From the
We identified two Drosophila genes (dgc
Guanylyl cyclases (GTP pyrophosphate-lyase (cyclizing); EC
4.6.1.2) catalyze the conversion of GTP to cGMP, which is a common
second messenger that is utilized in a wide variety of cells and signal
transduction pathways. There are two guanylyl cyclase isoforms, a
particulate/membrane-bound form and a soluble form. The particulate
guanylyl cyclase is a single polypeptide that possesses an
extracellular receptor domain, a single hydrophobic membrane-spanning
domain, followed by an intracellular protein kinase regulatory domain
and the catalytic domain(1, 2, 3) . The protein
kinase domain is similar in amino acid sequence to other protein kinase
catalytic domains(4) . Deletion of this kinase domain in
chimeric particulate guanylyl cyclases leads to constitutive activation
and the inability of bound ligand to further stimulate the guanylyl
cyclase(5, 6) . The catalytic domain is very similar in
amino acid sequence to the catalytic domains of soluble guanylyl
cyclases and adenylyl cyclases(2) . Most particulate guanylyl
cyclases are stimulated by natriuretic peptides or by bacterial
heat-stable enterotoxins(7) . The notable exceptions are the
stimulation of vertebrate retinal particulate guanylyl cyclase by
submicromolar calcium concentrations through a guanylyl
cyclase-activating protein (8, 9, 10) and the
nitric oxide stimulation of some particulate guanylyl
cyclases(11, 12, 13) .
The soluble guanylyl
cyclases are heterodimers composed of a large (
While both the
While much is known about
the structure and regulation of the soluble guanylyl cyclases through
biochemical studies, a detailed analysis of their function in vivo is still lacking. Drosophila provides an opportunity to
genetically examine the role of guanylyl cyclase in neuronal
development and function. In this study, we isolated two genes that
encode the
To obtain the cytosolic protein
fraction, the cells were washed with phosphate-buffered saline and
harvested in 50 mM triethanolamine-HCl buffer (pH 7.4)
containing 1 mM dithiothreitol. The cells were homogenized
with 10-15 strokes in a chilled Potter-Elvejhem homogenizer with
the pestle rotating at 500-1000 rpm. The homogenate was centrifuged at
12,000
The predicted amino acid sequence for the soluble
guanylyl cyclase
The predicted amino acid sequence for
the Dgc
We assayed the cytosolic fraction from the stably
transfected cell lines for guanylyl cyclase activity both in the
presence and absence of sodium nitroprusside, a known stimulator of
soluble guanylyl cyclase activity (). In the absence of
sodium nitroprusside, only the stably transfected line coexpressing
Dgc
We identified a soluble guanylyl cyclase
Immunolocalization revealed that both the
The exact origin of the
155-kDa protein detected with the Dgc
The Dgc
While we could not
determine whether Dgc
Two lines of evidence strongly suggest that the dgc
The major difference between
our results and the results for Dgc1 pertains to the distribution of
the mRNA and corresponding protein in the adult head. Our RNA Northern
hybridization detected a predominant 2.4-kb Dgc
The
identification of both the soluble guanylyl cyclase
Cytosolic fractions were isolated from human kidney 293 cells that
were not transfected or stably transfected with either the Dgc
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank the Friemann Life Science personnel for
immunizing, bleeding, and caring for the mice in this work. We are
grateful to Doug McAbee for his interest and advice on these studies.
We thank William Christiansen for guidance in transfecting the human
kidney 293 cells. We thank Doug McAbee and members of the Hyde lab for
comments on this manuscript and Dr. W. Pak for communicating results
prior to publication.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 and dgc
1) that encode the soluble guanylyl cyclase
and
subunits, respectively. The putative Dgc
1 protein
is 76 kDa, has 35% amino acid identity with previously isolated
subunits, and was immunolocalized to the adult retina, to the optic
lobes, and throughout the brain neuropil. The Dgc
1 protein is 86
kDa and exhibits 59% amino acid identity with the rat
1 protein.
However, the Dgc
1 protein has an additional 118 amino acids
inserted near the amino terminus, which makes it significantly larger
than the rat
1. The Dgc
1 protein was immunolocalized to the
optic lobes and throughout the brain neuropil, with no detectable
expression in the retina. The Dgc
1 and Dgc
1 cDNAs were stably
transfected into human kidney 293 cells. Expression of the individual
subunits and mixing of the individually expressed subunits failed to
generate significant guanylyl cyclase activity. Only coexpression of
the subunits resulted in significant guanylyl cyclase activity. Our
results indicate that Dgc
1 and Dgc
1 are soluble guanylyl
cyclase
and
subunits that are capable of forming a
functional guanylyl cyclase heterodimer.
) and small (
)
subunit, which both contain a catalytic domain near the carboxyl
terminus that is similar in amino acid sequence between all guanylyl
cyclases and adenylyl cyclases(2) . The soluble cyclases are
activated by the gases nitric oxide and carbon monoxide and also by the
NO sources, sodium nitroprusside and
nitroglycerin(1, 14) . Presently, three different
subunit forms (
1,
2, and
3) and two
subunits
(
1 and
2) have been cloned from rats, bovines, and humans.
The
subunits share 48% amino acid identity, with
2
possessing an extra 13 and 2 amino acids at the amino and carboxyl
termini, respectively(15) . Based on the cDNA sequence, the
subunits share 34% amino acid identity.
2 has an extra 86
amino acids at the carboxyl terminus relative to
1 and possesses
an isoprenylation/carboxymethylation consensus sequence that
1
lacks (16).
and
subunits possess putative
catalytic domains, they must be coexpressed to generate guanylyl
cyclase activity. Expression of either subunit alone or combining the
individual subunits in vitro failed to generate guanylyl
cyclase activity (17-19). Additionally, mutations within the
subunit catalytic domain blocked guanylyl cyclase activity
without affecting heterodimer formation(20) . This suggests that
both catalytic domains must be functional to generate enzymatic
activity. The guanylyl cyclase activity can also be manipulated by
altering the subunit composition. While the
1 subunit generates a
functional dimer with either
1 or
2, the
1/
1 dimer
is approximately 6-fold more active than
2/
1(15) . In
contrast, the
2 subunit failed to yield guanylyl cyclase activity
with either
1 or
2(21) .
and
subunits of soluble guanylyl cyclase. The
subunit is structurally similar to the rat
subunit, while
the
subunit contains an extra 118 amino acids inserted near the
amino terminus relative to the rat protein. Both the
and
subunits immunolocalize to specific neuropil regions of the Drosophila brain. Coexpression of the Drosophila
and
subunits demonstrates that they possess a guanylyl
cyclase activity, which is stimulated over 30-fold by sodium
nitroprusside. Therefore, these two Drosophila proteins
correspond to an
and a
subunit due to their amino acid
homology and enzymatic activity.
Materials
Restriction enzymes and fluorescein
isothiocyanate-conjugated goat anti-mouse secondary antibody were
purchased from Fisher Scientific (Promega Biotech). Sequenase version
2.0 and Random Primed DNA labeling kits were purchased from U.S.
Biochemical Corp. T7 and T3 RNA polymerases and RNA in vitro transcription kits were from Stratagene. RNA molecular weight
markers (0.24-9.5-kb(
)ladder), and the
protein molecular weight markers were purchased from Life Technologies,
Inc. and Bio-Rad, respectively. Custom oligonucleotide primers for DNA
sequencing were purchased from Amber, Inc. The
[
-
P]dATP,
[
-
P]CTP, and
I-Protein A were
from ICN Radiochemicals, and [
-
S]dATP and
Hybond-N were from Amersham Corp. Cell culture reagents (DME media,
fetal bovine serum, and geneticin sulfate) and guanylyl cyclase assay
reagents (3-isobutyl-1-methylxanthine, creatine phosphate, sodium
nitroprusside, Type I creatine phosphokinase, and alumina (Type WN-3))
were purchased from Sigma.
Screening cDNA Libraries
The 8H7 cDNA was among a
collection of cDNAs that corresponded to visual system
mRNAs(22) . Two Drosophila head cDNA libraries in
ZAPII (one synthesized by ourselves and the other a gift of Tom
Schwarz, Stanford University) were screened using standard
procedures(23) . The 8H7 cDNA was restriction-digested, and then
the cDNA was gel-purified and labeled with
[
-
P]dATP using the Random Primed DNA
labeling kit. The hybridization and wash conditions were identical to
those described for DNA Southern hybridizations(24) .
DNA Sequencing and Analysis
Single- and
double-stranded DNA sequencing was performed with the Sequenase version
2.0 enzyme and custom oligonucleotide primers using procedures
recommended by U.S. Biochemical Corp. Multiple clones were isolated for
both Dgc1 and Dgc
1, and both strands of all the cDNA clones
were sequenced. The MacVector software package (International
Biotechnologies, Inc.) was used to search the GenBank
and
NBRF data bases and to align the amino acid sequences.
RNA Northern Hybridizations
Poly(A) mRNA was electrophoresed through a 1% formaldehyde-agarose gel
and transferred to Hybond-N nylon membrane with 20
SSPE(25) . The mRNA was cross-linked to the nylon membrane using
ultraviolet light, followed by hybridizing the membrane to an antisense
RNA probe. The RNA probes were transcribed in vitro from
Dgc
1 and Dgc
1 cDNA clones in the plasmid Bluescript
SK
. The synthesis of the probe and the hybridization
and wash conditions were as described(25) .
Polytene Chromosome in Situ
Hybridization
Biotinylated cDNA probes, representing the open
reading frame and 3`-untranslated sequence of Dgc1 and Dgc
1,
were hybridized in situ to polytene chromosomes and detected
by peroxidase staining as described (26). The hybridizations were
repeated with different cDNA and genomic clones to confirm the
chromosomal assignment of both genes. The previously mapped dgq cDNA (31) was used as a positive control to ensure the
consistency of the hybridization technique between trials.
Polyclonal Antiserum to the Guanylyl Cyclase
Proteins
To obtain an anti-Dgc1 polyclonal antiserum, a
675-base pair EcoRI-BamHI fragment (Fig. 1, amino acids 143-369) was subcloned into the pGEMEX-1
plasmid (Stratagene) and expressed as a T7 gene 10 fusion protein. The
orientation and open reading frame of the construct was confirmed by
DNA sequence analysis. SDS-polyacrylamide gel electrophoresis analysis
of bacterial extracts confirmed an IPTG-induced fusion protein at the
appropriate molecular mass. The fusion protein band was excised from
the gel, purified by electroelution(27) , and used as an
immunogen in mice(28) .
Figure 1:
Amino acid
sequence comparison of Dgc1 and the rat GC
1. The amino acid
single letter codes for Dgc
1, soluble rat lung GC
1 (rGC
1) (18), and the soluble Drosophila Dgc1 proteins (33) are shown. The Dgc
1 presumed initiation
methionine codon was selected based on six of seven nucleotides
matching the Drosophila consensus translational initiation
site (37) and its being the first methionine codon after an in-frame
stop. This initiation codon also corresponds to the initiation
methionine in Dgc1 (33) and yields a protein whose predicted molecular
weight is very close to that calculated from immunoblots (Fig. 5).
Identical amino acids between Dgc
1 and the rat GC
1 are
labeled with filledcircles, while conserved amino
acid substitutions (D = E, F = Y, I = L =
V, K = R, and S = T) are labeled with opencircles. Dashes have been inserted to maximize
the extent of the amino acid identity. The Dgc
1 and Dgc1 sequences
differ in only three regions, which are underlined in the Dgc1
sequence. The amino acids that are highly conserved among all guanylyl
cyclases (2) are labeled with a starabove the Dgc
1 sequence. The Dgc
1 amino acid numbers
are shown to the right .
To obtain an anti-Dgc1
polyclonal antiserum, a 894-base pair BstYI/XhoII
fragment (Fig. 2, amino acids 252-550) was
subcloned into the BamHI site of the pGEX-1 plasmid to produce
a hybrid protein with the glutathione S-transferase protein of Schistosoma(27) . The orientation and open reading
frame of the construct was confirmed by DNA sequence analysis.
SDS-polyacrylamide gel electrophoresis analysis of bacterial extracts
confirmed an IPTG-induced production of the fusion protein at the
appropriate molecular mass. The fusion protein was purified from
IPTG-induced DH5
cells containing the expression construct by
affinity chromatography on immobilized glutathione and used to immunize
mice(28) .
Figure 2:
Amino acid sequence comparison of
Dgc1 and the rat lung GC-S
. The amino acid single
letter codes for Dgc
1 and the soluble rat lung GC-S
subunit (rGC
1) (18) are shown. The Dgc
1
presumed initiation methionine codon was selected based on six of seven
nucleotides matching the Drosophila consensus translational
initiation site (37) and its being the first methionine codon after an
in-frame stop. This initiation codon yields a protein whose predicted
molecular weight is very close to that calculated from immunoblots
(Fig. 5), and it generates the maximum amino acid homology at the
amino-terminal end with the other soluble guanylyl cyclase
subunits. Identical amino acids between Dgc
1 and GC-S
are labeled with filledcircles, while
conserved amino acid substitutions (D = E, F = Y, I
= L = V, K = R, and S = T) are labeled with opencircles. Dashes are inserted to
maximize the extent of the amino acid identity. The amino acids that
are highly conserved among all guanylyl cyclases (2) are labeled with a
star above the Dgc
1 sequence. The histidine at position 105
(labeled with an exclamationpoint) corresponds to
the nitric oxide insensitive His-to-Phe mutation, which prevents heme
binding to the
1 subunit (35). The Dgc
1 amino acid numbers
are shown to the right.
Based on several criteria, both polyclonal
antisera demonstrated the proper specificity. First, both antisera
recognized the respective purified fusion proteins in immunoblot
analyses. Second, both antisera elicited a positive signal in Drosophila head sections using standard
procedures(29) , while the preimmune sera did not stain Drosophila head sections. Third, both antisera detected
proteins from Drosophila heads with the predicted molecular
mass on immunoblots, with the exception of the additional 155-kDa
protein recognized by the anti-Dgc1. Fourth, both antisera
detected proteins of the expected molecular mass in the transfected
cells (Fig. 7).
Figure 7:
Immunoblot analysis of the Dgc1 and
Dgc
1 proteins in stably transfected human kidney 293 cells. Human
kidney 293 cells that were not transfected (293 cells) or
stably transfected with Dgc
1 (293 cells w/Dgc
1),
Dgc
1 (293 cells w/Dgc
1), or both (293 cells
w/Dgc
1 & Dgc
1) were harvested and homogenized. The
soluble fractions were isolated and equivalent amounts of total protein
were electrophoresed through a 10% SDS-polyacrylamide gel. As a
control, Drosophila head extracts (Oregon-R heads)
were run adjacent to the transfected cell extracts to confirm that the
guanylyl cyclase proteins were correctly migrating. The proteins were
transferred to nitrocellulose and incubated with either the Dgc
1
antiserum or the Dgc
1 antiserum (A and B,
respectively). Detection was performed with an alkaline
phosphatase-conjugated goat anti-mouse secondary antibody. No
detectable guanylyl cyclase was observed in the 293 cells. The 74-kDa
Dgc
1 protein was detected in both of the lines that were
transfected with the Dgc
1 cDNA. The 84-kDa Dgc
1 protein was
also detected in both of the lines that were transfected with the
Dgc
1 cDNA. The molecular masses were based on the Bio-Rad protein
markers and the corresponding Oregon-R head proteins in Fig.
5.
Immunoblot
Protein extracts were obtained by
homogenizing either 15 fly heads or two bodies in 30 µl of
extraction buffer (60 mM Tris, pH 7.0, 2% SDS, 10% glycerol,
0.001% bromphenol blue, and 1% -mercaptoethanol or 5 mM dithiothreitol). The samples were boiled for 5 min, and 20 µl
were separated by 10% SDS-polyacrylamide gel electrophoresis and
electroblotted onto nitrocellulose (Nitro ME, Micro Separations, Inc.).
The membrane was blocked in 5% non-fat dry milk in TBS (20 mM Tris (pH 7.5), 500 mM sodium chloride) overnight at room
temperature and then rinsed in TBS containing 0.05% Tween-20 (TTBS).
Primary antibody was diluted 1:500 in 2% non-fat dry milk/TTBS and
incubated with the membrane for 2 h at room temperature. Following
three 10-min washes in TTBS, the nitrocellulose was incubated with
either
I-Protein A (specific activity >30
µCi/µg) at 1
10
dpm/ml or alkaline
phosphatase-conjugated goat anti-mouse secondary antibody in 2% non-fat
dry milk, TTBS for 2 h. After washes in TTBS as above, the membrane was
dried and exposed to x-ray film for at least 18 h or developed using
Bio-Rad alkaline phosphatase conjugate substrate kit.
Immunohistochemistry
Immunofluorescent detection
of antibody staining Drosophila heads was performed
essentially as described(29) . White-eyed (w) wild-type adult heads were frozen in
Tissue-Tek OCT compound (Miles Laboratories), and 8-µm sections
were cut on a Zeiss Microm cryostat. Sections were retrieved onto
Superfrost Plus slides (Fisher) and allowed to dry for at least 30 min
at room temperature before staining. Sections were fixed for 30 min in
phosphate-buffered 2% formalin and washed for 5 min in 0.05%
Tween-20/TBS. Primary antibody was diluted 1:750 in TBS and incubated
with the slides in a humidified chamber for 20 min at room temperature.
The slides were washed for 5 min in 0.05% Tween-20/TBS and incubated
with a 1:50 dilution of fluorescein isothiocyanate-conjugated goat
anti-mouse secondary antibody for 15 min at room temperature and then
washed for 5 min in 0.05% Tween-20/TBS. Coverslips were mounted with
90% glycerol and 0.1% phenylenediamine/TBS.
Transfection
The Dgc1 cDNA, in Bluescript
SK
, was digested with XbaI and EcoRV to generate a restriction fragment containing the entire
Dgc
1 cDNA. This fragment was then cloned into a XbaI/HpaI-digested pCIS2M vector(30) , which
places the guanylyl cyclase cDNA transcription under the control of the
human cytomegaloviral major immediate early promoter and
enhancer(30) . A similar construct was made by isolating the BanIII/XbaI restriction fragment of Dgc
1 in
Bluescript SK
and subcloning it into the BanIII/XbaI sites of pCIS2M. 10
293 cells
(transformed primary human embryonic kidney, American Type Culture
Collection CRL 1573) were split in 60-mm plates and incubated with 3.5%
CO
at 36.5 °C in a humidified incubator. After
16-20 h, at about 70% confluency, the DME/F-12/FBS medium was
replaced with a fresh 5 ml, and the cells were incubated under the same
conditions for an additional 3 h. The cells were transfected with 5
µg of pCIS2M construct (or 5 µg of both pCIS2M constructs in
the cotransfection) and 0.5 µg of pRSVneo (which confers neomycin
resistance as a selectable marker for transfection(23) ) per
plate by the calcium phosphate method followed by glycerol
shock(23) . Following a 48-h incubation, the cells were replated
in selective medium containing 400-800 µg/ml G-418 Sulfate
(Sigma). Individual colonies of resistant cells were cloned and
propagated. Guanylyl cyclase protein expression was examined in the
lines that were stably transfected with the Dgc
1 and Dgc
1
cDNAs individually and together by immunoblots. The transfected lines
expressed the expected guanylyl cyclase protein(s) on immunoblots,
while no cross-reacting proteins were detected on immunoblots from
either the untransfected 293 cells nor 293 cells that were transfected
with only the pCIS2M vector that did not contain a guanylyl cyclase
cDNA insert (data not shown).
g for 5 min, and the supernatant was
recentrifuged for 20 min at 200,000
g. The protein
concentration in the cytosolic fraction was determined using the BCA
protein assay protocol (Pierce), with the supplied bovine serum albumin
as the protein standard.
Guanylyl Cyclase Assay
Guanylyl cyclase activity
of the cytosolic fraction (45 µg of protein/assay tube) was
determined by incubation at 37 °C in 50 mM
triethanolamine-HCl buffer (pH 7.4) containing 3 mM MgCl, 3 mM dithiothreitol, 1 mM 3-isobutyl-1-methylxanthine, 1 mM cGMP, 0.1 mM GTP, 5 mM creatine phosphate, 4.6 units/tube of creatine
phosphokinase Type I (EC 2.7.3.2), and 0.25 µCi
[
-
P]GTP with or without 0.1 mM
sodium nitroprusside in a total volume of 0.1 ml(31) . The
remaining [
-
P]GTP and other side products
were precipitated using zinc carbonate. The
P-labeled cGMP
remaining in the supernatant was further purified by anion exchange
chromatography over an acid alumina column(31) . The
P-labeled cGMP was eluted with sodium acetate, and
Cerenkov radiation was determined by liquid scintillation counting.
Initially, we examined cGMP production at 0, 5, 15, 30, and 40 min. We
determined that the 15-min time point was consistently in the linear
range of the assay, and we repeated all the assays using only the 0-
and 15-min time points for .
Isolation of Guanylyl Cyclase cDNAs
We are
interested in molecules that are important in Drosophila neuronal function. To identify such molecules, we isolated several
hundred cDNAs from a subtracted library that corresponded to mRNAs that
are expressed in the Drosophila head but not in the
body(25) . A subset of these corresponded to molecules involved
in visual transduction, such as rhodopsin, arrestin, and a G
protein(22, 32) . Another cDNA, 8H7, identified two
mRNAs by Northern hybridization, one retina-specific and the other
head-specific. We screened two Drosophila head cDNA libraries
with the 8H7 cDNA as a probe and identified several cDNA clones that
fell into three classes. All the cDNAs were similar in nucleotide
sequence in the 3` region of the open reading frame. However, cDNAs
within each class had identical nucleotide sequences throughout the
entire open reading frame. Based on the predicted amino acid sequences,
two of the classes corresponded to soluble guanylyl cyclase
and
subunits and the third class corresponded to a particulate
guanylyl cyclase.
subunit, Dgc
1, is shown in Fig. 1.
The putative protein has a predicted molecular mass of 76 kDa and lacks
potential hydrophobic membrane-spanning domains. This is similar to
other guanylyl cyclase
subunits. The Dgc
1 and rat soluble
guanylyl cyclase
1 subunits share 35% identity and an additional
8% conserved amino acid substitutions (Fig. 1). The Dgc
1
sequence also exhibits 35% amino acid identity with the rat
2
sequence (data not shown). Dgc
1 is 93% identical with the 46
invariant amino acids in the guanylyl cyclase catalytic
domain(2) . The Dgc
1 amino acid sequence is also 94%
identical to the previously reported Drosophila Dgc1 soluble
guanylyl cyclase
subunit sequence (Fig. 1)(33) . All
42 amino acid differences between the two Drosophila
sequences fall within three regions, which are due to seven nucleotides
that are present in Dgc
1 and absent in Dgc1. Thus, the Dgc
1
and Dgc1 cDNA sequences are 99.4% identical at the nucleotide level
within the open reading frame.
1 soluble guanylyl cyclase subunit is shown in Fig. 2. The Dgc
1 protein and the rat soluble guanylyl
cyclase
1 subunits share 59% identity and another 8% conserved
amino acid substitutions (Fig. 2). The Dgc
1 sequence
possesses less identity with the rat kidney soluble
2 sequence
(27%; data not shown, Ref. 16). However, Dgc
1 is 98% identical
with the 46 invariant amino acids in the guanylyl cyclase catalytic
domain(2) . The putative Dgc
1 protein has a molecular mass
of 86 kDa and lacks hydrophobic regions. While most guanylyl cyclase
subunits are larger than the
subunits, Dgc
1 is larger
than any of the known
subunits. This is due to an additional 118
amino acids located near the amino terminus of Dgc
1 that are not
present in any previously characterized
subunits (Fig. 2, amino acid residues 169-286). Because three different
cDNA clones from two different cDNA libraries had the 118-amino acid
insertion in the highly conserved
sequence, this region is not
likely a cloning artifact.
Localization of Guanylyl Cyclase Genes
We
localized the dgc1 and dgc
1 genes in
situ to polytene chromosomes. Both genes are located on the third
chromosome, with the dgc
1 gene at 99B and the dgc
1 gene at 100B (Fig. 3, panelsA and B, respectively). Two lethal mutations and a Minute mutation were previously mapped to the 99B region
(FLYBASE Project, Indiana University Gopher). The 100B region contains
5 previously identified lethal mutations. Because both the Dgc
1
and Dgc
1 proteins are expressed throughout the brain neuropil (see
below), it is possible that a mutation in either of the two genes could
result in a lethal phenotype.
Figure 3:
In situ hybridization of the Dgc
cDNAs to polytene chromosomes. The Dgc1 and Dgc
1 cDNAs were
biotinylated, hybridized to third instar larval chromosomes, and
detected as described under ``Experimental Procedures.'' The
Dgc
1 cDNA hybridized to the 99B region on the third chromosome (arrow in A), while the Dgc
1 cDNA hybridized to
the 100B region on the third chromosome (arrow in B).
Expression of Guanylyl Cyclase Genes
To determine
the broad tissue distribution and the sizes of the dgc1 and dgc
1 mRNAs, we examined their mRNA expression
using Northern blots. We synthesized in vitro antisense RNA
probes to the Dgc
1 and Dgc
1 cDNAs and hybridized them to
Northern blots containing poly(A)
mRNA isolated from
wild-type Drosophila heads, wild-type bodies, and eyes
absent (eya) heads. The eya mutant lacks
compound eyes and most of the underlying optic lobes(34) . Using
the Dgc
1 open reading frame as a probe, we detected three mRNAs (Fig. 4A). A predominant 2.4-kb mRNA and a less abundant
2.8-kb mRNA were both identified in wild-type and eya heads
but not in bodies. The detection in eya heads suggests that
the mRNA is expressed in non-retinal head tissues, although not
exclusively. The third mRNA is 4.1 kb and may be retina-specific
because it is not detected in either eya heads or bodies.
Because the longest original Dgc
1 cDNA was 2,352 base pairs and
could correspond to either the 2.4- or 2.8-kb mRNA, we rescreened the Drosophila head cDNA libraries to identify a Dgc
1 cDNA
that corresponded to the 4.1-kb mRNA. We isolated a 3.8-kb cDNA whose
sequence was identical with the open reading frame and the
3`-untranslated sequence of the smaller Dgc
1 cDNAs (data not
shown). However, the 3.8-kb cDNA had a different 5`-untranslated
sequence than the other Dgc
1 cDNAs, which could be generated
through alternative splicing. We repeated the Northern blot using the
5`-untranslated region of the 3.8-kb cDNA as a probe and detected the
4.1-kb mRNA and not the smaller Dgc
1 mRNAs (Fig. 4B). Therefore, the dgc
1 gene is
likely to be alternatively spliced, yielding three mRNAs that encode
the same 76-kDa protein. The Dgc
1 probe detected a 4.6-kb mRNA
(see Fig. 6B), which was found in both wild-type and eya heads but not in bodies.
Figure 4:
Northern blot analysis of the Dgc1
and Dgc
1 mRNAs. Poly(A)
mRNA isolated from
wild-type Oregon-R heads (O-R heads), Oregon-R bodies (O-R
bodies), and eyes absent heads (eyaheads) was electrophoresed through a formaldehyde-agarose
gel. The mRNA was transferred to Hybond-N membranes and hybridized with
an antisense RNA probe generated from either the Dgc
1 open reading
frame (A), the 5`-untranslated region of the 3.8-kb Dgc
1
cDNA (B), or the Dgc
1 open reading frame (C).
After autoradiography, the probes were stripped, and the membranes were
rehybridized with a ribosomal RNA probe to confirm that similar amounts
of mRNA were present in each lane (data not shown). The sizes of some
of the RNA molecular weight markers (dashes) and the soluble
guanylyl cyclase mRNAs (arrows) are shown in
kilobases.
Figure 6:
Protein localization of the Dgc1 and
Dgc
1 proteins in Drosophila heads. Frozen Drosophila head sections (8 µm) were stained with either the Dgc
1 (A and B) or Dgc
1 (C and D)
polyclonal antiserum, followed by detection with a fluorescein
isothiocyanate-conjugated goat-anti-mouse antibody. Mouse preimmune
sera failed to generate any detectable signal on the Drosophila sections (data not shown). A, the Dgc
1 antiserum
stains the retina and underlying optic lobes (lamina, medulla, lobula,
and lobula plate) of the adult visual system. The regions are labeled
as follows: r, retina; l, lamina; lo,
lobula; lp, lobula plate; m, medulla. B, the
Dgc
1 antiserum stains throughout the neuropil (n) of the
adult brain. The brain cortex (c) has very little if any
detectable signal. C, the Dgc
1 antiserum stains the
visual system's optic lobes. However, there is very little, if
any, detectable signal in the retina. The retina and optic lobes are
labeled as described in A. D, the Dgc
1 antiserum
stains much of the adult brain neuropil, with very little staining in
the cortex.
Generation of Antisera against Drosophila Guanylyl
Cyclase Proteins
We generated mouse polyclonal antisera against
Dgc1 (amino acids 143-369) and Dgc
1 (amino acids
252-550) bacterial fusion proteins. These fusion proteins contain
very little of the conserved guanylyl cyclase catalytic domains, which
should reduce cross-reactivity with other guanylyl cyclases. We
examined the specificity of these antisera by immunoblots. Homogenates
of wild-type heads, eya heads, and wild-type bodies were
electrophoresed under reducing conditions, transferred to
nitrocellulose, and incubated with the antisera. The Dgc
1
antiserum detected a 74-kDa protein in both wild-type and eya heads and a 155-kDa protein in only wild-type heads (Fig. 5A). The 74-kDa protein is in close agreement with
the 76 kDa predicted from the conceptual translation of the cDNAs. The
74-kDa protein must be expressed in both the retina (due to the
retina-specific 4.1-kb mRNA) and in regions of the brain (due to all
three mRNAs). The nature of the 155-kDa protein, which appears to be
retina-specific, is unknown. However, it is not encoded by any of the
three dgc
1 mRNAs (see ``Discussion''). The
Dgc
1 antiserum detected an 84-kDa protein (Fig. 5B), which is in close agreement with the
conceptually translated 86-kDa protein. Consistent with the Northern
blot, the Dgc
1 protein is expressed in wild-type and eya heads but not detected in wild-type bodies.
Figure 5:
Immunoblot analysis of the Dgc1 and
Dgc
1 proteins in Drosophila. Wild-type Oregon-R heads (O-R heads), eyes absent heads (eyaheads), and Oregon-R bodies (O-R bodies) were
isolated, homogenized in SDS sample buffer (36), electrophoresed
through a 10% SDS-polyacrylamide gel, and then transferred to
nitrocellulose. The nitrocellulose was incubated with a mouse
polyclonal antiserum generated against either a Dgc
1 or a
Dgc
1 fusion protein (A and B, respectively),
followed by detection using either alkaline phosphatase-conjugated goat
anti-mouse antibody or
I-labeled Protein A (A and B, respectively). Mouse preimmune sera failed to
detect either of the proteins (data not shown). 74-kDa head-specific
and 155-kDa retina-specific proteins were detected with the Dgc
1
antiserum, while the Dgc
1 antiserum detected only an 84-kDa
protein in both the Oregon-R and eya head samples. The
molecular masses of the Bio-Rad protein markers are shown to the left of each panel (dashes).
Localization of Guanylyl Cyclase Subunits in Drosophila
Head
We used the Dgc1 and Dgc
1 polyclonal antisera to
localize the soluble guanylyl cyclase subunits in the adult head. The
Dgc
1 antiserum stained the retina and underlying optic lobes
(lamina, medulla, lobula, and lobula plate) of the adult visual system (Fig. 6A). In addition, the Dgc
1 antiserum stained
a diffuse pattern throughout the adult brain neuropil (Fig. 6B). No obvious brain structures, such as the
mushroom bodies or the central complex, were preferentially stained.
The Dgc
1 immunodetection is consistent with the predicted
localization based on the Northern blots and immunoblots. While the
Dgc
1 antiserum failed to stain the adult retina, it did stain the
optic lobes (Fig. 6C). This suggests that the Dgc
1
protein is localized to either the photoreceptor axons or the secondary
and tertiary neurons of the visual system. Similar to the Dgc
1
pattern, the Dgc
1 antiserum also stained much of the adult brain
neuropil (Fig. 6D).
The Dgc
We examined whether
Dgc1 and Dgc
1 Proteins Function as a
Soluble Heterodimeric Guanylyl Cyclase
1 and Dgc
1 possessed guanylyl cyclase activity. Because
soluble guanylyl cyclase proteins function as heterodimers, we expected
the activity would only be found when both proteins were coexpressed.
We stably transfected human embryonic kidney cell line 293 with either
the Dgc
1 or the Dgc
1 cDNAs in the pCIS2M vector(30) .
We also stably cotransfected both the Dgc
1 and Dgc
1 cDNA
constructs into 293 cells. Immunoblots revealed that the 293 cells did
not express a protein that cross-reacts with either polyclonal
antiserum (Fig. 7). Lines transfected with the Dgc
1 cDNA
expressed only the 74-kDa Dgc
1 protein (Fig. 7A),
while the Dgc
1 transfected lines correctly expressed the 84-kDa
protein (Fig. 7B). It is interesting to note that the
cotransfected lines expressed more Dgc
1 and Dgc
1 protein than
the singly transfected lines (Fig. 7). This was observed in at
least three different stably transfected lines for each construct (data
not shown).
1 and Dgc
1 demonstrated any significant guanylyl cyclase
activity. This line possessed nearly 40-fold more guanylyl cyclase
activity than did a mixture containing the soluble fractions from the
individually transfected lines (5.61 and 0.144 pmol of cGMP
produced/mg/min, respectively). Mixing the individual Dgc
1 and
Dgc
1 soluble fractions resulted in guanylyl cyclase activity that
was not significantly different from that of either of the individual
subunits. Sodium nitroprusside increased the cotransfected line's
guanylyl cyclase activity approximately 35-fold, while it had only a
minimal effect on the four other cell preparations. Previously
characterized soluble guanylyl cyclases were stimulated
10-50-fold by sodium
nitroprusside(15, 17, 18) . Taken together,
these data demonstrate that the Dgc
1 and Dgc
1 proteins
possess guanylyl cyclase activity and are stimulated like the
previously characterized soluble guanylyl cyclase subunits.
and
subunit from Drosophila melanogaster. The Dgc
1 protein
closely resembles the previously identified
subunits in its
molecular mass and its similarity in amino acid sequence, particularly
in the catalytic domain. While Dgc
1 possesses a greater level of
amino acid identity with the other
subunits than Dgc
1 has
with the other
subunits, the additional 118 amino acids in the
amino-terminal half of Dgc
1 is unique among the soluble guanylyl
cyclase subunits. This 118-amino acid sequence does not appear to be an
artifact based on two independent lines of evidence. First, several
independent cDNA clones were isolated from two different libraries and
they all possessed the nucleotide sequence encoding the 118 amino
acids. Second, immunoblots identified a Drosophila head
protein that very closely matched the predicted molecular mass of the
Dgc
1 protein. These amino acids did not block guanylyl cyclase
activity of the heterodimeric protein or prevent sodium nitroprusside
stimulation. Analysis of this 118-amino acid domain with the MacVector
software identified only a few potential functional motifs. Three
potential casein kinase sites ((S/T)X(D/E)) are present at
amino acid positions 210, 267, and 286. Two potential casein kinase II
phosphorylation sites ((S/T)XX(D/E)) are present at amino
acids 212 and 250. Last, a potential cAMP or cGMP-dependent protein
kinase phosphorylation site ((R/K)XX(S/T)) is present at amino
acid 284. It is not clear if any of these sites or any other
unidentified motifs play a functional role in either the activity or
regulation of the Dgc
1 protein. Therefore, the function of this
region must await further biochemical or genetic analyses.
and
subunits
are expressed in the Drosophila brain; however, their
distribution is not completely overlapping. Most notably, the 74-kDa
Dgc
1 protein is expressed in the retina, while no significant
level of Dgc
1 is detected in the retina (Fig. 6). The 74-kDa
protein must be expressed in the retina because the 4.1-kb mRNA, which
encodes the 74-kDa protein, is expressed in wild-type heads and not
detected in eya heads. By comparison, this mRNA must be
expressed in the tissue that is missing in the eya mutant,
which is primarily the compound eyes(34) . Because the 84-kDa
Dgc
1 protein is not expressed in the retina, an additional soluble
guanylyl cyclase
subunit must exist to generate a functional
heterodimer with the 74-kDa Dgc
1 in the retina. Even though
Dgc
1 and Dgc
1 are not both expressed in the retina, they may
still be coexpressed and form a functional heterodimer in the optic
lobes or elsewhere in the brain (Fig. 6). However, we could not
determine whether the two subunits are expressed in the same neuron
because of the diffuse staining in the brain. It is also possible that
Dgc
1 interacts with Dgc
1 in some neurons and with an
unidentified
subunit in others.
1 antiserum remains unknown.
It is not likely an artifact of the antiserum because two different
mice produced polyclonal antisera that detected the 74- and 155-kDa
proteins on immunoblots. However, this protein is not encoded by the dgc
1 gene based on two criteria. First, cDNAs
representing the different Dgc
1 mRNAs have an identical open
reading frame, which encodes the 74-kDa protein. Second, the 155-kDa
protein is not a dimer of the 74-kDa protein because the samples were
electrophoresed under reducing conditions and it was not detected in
the Dgc
1 transfected cell lines (Fig. 7). This suggests that
the retina-specific 155-kDa protein shares a common epitope with
Dgc
1. This epitope is most likely not within the highly conserved
catalytic domain for three reasons. First, the Dgc
1 fusion protein
used to generate the polyclonal antiserum contained very little of the
catalytic domain. Second, the Dgc
1 antiserum did not detect the
Dgc
1 protein on immunoblots, which also possesses the highly
conserved catalytic domain. Third, an antiserum generated against the
Dgc
1 guanylyl cyclase catalytic domain failed to detect the
155-kDa protein on immunoblots and failed to stain the retina (data not
shown).
1 and Dgc
1 subunits behaved biochemically
like the previously characterized guanylyl cyclase subunits, with
activity requiring coexpression of both Dgc
1 and Dgc
1 and its
stimulation by sodium nitroprusside. It was previously shown that the
and
subunits must be coexpressed to generate a detectable
enzymatic activity(18) . Expression of either subunit alone or
the mixing of the subunits prior to the in vitro assay failed
to generate any detectable enzymatic activity(17) . This
suggests that the
and
subunits require an assembly that is
associated with translation or that some post-translational
modification occurs that requires the presence of both subunits.
Coexpressing Dgc
1 and Dgc
1 resulted in higher levels of the
cyclase subunits than individually expressing the subunits (Fig. 7A, lanes2 and 3; Fig. 7B, lanes2 and 3). This
increased expression in the cotransfected lines was observed for
several independent lines, which suggests that the subunits enhance the
transcription or translation of the other subunit or that the subunits
somehow stabilize their heterodimeric partner. This stabilization could
be the mechanism that permits coexpressed subunits to form
enzymatically active heterodimers and blocks the mixture of
individually expressed subunits from demonstrating guanylyl cyclase
activity. The sodium nitroprusside stimulation further suggests that
the Drosophila protein has a heme moiety similar to previously
characterized soluble guanylyl cyclases. Consistent with this idea,
Dgc
1 possesses a histidine residue at position 105 (Fig. 2)
that is conserved in the rat
1 sequence. This histidine was shown
by site-specific mutagenesis to be required for heme binding and
subsequent guanylyl cyclase activity(35) . Thus, regulation of
the Drosophila soluble guanylyl cyclase activity may be
similar to other soluble guanylyl cyclases.
1 and Dgc
1 are coexpressed in the same
cells within the Drosophila head, they produced a functional
heterodimer in human kidney 293 cells. Not all heterodimer combinations
yield guanylyl cyclase activity. For example, the rat
2 does not
exhibit activity when coexpressed with either
1 or
2(21) . Formation of a Dgc
1 and Dgc
1 functional
heterodimer in transfected cells suggests that their expression
coincides in Drosophila and they form a functional heterodimer in vivo. If Dgc
1 interacts with Dgc
1 in some neurons
and with a different
subunit in other neurons, this could provide
a mechanism to alter the kinetics or regulate the guanylyl cyclase
activity in different cells. Even when different
and
combinations produce functional protein, their relative activities can
be drastically different. For example, the rat
1 subunit generates
a 3-6-fold more active heterodimer with
1 than with
2(15) .
1 and the previously isolated dgc1 genes are
the same. First, the Dgc
1 subunit possesses greater than 94% amino
acid identity with the Drosophila Dgc1 guanylyl cyclase
subunit(33) , with all the amino acid differences between the
two proteins localizing to three regions (Fig. 1). Similarly, the
two cDNA sequences revealed greater than 99% nucleotide sequence
identity. The Dgc
1 sequence possesses seven additional nucleotides
not present in dgc1 (GG-637, GG-638, C-711, A-1598, G-1603,
C-1615, G-2021), which accounts for all the amino acid differences by
shifting the reading frame. Second, both genes localize to the same
region on the right arm of the third chromosome. While the dgc1 gene was originally localized to 63A on the left arm of the third
chromosome, careful examination of the dgc1 chromosomal in
situ hybridization ( Fig. 3in Ref. 33) revealed that the
signal is actually in the distal region of the right arm of the third
chromosome. This is very close, if not identical, to our localization
of dgc
1 to the 99B region. Liu et al.(38)
independently isolated the dgc
1 gene and their DNA
sequence and chromosomal map position are consistent with our results.
Taken together, the previously isolated dgc1 and our dgc
1 genes are identical.
1 mRNA that was
equally abundant in both wild-type and eya heads, while the
2.5-kb Dgc1 mRNA was dramatically more abundant in wild-type whole
flies than eya whole flies(33) . While the mRNA size
difference is not significant, the relative mRNA expression in
wild-type versuseya heads is important because it
suggests that the corresponding protein is either expressed throughout
the head (Dgc
1) or is predominantly expressed in the retina
(Dgc1). Two lines of evidence with the guanylyl cyclase protein's
expression are consistent with our RNA analysis. First, immunoblots
revealed equivalent amounts of Dgc
1 protein in both wild-type and eya heads. Second, immunostaining demonstrated that the
Dgc
1 protein was expressed abundantly in the optic lobes and brain
neuropil and to a lesser extent in the retina. Liu et al.(38) independently detected significant Dgc
1 protein
expression in the optic lobes and central brain neuropil.
and
genes in Drosophila will permit a molecular and genetic
analysis of the role of these proteins. Because the Dgc
1 and
Dgc
1 subunits are expressed throughout the adult brain, it is
possible that the corresponding dgc
1 and dgc
1 mutations may be lethal. Lethal mutations were already identified
in the 99B and 100B regions. Germ line transformation of wild-type dgc
1 and dgc
1 genes and the subsequent
suppression of lethality would demonstrate that the lethal mutation is
in the dgc gene. The dgc
1 and dgc
1 mutations would also enhance our analysis of the functional
interactions between these two subunits in vivo and would
expedite our study of the role of the extra 118-amino acid domain in
Dgc
1.
Table: Guanylyl
cyclase activity in stably transfected human embryonic kidney 293 cells
1 or
Dgc
1 cDNA or with both cDNAs and assayed for guanylyl cyclase
activity as described under ``Experimental Procedures.''
Heat-denatured 293 cytosolic fractions had the same guanylyl cyclase
activity as the 293 cytosolic fraction. Activity was determined as
previously described (31), in the presence and absence of 0.1 mM sodium nitroprusside (SNP). Each value represents the mean
± S.D. (n = 9).
/EMBL Data Bank with accession number(s) U27117 and
U27123.
-D-galactopyranoside; TBS,
tris-buffered saline.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.