(Received for publication, June 5, 1995)
From the
A variant of the subunit of soluble guanylyl
cyclase (
) containing 31 additional amino acids was
identified in a number of cell lines and tissues. The in-frame sequence
of the insert was within the proposed catalytic domain of guanylyl
cyclases and was homologous to a region within the putative catalytic
domain of adenylyl cyclases. Messenger RNA for the new variant was
detected in some but not all cell lines and tissues expressing the
subunit. The novel form, as well as the
subunit lacking the insert, were coexpressed with the
subunit in Sf9 and COS-7 cells;
/
coexpression yielded a NO-sensitive recombinant protein, whereas
the coexpressed
/
subunits exhibited
no guanylyl or adenylyl cyclase activities. Because both subunits
(
/
) copurified, the novel variant
retains its ability to heterodimerize. In coexpression experiments, the
subunit competed with the
subunit
for dimerization with the
subunit, thereby reducing
/
-catalyzed guanylyl cyclase
activity. These data show that the novel variant functions as a
dominant negative protein and that post-transcriptional mRNA processing
represents a potential mechanism for regulation of NO-sensitive
guanylyl cyclase acitivity.
Guanylyl cyclases (GTP pyrophosphate-lyase (cyclizing), EC
4.6.1.2) exist in membrane-bound and cytoplasmic forms(1) . The
known membrane-spanning forms are cell surface receptors for
extracellular peptides and appear to exist as homodimers or other
higher ordered structures. The cytoplasmic or soluble forms represent
heme-containing heterodimers that are directly activated by nitric
oxide. The cDNAs of two and two
subunits
(
,
,
, and
) have been
identified(2, 3, 4, 5, 6, 7) .
All subunits contain a putative catalytic domain homologous to the
membrane-bound guanylyl cyclases and to the two putative catalytic
domains of adenylyl cyclases (C
and
C
)(2, 4, 5) . A heterodimer
between an
and a
subunit appears to be required for enzyme
activity(8, 9) .
The mechanisms by which the
soluble or membrane forms of guanylyl cyclase are regulated have been
explored at both the protein and the gene level. In the case of the
plasma membrane guanylyl cyclase A, an adenine nucleotide is required
for hormonal stimulation of cyclase
activity(10, 11, 12) . Guanylyl cyclase A, as
well as other membrane forms of guanylyl cyclase, is also known to be
regulated by
phosphorylation/dephosphorylation(13, 14, 15) .
A soluble form of guanylyl cyclase has been shown to be phosphorylated,
but the functional significance remains unclear(16) . At the
transcriptional level, regulation of expression or differential
expression of the and
subunits of soluble guanylyl cyclase
has been demonstrated(17, 18) .
Differential RNA
splicing can serve to switch an active to an inactive protein, the most
impressive example being the cascade of alternative splicing events
that control somatic sexual differentiation in Drosophila(19) . A naturally occurring variant of FosB
generated by alternative splicing (FosB) has been shown to inhibit
Fos/Jun transcriptional activity(20) . This dominant negative
effect is presumably due to competition with FosB at the step of
heterodimer formation with Jun. Similarly, helix-loop-helix proteins
have been described that negatively regulate other helix-loop-helix
proteins through the formation of nonfunctional heterodimeric
complexes(21) . Modulation of enzyme activity by alternative
splicing within the catalytic region has been suggested for
protein-tyrosine phosphatases (22, 23) .
Here, we
present a variant form of the subunit of soluble
guanylyl cyclase that contains an in-frame insert of 31 amino acids
within the catalytic domain. The insert is homologous to a region
within the apparent catalytic domain of adenylyl cyclases. Although the
new variant heterodimerizes with the
subunit, an
active enzyme is not formed. Thus, the
subunit
effectively competes with
lacking the insert, thereby
functioning in a dominant negative manner. This suggests that guanylyl
cyclase activity can be regulated at the level of RNA processing.
HL-60 cell cDNA was amplified in the polymerase chain
reaction using degenerate primers (A and B) corresponding to highly
conserved regions of guanylyl cyclases. Among the subcloned PCR
products, one clone () corresponded to the
subunit with an additional in-frame insert of 93 base
pairs. Independently, an identical clone was isolated using Y-79 cell
cDNA. In both cell lines, cDNAs with or without insert were found. The
nucleotide sequence did not reveal branch site nor donor or acceptor
pre-mRNA consensus splicing signals, excluding the possibility of
unprocessed pre-mRNA(32) . Identification of the remnants of
the donor consensus that remain in the mRNA after splicing strongly
suggests alternative inclusion of an additional exon (32) (Fig. 1A).
Figure 1:
Nucleotide sequence of the
additional exon in the subunit and amino acid
alignment. A, given is the nucleotide sequence and the deduced
amino acid sequence of the additonal exon in the
subunit (underlined). The letters printed in bold correspond to the remnants of the donor consensus sequences that
remain in the mRNA after splicing ((C/A)AG). B, shown is a
comparison of a sequence within the novel variant exon of the
subunit (sGC
) to the analogous
region within the apparent catalytic domain of adenylyl cyclases (AC I-VIII, C
-domain). Identical residues are shaded. The accession numbers with the position of the aligned
amino acids are as follows: sGC
, 620-639; type
I, Swissprot Q08828, 674-693; type II, Swissprot Q08462,
314-333; type III, Swissprot P21932, 1019-1038; type IV,
Swissprot P26770, 951-970; type V, Swissprot Q04400, 991-1010;
type VI, PIR A46187, 1059-1078; and type VIII, PIR PQ0227,
490-509.
The 31 additional deduced
amino acids are inserted after amino acid 612 of the subunit within the apparent catalytic region. Amino acid
alignments of the catalytic regions of guanylyl and adenylyl cyclases
show a gap in the same position(33) . This prompted us to look
for further sequence homologies. Within the same region of the
catalytic consensus sequence of adenylyl cyclases (C
domain), a sequence homologous to the insert was apparent (Fig. 1B). The homology was further evaluated by the
method of Needleman and Wunsch(34) , as implemented by Dayhoff (35) and Doolittle et al.(36) using the
computer program PCOMPARE (PCGENE 6.60, IntelliGenetics Inc.). The
comparisons of the insert in the
subunit with the
analogous sequences in human type 1 and type 2 adenylyl cyclases
yielded statistically significant alignment scores (4.3 and 3.9,
respectively, using the genetic code matrix and a number of random runs
of 100).
Preliminary reverse transcription PCR studies showed an
almost ubiquitous expression of the subunit along
with the
and
subunits (data not
shown). To test whether alternative splicing of
pre-mRNA occurs in all or only in some cell lines or tissues,
cDNAs containing
mRNA were chosen (Fig. 2, upper panel) and analyzed with respect to the presence of
mRNA. Messenger RNA coding for the
subunit could be demonstrated in human cDNA libraries from liver,
colon, and endothelium (Fig. 2, lower panel). The
alternatively spliced transcript
was also present in
the cDNAs of the human promyelocytic leukemia cell line HL-60, in the
human retinoblastoma cell lines Y-79 and WERI, and in the human
megakaryocytic leukemia cell line MEG01. In contrast, mRNA coding for
the
subunit was neither detectable in human cDNA
libraries from fetal brain, testis, and the Jurkat T-cell line nor in
the human erythroleukemia cell lines HEL and K562.
Figure 2:
Comparison of and
expression in various human tissues and cell lines.
PCR was performed using
-specific primers C and D (upper panel) and
-specific primers E and F (lower panel). Reverse transcribed mRNA (5 ng) of the
respective cell lines or cDNA library solution
(10
-10
plaque-forming units) of the
respective tissues were used as template. PCR reactions were carried
out as described under ``Experimental Procedures.'' Products
were separated on a 1% (w/v) agarose gel, blotted, and detected with an
- (upper panel) or an
-specific probe (lower panel). bp,
base pairs.
Expression of the
subunit in COS-7 cells yielded a protein with an
apparent molecular mass of 81 kDa that was recognized by polyclonal
antibodies against the carboxyl terminus of the
subunit (data not shown). However, coexpression of the novel
variant with the
subunit did not result in detectable
basal or nitric oxide-stimulated guanylyl cyclase activity in the COS
expression system (data not shown). Because higher expression levels
can be achieved in the Sf9-baculovirus expression system, coexpression
experiments of the
/
,
/
, and
/
subunits were performed. As shown in Table 1, the highest
guanylyl cyclase activity was demonstrated in the cytosol from Sf9
cells coinfected with the viruses coding for the
and
subunits. In comparison, the
/
heterodimer exhibited
3-9-fold lower activity, whereas no guanylyl cyclase activity
could be detected in Sf9 cells infected with the
and
viruses. Because formation of cAMP has been reported
for guanylyl cyclase prepared from rat liver (37) , adenylyl
cyclase activity was also assessed (see Table 1). Adenylyl
cyclase activity was at least 1 order of magnitude lower than guanylyl
cyclase activity. It was highest in the presence of Mn
and NO. Again, the
/
enzyme
displayed higher activity than the
/
form, whereas no adenylyl cyclase activity could be detected for
the coexpressed
and
subunits.
To test whether the subunit retains its ability
to form a heterodimer, the
subunit was coexpressed
with a modified
subunit containing an amino-terminal
hexahistidine tag. Subsequently, a two-step purification over DEAE
anion exchange and Ni
-nitrilotriacetic acid-agarose
columns was performed. The purified protein was analyzed on a 8% (w/v)
polyacrylamide gel and revealed two major bands (Fig. 3, lane d). The apparent molecular masses corresponded well with
those of the
and the
subunits. The
identity of the purified proteins as
and
was additionally verified by immunoblotting (data not shown).
Figure 3:
SDS-polyacrylamide gel of the purified
/
heterodimer. Shown is a Coomassie
Blue-stained 8% (w/v) polyacrylamide gel. Lane a, molecular
mass markers; lane b, 10 µg of protein of a 100,000
g supernatant of recombinant baculovirus-infected Sf9
cells; lane c, 10 µg of protein of pooled fractions after
DEAE chromatography; lane d, 2 µg of protein of purified
/
heterodimer.
The subunit can apparently form heterodimers with
the
subunit. Therefore, we investigated whether the
novel variant could compete with the
subunit for
dimerization with the
subunit, thereby exerting a
dominant negative effect. In fact, coexpression of increasing amounts
of the
subunit with constant amounts of the
and
subunits in Sf9 cells resulted
in a decrease of both basal and nitric oxide-stimulated enzyme activity (Fig. 4A). To rule out that this inhibition was merely
due to a decrease in the expression level of the
or
subunits, the expression levels were monitored by
immunoblotting. Fig. 4B shows the constant expression
of the
(79 kDa, upper panel) and
subunits (61 kDa, lower panel), which is not affected by
the increasing expression of the
subunit. Due to its
slightly higher molecular mass (81 kDa), the band representing the
subunit can be identified closely above the
subunit (see Fig. 4B, lane
2-7, upper panel). The lower apparent molecular
mass of the
subunit (61 kDa versus 70 kDa in Fig. 3) is caused by the addition of 4 M urea to the 8%
(w/v) polyacrylamide gel that had to be used to separate the
and
subunits.
Figure 4:
Dominant negative effect of the
subunit on basal and NO-stimulated guanylyl cyclase
activity. Sf9 cells (15
10
) were coinfected with
recombinant baculoviruses coding for the
and
subunits (1 M.O.I. each) and increasing amounts of
-virus (0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 M.O.I.). A, enzyme activity in the cytosol of the coinfected cells was
determined as described under ``Experimental Procedures.''
Shown are data of one representative experiment out of three.
Determinations were performed in duplicate with cytosol from
independent Sf9 cell infections. Basal activity was determined in the
presence of 3 mM Mn
(open circles, right ordinate) or nitric oxide-stimulated activity in the
presence of 3 mM Mg
and 10 µM DEA-NO (filled circles, left ordinate).
Endogenous enzyme activity was determined in cytosol from Sf9 cells
infected with 5 M.O.I. of
(see also lane c in B). It was below 10 pmol
min
mg
and was subtracted as nonspecific
activity. B, shown are immunoblots of the same cytosolic
protein preparations that were used for the determination of enzyme
activity. Cytosolic protein (25 µg/lane) was separated on a 8%
(w/v) polyacrylamide gel containing additional 4 M urea. After
transfer to nitrocellulose membranes, guanylyl cyclase subunits were
detected using an antibody against the carboxyl-terminal peptide of the
and
subunit (upper panel)
and an antibody against the carboxyl-terminal peptide of the
subunit (lower
panel).
A novel variant cDNA coding for a guanylyl cyclase subunit
() was obtained from different human cell lines using
PCR with degenerate primers based on conserved sequences of the
catalytic domain of mammalian guanylyl cyclases. The newly identified
sequence represents an alternatively spliced transcript of the
subunit of soluble guanylyl cyclase, which is present
in some but not all tissues and cell lines containing
mRNA. On comparison with mammalian adenylyl cyclases, the variant
exon shows homology with the catalytic C
domain.
Coexpression experiments in Sf9 cells suggest that the
subunit acts as a negative regulator of NO-sensitive guanylyl
cyclase activity. This dominant negative effect of the
subunit is due to competition with
lacking the
insert and the formation of nonfunctional heterodimers. Our data
support the hypothesis that NO-sensitive guanylyl cyclase activity can
be regulated by post-transcriptional modification of the
pre-mRNA.
In a previous study, we showed that the subunit can form heterodimers with alternative partners
(
/
)(7) . Highest enzyme
activity was found in COS-7 cells expressing the
/
heterodimer. In contrast, the
/
heterodimer was shown to be
3-6 times less active. Here, the lower enzyme activity of the
/
form is confirmed in the
Sf9-baculovirus expression system. In addition, tissue screening by
reverse transcription PCR indicates virtually ubiquitous distribution
of the
,
, and
subunits, arguing against exclusive expression of either
heterodimer. Thus, it is conceivable that a given cell modulates its
degree of NO responsiveness by the expression of differing amounts of
alternative
subunits. In the present study, we show that
alternative splicing of the
pre-mRNA converts the
translational product into a dominant negative protein. This
dramatically increases the regulatory potential of alternative
heterodimer formation. Differential splicing leading to a dominant
negative protein in heterodimeric complexes is not without precedence:
FosB, a member of the AP-1 family of transcription factors, is a
truncated form of FosB that arises by alternative splicing of the FosB
transcript(20) .
FosB retains its ability to form
heterodimers with each of the Jun proteins but does not activate a
target gene, thus acting as a negative regulator.
The cyclase
catalytic consensus sequence is conserved across the guanylyl and
adenylyl cyclase families(38) . In particular, there is analogy
between soluble guanylyl cyclases and adenylyl cyclases; in both cases,
there is evidence that catalytic activity is dependent on the
interaction of two homologous but not identical catalytic consensus
sequences (located on /
and C
/C
,
respectively)(8, 9, 39) . Beyond this
structural resemblance, soluble guanylyl cyclase preparations exhibit
adenylyl cyclase activity(37) . Because a sequence within the
additional exon of the
subunit shows homology with
the C
domain of adenylyl cyclases, our initial hypothesis
was that alternative inclusion of the additional exon could convey
higher adenylyl cyclase activity upon the soluble enzyme. Measurement
of adenylyl cyclase in
/
-coinfected
Sf9 cells shows that on the contrary, adenylyl cyclase activity is
abolished (see Table 1). Therefore, whereas the novel sequence
strengthens the similarity between the two enzyme families, inclusion
of the optional exon does not lead to a transition from guanylyl to
adenylyl cyclase activity.
In a recent study, Ujiie et al. showed desensitization of soluble guanylyl cyclase in cultured rat
medullary interstitial cells (18) . Pretreatment of these cells
with the NO-releasing compound sodium nitroprusside resulted in a
decrease in guanylyl cyclase activity and a concomitant slight decrease
in the mRNAs coding for the and
subunits as detected by Northern blotting. In view of our
results, it is also important to consider desensitization caused by a
post-transcriptional mechanism. Thus, alternative processing of the
premRNA in human tissues could contribute to the
development of tolerance.
In conclusion, we propose a model where
differential expression of isoforms (
,
, and
) modulates NO-sensitive
guanylyl cyclase activity. Expression of the
/
heterodimer yields high guanylyl
cyclase activity. Increasing transcription of the
mRNA at the expense of the
mRNA gradually
decreases enzyme activity. On the post-transcriptional level,
alternative splicing of the
pre-mRNA offers an
additional potent mechanism to down-regulate NO-sensitive guanylyl
cyclase activity.