From the Arizona Research Labs, Division of
Neurobiology, The University of Arizona, Tucson, Arizona 85721 and
the § Department of Biological Structure and Function,
Oregon Health Sciences University, School of Dentistry,
Portland, Oregon 97201-3097
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ABSTRACT |
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Previously characterized soluble guanylyl
cyclases form The intracellular messenger cGMP mediates a wide variety of
cellular and physiological processes. It functions as the primary messenger in visual transduction (1, 2), is an important regulator in
vascular smooth muscle and kidney function (3, 4), and has been
implicated in a number of forms of neuronal plasticity (5-7).
The enzymes that regulate the synthesis of cGMP fall into two major
classes: the cytoplasmically localized, soluble guanylyl cyclases and
the membrane associated, receptor guanylyl cyclases (4). Soluble
guanylyl cyclases are obligate heterodimers composed of an We have been using the insect Manduca sexta as a model
system to study cGMP regulation and have previously shown that a
neuropeptide, eclosion hormone, is a potent stimulator of cGMP levels
in the central nervous system (15). Biochemical characterization of the
eclosion hormone-stimulated cGMP increase suggested that eclosion hormone might activate a NO-insensitive, soluble guanylyl cyclase (15-18). In an attempt to identify the pathway for eclosion
hormone-stimulated cGMP levels, we used reverse
transcription-polymerase chain reaction (RT-PCR) to identify guanylyl
cyclases from Manduca nervous tissue. This approach has
yielded a number of different guanylyl cyclases. We have previously
described the cloning and expression characteristics of the
Manduca homologues of the Animals--
The rearing and staging of M. sexta
(Lepidoptera: Sphingidae) has been described previously (17).
RNA Isolation and Degenerate Oligonucleotide
RT-PCR--
Poly(A)+ RNA was isolated from prepupal
M. sexta abdominal central nervous systems using Trizol
reagent (Life Technologies, Inc.) and oligo(dT)-cellulose columns (Life
Technologies, Inc.). First strand cDNA was generated from 5 µg of
poly(A)+ RNA using oligo(dT) primers and Superscript II RT
(Life Technologies, Inc.) and resuspended in 40 µl of water.
Degenerate oligonucleotide primers were designed against the amino acid
sequences DVYKVETI (CCRAAIARRCARTAICKNGGCAT) and MPRYCLFG
(GAYGTITAYAARGTIGWIACNAT). These sequences are in the catalytic domain
(Fig. 1) and are highly conserved in both receptor and soluble guanylyl
cyclases. PCR was performed in a 20-µl reaction containing 1 µl of
cDNA, 200 pmol of each degenerate primer, 2 mM
MgCl2, 1× PCR buffer II (Perkin-Elmer), all four
deoxynucleotides at 200 µM, 12.5 µCi of
[35S]dATP, and 2 units of Amplitaq (Perkin-Elmer). Thirty
cycles of 94 °C for 20 s, 50 °C for 20 s, and 72 °C
for 30 s were performed. The resulting PCR products were analyzed
on an 8% polyacrylamide sequencing gel, and bands below 235 base
pairs, the expected size of receptor cyclases, were cut out, eluted,
reamplified, T/A cloned into pCRII (InVitrogen), and manually sequenced.
cDNA Library Construction and Screening--
cDNA
libraries were constructed from 5 µg of poly(A)+ RNA
isolated from prepupal abdominal central nervous systems.
Oligo(dT)-primed, double-stranded cDNA was generated using a
Superscript Choice cDNA construction kit (Life Technologies, Inc.)
according to the manufacturer's instructions, except that the reverse
transcription reaction was performed in a thermocycler at 37 °C for
15 min followed by a slow rise to 50 °C over the subsequent 45 min.
Adapted cDNA was then ligated into EcoRI-cut
Lambda-ZAPII (Stratagene) and packaged using Gigapack Gold III
(Stratagene) packaging extract. The library was screened using
nitrocellulose filters (Schleicher & Schuell), hybridized, and washed
according to the manufacturer's instructions.
DNA Sequencing and Sequence Analysis--
Manual DNA sequencing
was performed using Sequenase kit, Version 2.0 (Amersham Pharmacia
Biotech). Most sequencing was carried out at an automated sequencing
facility running an ABI model 377 sequencer. Sequence analysis was
performed using Geneworks (Intelligenetics) DNA analysis software, and
protein sequence alignments used the ClustalW program through the
Baylor College of Medicine search launcher.
Cell Expression and Guanylyl Cyclase and cGMP Assays--
The
open reading frame of MsGC- Northern Blot Analysis--
Ten µg of total RNA was separated
on a formaldehyde-agarose (1%) gel and blotted onto Zetaprobe membrane
(Bio-Rad). The blot was UV cross-linked, dried, and hybridized
overnight at 42 °C with a hybridization solution consisting of 50%
formamide, 5× saline/sodium phosphate/EDTA, 5× Denhardt's solution,
1% SDS, 10% dextran sulfate, 100 µg/ml sonicated salmon sperm DNA,
and 32P-labeled probe at 106 cpm/ml. Probes
were generated by random priming of a gel-purified fragment, containing
the entire open reading frame, as a template.
RT-PCR--
Total RNA was isolated using Trizol reagent, and
5-µg aliquots were treated with DNaseI (Life Technologies, Inc.) for
30 min at 37 °C. cDNA was synthesized using oligo(dT) primers
and Superscript RT in 20 µl for 1 h at 37 °C. The cDNA
was diluted to 40 µl with water, and PCR was performed in 20 µl
containing 1 µl of cDNA, 200 pmol of each primer, 2 mM MgCl2, PCR buffer II (Perkin-Elmer), all
four deoxynucleotides at 200 µM each, and 2 units of
Amplitaq (Perkin-Elmer). Thirty cycles of 94 °C for 20 s,
66 °C for 20 s, and 72 °C for 30 s were performed.
Products were analyzed on a 1% agarose gel and stained with ethidium
bromide. The primers for MsGC- Antibody Production and Western Blot Analysis--
A glutathione
S-transferase (GST) fusion protein containing amino acids
178-940 of MsGC-
COS-7 cells or nervous tissue was homogenized in phosphate-buffered
saline containing protease inhibitors (Complete Mixture, Boehringer
Mannheim) and SDS sample buffer added (final concentrations: 20 mM Tris-HCl, pH 6.8, 1.6% SDS, 4% Cloning of MsGC-
BLAST analysis of the protein encoded by the open reading frame showed
that it was most similar to
Several different functional domains have been identified in mammalian
soluble guanylyl cyclases (21-23). These consist of the N-terminal
putative heme-binding domain, a putative dimerization domain (based on
analogy with receptor guanylyl cyclases), and the C-terminal catalytic
domain. Table I shows the percentage of amino acid identities in these
domains when MsGC-
Further analysis of the predicted amino acid sequence for MsGC-
MsGC- Expression of MsGC-
Friebe et al. (23) report that mutant
These experiments suggest that MsGC-
The C terminus of MsGC-
In addition to assessing the enzyme activity of MsGC- Tissue Distribution of MsGC-
To demonstrate that the MsGC- This paper describes the cloning and initial characterization of a
novel In addition to the sequence information, expression data also show that
MsGC- An important question raised by these finding is whether or not
MsGC- It will also be interesting to determine whether MsGC- Northern blot analysis suggests that MsGC--
heterodimers that can be activated by the gaseous
messenger, nitric oxide. In mammals, four subunits have been cloned,
named
1,
2,
1, and
2. We have identified a novel soluble
guanylyl cyclase isoform from the nervous system of the insect
Manduca sexta that we have named M. sexta
guanylyl cyclase
3 (MsGC-
3). It is most closely related to the
mammalian
subunits but has several features that distinguish it
from previously identified soluble cyclases. Most importantly,
MsGC-
3 does not need to form heterodimers to form an active enzyme
because guanylyl cyclase activity can be measured when it is expressed
alone in COS-7 cells. Moreover, this activity is only weakly enhanced
in the presence of the nitric oxide donor, sodium nitroprusside.
Several of the amino acids in rat
1 subunits, previously identified
as being important in heme binding or necessary for nitric oxide
activation, are substituted with nonsimilar amino acids in MsGC-
3.
There are also an additional 315 amino acids C-terminal to the
catalytic domain of MsGC-
3 that have no sequence similarity to any
known protein. Northern blot analysis shows that MsGC-
3 is primarily
expressed in the nervous system of Manduca.
INTRODUCTION
Top
Abstract
Introduction
References
and a
subunit. Two
(
1 and
2) and two
(
1 and
2) subunits have been cloned from mammalian tissues (8-13). The active heterodimer requires and binds heme as a prosthetic group and can be
potently activated by the gaseous messenger, nitric oxide (NO)1 (14). The soluble
cyclases are found in the cytosol of cells, although the mammalian
2
subunit has a consensus isoprenylation site, suggesting that it might
be associated with membranes. The receptor guanylyl cyclases, by
contrast, are integral membrane proteins with transmembrane and
extracellular domains (4).
1 and
1 subunits, named
MsGC-
1 and MsGC-
1 (19). Here we describe the cloning and
preliminary characterization of a novel
subunit, MsGC-
3. This
new subunit shows significant basal activity when expressed alone and
shows only modest stimulation in the presence of NO.
MATERIALS AND METHODS
3 was cloned into pcDNA3.1
(Invitrogen), and an 8-µg aliquot was transfected into a 10-cm dish
of COS-7 cells using LipofectAMINE (Life Technologies, Inc.). Three
days after transfection, the cells were harvested and homogenized in 1 ml of 25 mM Tris-HCl, pH 7.4, containing 100 µM phenylmethylsulfonyl fluoride. Cell extracts were
assayed for guanylyl cyclase activity by monitoring the conversion of
[
-32P]GTP to [32P]cGMP as described
previously (20). Under the conditions used, the production of cGMP was
linear with respect to time. To assay cGMP levels in intact COS-7
cells, cells were plated onto 24-well plates, and each well was
transfected with 0.2 µg of each plasmid. After 3 days, the cells were
incubated in saline (composition: NaCl (120 mM), KCl (5.4 mM), CaCl2 (1.8 mM), Tris-HCl (25 mM) and glucose (15 mM), pH 7.4) for 30 min at
37 °C, followed by a further 5 min in the presence or absence of 1 mM sodium nitroprusside (SNP). The saline was then removed,
and the cells were lysed with ethanol:1 M HCl (100:1).
Following centrifugation, the supernatant was lyophilized, redissolved
in 50 mM sodium acetate, pH 6.2, and assayed for cGMP
content with a commercial radioimmunoassay kit (NEN Life Science Products).
3 and MsGC-
1 were designed and
checked for false hybridization to other guanylyl cyclases,
using Oligo 4.0 (Molecular Biology Insights). The primers for MsGC-
1
were TCACTGCTGTGTTCCGAT and ATAGAAGGCCGTGGTCTT, corresponding to
nucleotides 5012-5029 and 5621-5638 that are located in the
3'-untranslated region. The primers for MsGC-
3 were
AAGAGAGGTAGCCCGCCA and CATCATTCGCCAGTTGTC corresponding to amino
acids REVARH (149-154) and DNWRMM (314-319).
3 was made by ligating the
MunI/SalI fragment of SGC4 and the
SalI/XbaI fragment of SGC25 (see Fig. 1) into the
GST fusion vector, pGEX-3X (Amersham Pharmacia Biotech), digested with
EcoRI and XbaI. The resulting GST-MsGC-
3
fusion protein was purified using glutathione-coupled Sepharose
(Amersham Pharmacia Biotech) and sent to HTI Bio-Products (Ramona, CA)
for antibody production. Antisera were screened using Western blots of
COS-7 cells transfected with MsGC-
3 and affinity purified using
GST-MsGC-
3 coupled to NHS-activated Sepharose (Amersham Pharmacia Biotech).
-mercaptoethanol,
4% glycerol and 20 µg/ml bromphenol blue). The proteins were
separated on a 10% polyacrylamide gel and transferred to
polyvinylidene difluoride membrane. Primary antisera were used at 1:100
for whole sera or 1:10 for affinity purified antisera and detected
using 1:10,000 horseradish peroxidase-conjugated goat anti-rabbit
antibodies (The Jackson Laboratory) and visualized with
chemoluminescence and a digital imaging system (ChemiImager, Alpha Innotech).
RESULTS
3--
Using RT-PCR with degenerate
oligonucleotide primers, we isolated cDNA fragments of a variety of
different guanylyl cyclases from the abdominal central nervous system
of M. sexta. Two of these have been described previously
(19), appear to be the Manduca homologues of the mammalian
soluble guanylyl cyclase subunits,
1 and
1, and have been named
MsGC-
1 and MsGC-
1, respectively. In the course of this earlier
study, we isolated an additional cDNA fragment that also had high
sequence similarity to mammalian
subunits. Using this fragment, we
screened a cDNA library and isolated five overlapping cDNA
clones. These clones, when sequenced and compared, could be combined to
form a cDNA construct of 5271 base pairs (Fig.
1A). The size of this cDNA
construct approximately matched the 5.1-kb band seen on Northern blots
(see Fig. 4A). This composite clone has an open reading
frame of 940 amino acids, beginning with a consensus ribosome binding
site. There are multiple stop codons in all frames both 5' and 3' to
this open reading frame. The translated N terminus of this clone also
shows a significant level of identity to other cloned
subunits of
guanylyl cyclases suggesting that this construct represents the
full-length sequence of MsGC-
3. Using a unique BamHI
site, a composite clone was created from clones SGC4 and SGC25, which
contained the entire open reading frame. Both strands of this composite
clone were analyzed and the resulting sequence placed into
GenBankTM under accession number AF064514. This clone was
then used for further expression studies.
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Fig. 1.
A, schematic diagram of the five
overlapping cDNA clones that combine to make up the 5271-base pair
cDNA clone of MsGC- 3. The open reading frame of the construct is
shaded in black (ORF), and the unique
BamHI site used to create the SGC4-SGC25 composite clone is
noted. B, predicted amino acid sequence of MsGC-
3 and
alignment with a C. elegans guanylyl cyclase gene
(GenBankTM accession number 1109803, Ref 20)
(CeGC-
3), MsGC-
1, and the rat
1 subunit. Residues
shaded in gray are conserved between MsGC-
3 and any of
the other sequences. In the regulatory region, those residues conserved
between all guanylyl cyclases are shaded in black, and those
conserved in all guanylyl cyclases except MsGC-
3 are marked with an
asterisk. In the C-terminal domain, unique to MsGC-
3 and
C. elegans, the residues marked with a double
underline show potential phosphorylation sites, and those marked
with a single underline show the potential isoprenylation
site. The sequences used for designing primers used in the RT-PCR for
cloning MsGC-
3 are marked P1 and P2. The
nucleic acid sequence of the SGC4/SGC25 composite clone including the
entire open reading frame of MsGC-
3 has been deposited with
GenBankTM, accession number AF064514.
subunits of soluble guanylyl cyclases.
The most closely related sequence was a Caenorhabditis elegans clone (GenBankTM accession number 1109803, Ref. 20). Of previously identified and characterized guanylyl cyclases,
it had the most similarity to vertebrate
1 subunits, with a 37%
overall sequence identity to the rat
1 subunit compared with 29%
sequence identity to the rat
2 subunit (Table
I). This is, however, much lower than the 59% identity seen between MsGC-
1 and rat
1 (Ref. 19 and Table I). In addition, the newly identified Manduca sequence has a 315 amino acid region C-terminal to its catalytic domain that is not
present in any other guanylyl cyclase of any species cloned to date.
The C. elegans clone also has a C-terminal extension but has
very low similarity to the Manduca sequence (Table I). BLAST
analysis of the 315-amino acid sequence from the Manduca sequence did not reveal any significant similarity to any other sequence in the data base. The clone does not appear to be the Manduca homologue of a
2 subunit, and as it has novel
features not seen in previously cloned and characterized guanylyl
cyclases, we propose to name this cyclase MsGC-
3. Fig. 1B
shows an alignment of the amino acid sequences of MsGC-
3 with the
C. elegans sequence, MsGC-
1, and the rat
1
subunit.
Comparison of amino acid identities in the functional domains of
Manduca, a C. elegans gene (GenBankTM accession number 1109803;
Ref. 20) and rat and
subunits
1 subunit), the putative dimerization domain
(312-377), and the catalytic domain (378-619).
3 is compared with other
subunits. The level
of sequence identity in the catalytic domain is very similar (~40%)
whether MsGC-
3 is compared with the C. elegans gene, rat
1, rat
2, or MsGC-
1. MsGC-
3 is most similar, however, to
the C. elegans gene and the rat
1 subunit in its
heme-binding and dimerization domains. Although MsGC-
3 is similar to
rat
1, the degree of similarity is considerably lower (< 40%)
compared with the sequence identities seen when MsGC-
1 is compared
with rat
1 or when MsGC-
1 is compared with rat
1(50-72%).
These comparisons again suggest that MsGC-
3 is a novel
subunit and not a Manduca homologue of an identified vertebrate guanylyl cyclase.
3
reveals a number of other novel features. Several studies of mammalian
guanylyl cyclases, using sequence comparison and mutational analysis,
have identified 23 amino acids proposed to be involved in heme binding,
NO activation, and/or dimerization (23-25). These residues are marked
in Fig. 1B, and the sequence alignment shows that six of
these residues are not conserved in MsGC-
3, and five of those six
are also substituted in the C. elegans gene (although with
different substitutions). Most notable of these are the cysteines found
in positions 78 and 214 in rat
1, which are absent in both MsGC-
3
and the C. elegans gene and are replaced with valine and
glutamic acid, respectively, in MsGC-
3. Friebe et al.
(23) have shown that mutating these cysteines to serines in the rat
1 subunit produces a guanylyl cyclase that, when co-expressed with a
wild-type
1 subunit, has detectable (but low) basal activity in the
presence of manganese but cannot be activated by NO. Interestingly,
histidine-105 (position based on rat
1 sequence), which has been
suggested to be the axial ligand for heme binding and also shown to be
required for NO-stimulated guanylyl cyclase activity (24), is present
in MsGC-
3 and the C. elegans gene. The high degree of
similarity between MsGC-
3 and the C. elegans gene
suggests that it is also a novel
subunit and could also be
considered a
3 subunit.
3 has a C-terminal 315-amino acid domain that is not present in
any other guanylyl cyclase. This region shows no significant similarity
to any other protein in the data base. A search for protein motifs
revealed that there are a number of possible protein phosphorylation
sites within this C-terminal domain, and a potential C-terminal
isoprenylation site was also found. Interestingly, the rat
2 subunit
also has a potential C-terminal isoprenylation site, although the
modification has not yet been demonstrated in vivo (4). None
of the phosphorylation sites are conserved in the C. elegans
gene, nor is the isoprenylation site.
3 in COS-7 Cells--
To examine the enzyme
properties of MsGC-
3, we subcloned its open reading frame into the
mammalian expression vector pcDNA3.1. We then transiently
transfected this construct into COS-7 cells, either alone or in
combination with plasmids containing the other Manduca-soluble guanylyl cyclase subunits. Cell extracts
were then assayed for guanylyl cyclase activity in the presence or absence of SNP as a NO donor (Fig. 2). As
guanylyl cyclases have been shown to vary in their activity depending
on whether magnesium or manganese is present (26), we tested each cell
extract in the presence of each metal ion. In the presence of
magnesium, only cells co-transfected with both MsGC-
1 and MsGC-
1
showed guanylyl cyclase activity above that seen for cells transfected with control plasmid. As reported previously, this activity could be
stimulated by SNP (19). MsGC-
3 showed no guanylyl cyclase activity
when expressed alone, and it did not appear to form an active
heterodimer when co-expressed with either MsGC-
1 or MsGC-
1. In
the presence of manganese, cells co-transfected with MsGC-
1 and
MsGC-
1 showed higher basal and lower SNP-stimulated activity when
compared with cell extracts assayed in the presence of magnesium. This
is a similar result to the enzymatic activity of mammalian soluble
guanylyl cyclases (23). In cells transfected with only MsGC-
3 and
assayed in the presence of manganese, we detected significant basal
guanylyl cyclase activity above that seen in cells transfected with
control plasmid. We did not, however, detect any stimulation of
activity in the presence of SNP. This suggests that MsGC-
3 does not
need to form heterodimers to form an active guanylyl cyclase and that
this active enzyme is NO-insensitive. When MsGC-
3 was co-expressed
with either MsGC-
1 or MsGC-
1, the total guanylyl cyclase activity
present in the cell extracts was less than when MsGC-
3 was expressed
alone and a small but significant increase in activity was detected in
the presence of SNP. It is not clear whether this increase was due to
the formation of low levels of NO-sensitive heterodimers. COS-7 cells
transfected with control plasmid alone also showed a small increase in
guanylyl cyclase activity in the presence of SNP, presumably due to the expression of low levels of endogenous soluble guanylyl cyclase.
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Fig. 2.
Guanylyl cyclase activity in COS-7 cells
transfected with Manduca-soluble guanylyl cyclase
subunits. COS-7 cells were transiently transfected with
pcDNA3.1 vectors containing the open reading frame of the
Manduca guanylyl cyclase subunits. Cell extracts were then
assayed for guanylyl cyclase activity in the presence of 4 mM Mg2+ (A) or 4 mM
Mn2+ (B) and in the absence (open
columns) or presence (closed columns) of 250 µM SNP. Each assay was carried out in triplicate, and the
columns show the means (± S.E.) of three separate
experiments.
subunits, missing
either cysteine-78 or cysteine-214, show a substantial reduction in
their ability to form NO-sensitive heterodimers. NO sensitivity was
restored, however, by reconstitution with heme. Table
II shows that this does not occur with
MsGC-
3. We transfected COS-7 cells with either MsGC-
3 alone or
MsGC-
3 together with MsGC-
1. We then assayed the cell extracts
for guanylyl cyclase activity after incubation with 5 µM
hematin and 100 µM dithiothreitol. In neither case did we
see an increase in NO-stimulated activity. By contrast, when cells
transfected with MsGC-
1 together with MsGC-
1 were treated in a
similar fashion, heme reconstitution did increase the NO-stimulated
activity, presumably because some endogenous heme was lost during cell
homogenization.
Incubation with hematin does not affect MsGC-3 activity
3 either does not bind heme, or
binds it in such a manner that renders the cyclase NO-insensitive. Recently, a compound,
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), has been identified as a potent inhibitor of mammalian guanylyl
cyclases (27). It appears to act as a heme site inhibitor, inhibiting
activity by irreversible oxidation of the heme group (28). We tested
whether ODQ could inhibit MsGC-
3 by assaying guanylyl cyclase
activity of transfected COS-7 cell extracts in the presence of
different concentrations of ODQ (Fig. 3).
ODQ has an IC50 of 0.72 µM for inhibiting
mammalian soluble guanylyl cyclase (28), yet 100 µM had
no inhibitory effect on MsGC-
3. As a control for the effectiveness
of ODQ, we also assessed its effects on Manduca NO-sensitive
soluble guanylyl cyclase (Fig. 3). These results showed that 100 µM ODQ inhibited Manduca NO-sensitive activity
by 95% with an IC50 of 2.9 µM.
Interestingly, basal activity of the Manduca NO-sensitive
cyclase showed no sensitivity to ODQ. This is in contrast to the
mammalian enzyme, which showed 80% inhibition of basal activity in the
presence of 30 µM ODQ (28).
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Fig. 3.
Effect of ODQ on Manduca guanylyl
cyclases. COS-7 cells were either transiently transfected with
MsGC- 3 (solid line) or co-transfected with MsGC-
1 and
MsGC-
1 (dotted line, MsGC-
1+
1 + 100 µM SNP; dashed line, control) and assayed for
guanylyl cyclase activity in the presence of different concentrations
of ODQ. MsGC-
3 was assayed in the presence of 4 mM
Mn2+ and MsGC-
1+
1 in the presence of 4 mM
Mg2+ and the presence and absence of 100 µM
SNP. Each data point was assayed in triplicate and
represents the mean ± S.E.
3 contains a potential isoprenylation site
(Fig. 1B). To determine whether MsGC-
3 is
membrane-associated, we separated transfected COS-7 cell homogenates
into particulate and cytoplasmic fractions and assayed each separately
for guanylyl cyclase activity. We found that the majority of MsGC-
3
activity was pelleted by ultracentrifugation (Table
III), suggesting that the enzyme was
associated with membranes. Whether this is a result of isoprenylation
or not will require further study.
MsGC-3 activity is primarily present in the pellet fraction of
COS-7 cell homogenates
3 in
homogenates of transfected COS-7 cells, we also measured the cGMP content of intact COS-7 cells after they had been transiently transfected with MsGC-
3. We found that in unstimulated COS-7 cells,
MsGC-
3 forms an active guanylyl cyclase capable of causing a much
larger accumulation of cGMP than when COS-7 cells are either co-transfected with MsGC-
1 and MsGC-
1, or transfected with vector alone (Table IV). Surprisingly, when the
COS-7 cells were stimulated with SNP, all of the transfected cells
showed a significant increase in cGMP content. Presumably, the increase
in cGMP in response to SNP in COS-7 cells transfected with vector was
due to the presence of endogenous soluble guanylyl cyclase. When this
background value was subtracted from the cGMP levels in cells
transfected with Manduca guanylyl cyclases, over a 100-fold
stimulation in cGMP content was observed with cells co-transfected with
MsGC-
1 and MsGC-
1, whereas cells transfected with MsGC-
3
showed less than a 3-fold stimulation.
Activity of MsGC-3 in intact COS-7 cells
3--
We examined the tissue
distribution of MsGC-
3 in larval tissues using Northern blot
analysis. We found a single 5.1-kb transcript present at high levels in
the abdominal central nervous system. The same transcript was found at
lower levels in brain and trachea (Fig.
4A). A signal was also
detected in the heart, although it could not be resolved into discrete
bands. No signal was detectable in fat body, muscle or gut. We also
examined the expression of MsGC-
3 in several tissues of adult
Manduca, just prior to ecdysis. No message could be detected
in any tissues (brain, abdominal central nervous system, antennae, or
muscle) at that stage (data not shown) suggesting that MsGC-
3 is
developmentally regulated. Because MsGC-
3 codes for a guanylyl
cyclase that is relatively insensitive to NO, we were especially
interested in determining whether its transcript could be detected in
tissues that are known to increase cGMP in response to eclosion
hormone. Previous studies have shown that the eclosion
hormone-stimulated cGMP increases are not mimicked by NO (16, 29).
These targets include the epitracheal glands (30) and the transverse
nerve (16). To determine whether MsGC-
3 could be detected in these
tissues, we used RT-PCR. We were able to detect a strong,
RT-dependent band in epitracheal glands and transverse
nerve samples, as well as the abdominal central nervous system, muscle,
and trachea (Fig. 4B). Because MsGC-
3 is expressed in
trachea, and it is possible that tracheal epithelial cells could have
contaminated both the epitracheal gland and transverse nerve samples,
we examined the expression of MsGC-
1 as a control. We found that
although both MsGC-
3 and MsGC-
1 were detectable in trachea, only
MsGC-
3 could be detected in the epitracheal gland and transverse
nerve samples.
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Fig. 4.
Tissue distribution of MsGC- 3.
A, Northern blot showing the expression of MsGC-
3 in
different Manduca tissues prior to pupal ecdysis. A 5.1-kb
band was detected in total RNA from trachea, brain, and abdominal
central nervous system. A hybridization signal in the region of 5.1 kb
was detected using RNA from heart, but it could not be resolved into a
discrete band. No signal was detectable in RNA from intersegmental
muscle, fat body, or gut. Hybridization to the M. sexta
eukaryotic elongation factor 4A (EEF) (1.6 kb) was used as a
loading control and to demonstrate the integrity of the RNA.
B, RT-PCR of MsGC-
1 and MsGC-
3 showing MsGC-
3
expression in abdominal central nervous systems, epitracheal glands,
transverse nerves, trachea, and intersegmental muscles. MsGC-
1 is
only detectable in abdominal central nervous systems, trachea, and
intersegmental muscle. A control experiment in which the reverse
transcriptase was omitted from the reaction was performed as a control
for genomic DNA contamination. No PCR products were detected in these
samples. C and D, Western blot analysis showing
that a protein of the predicted size for MsGC-
3 is expressed in
Manduca central nervous system. Samples consisted of either
COS cells transfected with control plasmid (lane a); plasmid
containing MsGC-
1 (lane b), MsGC-
1 (lane
c), or MsGC-
3 (lane d); or abdominal central nervous
tissue from Manduca (lane e). The blots were then
probed with either preimmune serum (C) or immune serum
affinity-purified with GST-MsGC-
3 fusion protein (D).
Molecular mass markers (left) are in kDa.
3 protein is indeed expressed in
Manduca nervous tissue, we constructed a GST fusion protein containing amino acids 178-940 of MsGC-
3 and immunized rabbits with
the resulting protein. MsGC-
3 has a predicted molecular mass of 106 kDa, and Fig. 4D shows that antisera that had been affinity
purified against the MsGC-
3 fusion protein recognized a protein of
approximately 106 kDa in COS-7 cells that had been transfected with the
MsGC-
3 expression plasmid (lane d). COS-7 cells
transfected with control plasmid (lane a) or plasmids
containing MsGC-
1 (lane b) or MsGC-
1 (lane
c) did not show the presence of any bands. Preimmune serum did not
show the presence of this 106 kDa band in COS-7 cells expressing
MsGC-
3 or any of the other plasmids (Fig. 4C). When
abdominal central nervous tissue was blotted and probed with the
affinity purified antisera, a single band was detected with a slightly
higher molecular mass compared with the band in COS-7 cells. This
demonstrates that a protein of the predicted size of MsGC-
3 is
expressed in Manduca central nervous system. It also
suggests that some processing takes place in vivo, resulting
in a protein with an apparently larger molecular mass compared with
MsGC-
3 expressed in COS-7 cells. The primary sequence of MsGC-
3
does not give any clues as to the nature of this processing.
DISCUSSION
isoform of soluble guanylyl cyclases from M. sexta that we have named MsGC-
3. Previously described guanylyl cyclases can be classified as either receptor guanylyl cyclases, which are
integral membrane proteins, or soluble guanylyl cyclases, which are
generally cytoplasmically localized (4). Soluble guanylyl cyclases are
thought to exist as heterodimers of an
and a
subunit, and two
and two
isoforms have been identified in mammals (8-13).
MsGC-
3 has a high degree of similarity to the rat
1 subunit
throughout all three of its domains, demonstrating that MsGC-
3
clearly belongs to the soluble guanylyl cyclase family rather than the
receptor guanylyl cyclase family. The principal difference between the
and
subunits is that the heme-binding domains of
subunits
are longer: 372 amino acids for rat
1 as compared with 311 for rat
1 (9, 11). The equivalent region of MsGC-
3 also contains 311 amino acids, which identifies it as a
subunit rather than an
subunit. Although sequence analysis shows that MsGC-
3 is most
similar to mammalian
1 subunits, there are a number of features that
suggest that it is a novel
subunit rather than the
Manduca homologue of
1 or
2. First, we have previously
cloned and characterized another Manduca
subunit that
has a higher degree of similarity to rat
1 than does MsGC-
3 (19).
Second, MsGC-
3 has a C-terminal extension of 315 amino acids not
seen in any other guanylyl cyclase cloned to date. Finally, MsGC-
3
lacks several amino acids that are conserved in all other soluble
cyclases, some of which have also been shown to be necessary for NO
activation (23). A search of the data bases also reveals a gene from
C. elegans that appears to be homologous to MsGC-
3. The
C. elegans gene is lacking five of the 6 highly conserved amino acid residues absent in MsGC-
3 and also has an extended C-terminal tail.
3 has novel features when compared with other guanylyl
cyclases. Previous studies have shown that soluble guanylyl cyclases
are obligate heterodimers (11). We have demonstrated that the
Manduca homologues of
1 and
1 subunits also conform to
this pattern (19) (Fig. 2). MsGC-
3, by contrast, shows significant guanylyl cyclase activity when expressed alone in COS-7 cells. This is
true both when the cells are homogenized and enzyme activity measured
in vitro and when guanylyl cyclase activity is assessed in
intact cells by measuring cGMP accumulation. Like other guanylyl cyclases, the activity measured in homogenates is higher when Mn-GTP is
used as a substrate compared with when Mg-GTP is used. Unlike
heterodimeric guanylyl cyclases, however, MsGC-
3 responds very
poorly to NO. When measured in COS-7 cell homogenates, in the presence
of either manganese or magnesium, the guanylyl cyclase activity of
MsGC-
3 shows no stimulation with SNP. When measured in intact COS-7
cells, MsGC-
3 did show significantly higher accumulations of cGMP
when treated with SNP. This increase was substantially less, however,
than that found in COS-7 cells that were co-transfected with MsGC-
1
and MsGC-
1. It will be very interesting to determine whether the
C. elegans homologue is also active as a homomer and whether
it is also NO-insensitive.
3 is capable of binding heme. Cysteine-78 and cysteine-214 have
been shown to be important in NO sensitivity in the rat
1 subunit
(23). When these cysteines were point-mutated to serines and the
recombinant subunit co-expressed with wild-type
1 subunits, the
resulting guanylyl cyclase was NO-insensitive, although NO-sensitivity could be restored by incubation with heme (23). This suggests that loss
of these two cysteines results in a guanylyl cyclase capable of binding
heme, but with a much lower affinity. MsGC-
3 lacks both of these
cysteines and is NO-insensitive. However, NO-sensitivity cannot be
restored with heme reconstitution, whether the activity is measured in
extracts of COS-7 cells transfected with MsGC-
3 alone or
co-transfected with MsGC-
1. The soluble guanylyl cyclase inhibitor,
ODQ, is thought to act by irreversible oxidation of the heme group
resulting in a NO-insensitive enzyme (28). ODQ has no effect on the
activity of MsGC-
3, suggesting either that MsGC-
3 does not bind
heme or that it binds heme in an oxidized state and hence is unaffected
by either NO or ODQ. The fact that a slight stimulation of activity by
SNP is seen in intact COS-cells suggests that MsGC-
3 does bind heme
in vivo, but that it is rapidly lost or irreversibly
oxidized during the homogenization process.
3 forms
homodimers, or if it is capable of forming heterodimers with other
soluble guanylyl cyclase subunits. The dimerization domain of soluble
guanylyl cyclases has not been investigated in detail. It is defined
primarily by comparison to receptor guanylyl cyclases (22) as the
region on the immediate N-terminal side of the catalytic domain and the
region in fact shows little amino acid conservation between soluble and
receptor guanylyl cyclases. When COS-7 cells co-transfected with
MsGC-
3 and either MsGC-
1 or MsGC-
1 were assayed, lower
guanylyl cyclase activity was detected compared with COS-7 cells
transfected with MsGC-
3 alone. This reduced activity could reflect
the formation of a less active heterodimer, although it could also be
the result of reduced transfection efficiency. It is also interesting
to note that although the rat
2 subunit will form heterodimers with
1 subunits, the
1/
2 heterodimer shows lower NO-stimulated
activity compared with the
1/
1 heterodimers (31).
3 is primarily
localized to the nervous system of Manduca, and Western
blots demonstrate that the MsGC-
3 protein is expressed in the
central nervous system. We have previously identified additional
guanylyl cyclases within the nervous system of Manduca,
which are either NO-sensitive heterodimeric cyclases (19) or
NO-insensitive cyclases (32). MsGC-
3 forms a complementary class of
guanylyl cyclase that is active as a homomer and only weakly activated
by NO. Previous studies have identified a number of sites in
Manduca that show cGMP elevations in a NO-independent manner
(16, 29), and RT-PCR analysis suggests that MsGC-
3 is present in two
of these sites: the epitracheal glands and the transverse nerve. A
detailed in situ hybridization or immunocytochemical
investigation will be required to confirm these results and to suggest
other possible physiological roles for this novel member of the
guanylyl cyclase family.
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FOOTNOTES |
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* This project was supported by NINDS, National Institutes of Health Grant NS29740 and National Science Foundation Grant 9604536.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF064514.
¶ To whom correspondence should be addressed: Dept. of Biological Structure and Function, Oregon Health Sciences University, School of Dentistry, 611 S.W. Campus Dr., SD, Portland, OR 97201-3097. Tel.: 503-494-8596; Fax: 503-494-8554; E-mail: mortonda{at}ohsu.edu.
The abbreviations used are:
NO, nitric oxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; RT, reverse transcriptase; PCR, polymerase chain reaction; SNP, sodium
nitroprusside; MsGC-3, M. sexta guanylyl cyclase
3; kb, kilobase; GST, glutathione S-transferase.
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REFERENCES |
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