very similar to that of nontagged rhsGC expressed
in the same system (17). In the presence of Mn2+, basal sGC
activity was increased, whereas NO-stimulated activity was decreased
(Table I), as reported for native (21) and recombinant sGC (9).
Specific basal sGC activity was dramatically increased in the GSH
eluate fraction (Table I), similar to sGC/ prepared by multi-step
purification procedures (8, 22-24). Moreover, NO sensitivity of
purified GST/ was preserved, because the enzyme was activated
30-fold by 100 µM sodium nitroprusside (Table I).
Specific activities of rhsGC dimers
or GST were coexpressed with or in Sf9 cells.
Basal and sodium nitroprusside (100 µM)-stimulated
specific sGC activities were determined in crude Sf9 cell
supernatant fractions in the presence of 3 mM Mg2+
or 3 mM Mn2+. sGC activities are given as mean ± S.E. of at least three experiments performed in duplicate. The
corresponding stimulation factors are provided (-fold). NA, not
applicable.
Heterodimerization of both GST/ and GST/ complexes was
dependent on coexpression of the respective subunits (Fig.
1B, lanes 5-8). Accordingly, no basal or
NO-stimulated sGC activity was detected in the load or eluate fractions
derived from separately expressed and mixed GST and (or GST
and ) subunits (data not shown), which was in agreement with
previous findings on nontagged and subunits (15)
When GST/ and GST/ were coexpressed in Sf9 cells,
none of the crude cell lysates contained any detectable sGC activity (i.e. <30 pmol cGMP mg1 min1;
Table I), neither with Mg2+ nor Mn2+. To
investigate whether sGC subunits homo-oligomerized, fractions were
analyzed by Western blot. Interestingly, nontagged efficiently copurified with GST, demonstrating a direct physical interaction between sGC subunits (Fig.
2A, lanes 3 and
4). This / homodimerization was dependent on
coexpression of both subunits (lanes 7 and 8). Similarly, the nontagged subunit copurified with GST. However, binding of GST to the GSH-Sepharose was weakened when nontagged was coexpressed (Fig. 2A, lanes 1 and
2), requiring longer exposure times during Western blot
analysis (Fig. 2B, upper panel). In contrast,
nontagged , which was expressed separately and mixed with GST,
had no effect on binding of GST to the beads (Fig. 2A,
lanes 5 and 6). This demonstrated that nontagged
interacted with GST upon coexpression, leading to an
interference with binding to GSH-Sepharose, possibly because of steric
hindrance. Coelution of and with the respective GST-tagged
subunit did not result from unspecific binding to the GSH-Sepharose,
because the wash steps prior to elution did not contain any nontagged
sGC (Fig. 2B). Likewise, and did not coelute with
GST alone (Fig. 2C). Therefore, coelution of with GST
and of with GST represent specific protein-protein interactions
of identical rhsGC subunits.
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To analyze whether single rhsGC subunits form dimeric (as does /)
or multimeric complexes, crude Sf9 supernatant fractions containing rhsGC were subjected to size exclusion chromatography (Fig.
3). To exclude any potential artifacts
caused by the GST tag, nontagged subunits were applied in this set of
experiments. As shown in Fig. 3A, coexpressed rhsGC and
subunits perfectly co-eluted upon size exclusion chromatography,
with a Stoke's radius of 5.3 nm (Fig. 3A,
inset). A similar Stoke's radius (4.8 nm) was reported for
heterodimeric sGC purified from rat lung (3). Importantly, rhsGC and
rhsGC, when expressed separately, showed an elution pattern very
similar to heterodimeric sGC (Fig. 3B), suggesting that both
subunits indeed exist as homodimers. However, a fraction of rhsGC
(<20%) formed aggregates under these conditions (Fig. 3B,
fractions 2-6). The peaks of rhsGC and rhsGC were slightly separated, reflecting apparent differences in the Stoke's radii between the / and / homodimers (Fig.
3B).
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To further investigate whether sGC homodimer formation can occur also
in the presence of complementary subunits, Sf9 cells were
cotransfected with three viruses coding for GST, , and (or
GST, and ). Viruses coding for and were applied at a
high m.o.i. to ensure that nearly all cells (>99%) expressing GST
(or GST) were simultaneously infected with both and viruses.
As shown in Fig. 2D, heterodimer (i.e. GST/
or GST/), formation is preferred under these conditions in insect
cells. However, about 10% of the recombinant protein formed GST/
or GST/ complexes even in the presence of the respective
complementary subunits. This demonstrated the existence of an
equilibrium between homo- and heterodimeric sGC.
Based on these data, sGC and subunits are in fact capable of
forming homodimeric complexes in intact Sf9 cells. It has to be
clarified whether classical purification methods efficiently remove
homodimeric sGC from crude preparations of recombinant sGC or whether
apparently homogenous sGC preparations contain "silent" homodimeric
sGC. With the novel purification method presented here, only GST/
heterodimers will be purified, because / does not bind to the column.
There are different potential mechanisms that might underlie the
observed preferential heterodimer formation in Sf9 cells. Homodimer formation may be suppressed in intact cells because of a much
higher affinity between complementary subunits. So far, no data on sGC
dimerization kinetics and apparent KD values are
available. However, the high stability of homodimeric sGC upon mixing
of separately expressed subunits, which seems to be similar to that of
heterodimeric sGC, argues against substantial affinity differences. On
the other hand, sGC dimerization might be a regulated process in living
cells. The existence of at least two different isoforms of each subunit
(1, 2, 1, and 2) led to the concept that sGC activity is
regulated in vivo by alternative heterodimerization (4, 5).
It is an intriguing possibility that regulation of sGC activity
in vivo might involve not only alternative
heterodimerization but also changes in the extent of homodimerization.
Based on our data, it cannot be excluded that a further protein is
associated with homodimeric sGC complexes, which may be involved in
complex formation and therefore be a functional inhibitor of sGC
activity. An endogenous inhibitor of sGC has been described in Ref. 25.
Interestingly, both homo- and heterodimerization depend on co- or
post-translational processes. It has been suggested that
chaperone-mediated formation of hetero-oligomeric protein complexes is
involved in the regulation of signaling pathways (26). A related
process may regulate sGC protein-protein interactions and thus NO/cGMP signaling.
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ACKNOWLEDGEMENTS |
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We thank Dr. Cornelius Krasel for helpful discussions and Dr. Christoph Weber for providing a baculovirus for GST expression.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB355/C7.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.
To whom correspondence should be addressed: Dept. of Pharmacology
and Toxicology, Julius-Maximilians-University, Versbacher Str. 9, D-97078 Wuerzburg, Germany. Tel.: 49-931-201-3992; Fax: 49-931-201-3539; E-mail: medk311{at}rzbox.uni-wuerzburg.de.
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ABBREVIATIONS |
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The abbreviations used are:
sGC, soluble
guanylyl cyclase;
rhsGC, recombinant human sGC;
cGMP, 3',5'-cyclic
guanosine monophosphate;
CHD, cyclase homology domain;
GSH, glutathione;
GST, glutathione S-transferase;
GST, GST-tagged rhsGC1;
GST, GST-tagged rhsGC1;
pGC, particulate
guanylyl cyclase;
bp, base pairs;
m.o.i., multiplicity of
infection.
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, rhsGC
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