From the
We have isolated, by interspecies hybridization, two classes of
Drosophila cDNA each encoding a different guanylate cyclase
(GC). One of them encodes an
Guanylate cyclase (GC)
Two
types of guanylate cyclase have been described, the receptor-type and
the soluble-type, which, in most cases, coexist in the same cell. The
soluble form of GC (sGC) has been purified from bovine and rat lung
tissues and has been shown to be a heme-containing protein of
approximately 150 kDa consisting of two subunits,
The diffusible second messenger, nitric oxide (NO), has been
shown to be a physiological activator for sGC (Palmer et al.,
1987; Waldman and Murad, 1987), and its activation has been found to
require a heme moiety. Thus, activation is thought to be triggered by
the complex formed between NO and the GC-associated heme (Waldman and
Murad, 1987). NO synthase (the enzyme required for NO production), sGC,
and cGMP have been localized immunocytochemically in many regions of
the mammalian central and peripheral nervous systems (Waldman and
Murad, 1987; Garthwaite, 1991; Vincent and Hope, 1992), indicating
their apparent importance to the nervous system.
Receptor-type GCs
identified to date all consist of a single polypeptide of about
150-200 kDa. cDNAs corresponding to three GC isoforms (GC-A, -B,
-C) in mammals and two in sea urchins have been isolated and
characterized (Chinkers et al., 1989; Schulz et al.,
1989, 1990; Singh et al., 1988). Proteins deduced from these
cDNAs all contain an extracellular ligand-binding domain, a single
transmembrane domain, and two intracellular regions, a kinase-like
domain and a cyclase domain.
Ligands for these rGCs vary. GC-A and
-B are activated by several natriuretic peptides responsible for
maintaining homeostasis of body fluids and electrolytes (Chang et
al., 1989; Chinker et al., 1989; Lowe et al.,
1989; Schultz et al., 1989; Drewett and Garbers, 1994), and
GC-C is stimulated by heat-stable enterotoxin and guanylin (Currie
et al., 1992; de Sauvage et al., 1992; Schulz et
al., 1992). In tissue in situ hybridization, both GC-A
and GC-B RNAs have been found in a wide range of tissues, including the
brain (Wilcox et al., 1991).
Little is known about
GC-cGMP-mediated biochemical pathways in invertebrates. Nevertheless,
cGMP has been implicated in a number of invertebrate neuronal
processes. Bacigalupo et al.(1991) have reported that cGMP can
activate light-dependent ion channels in membrane patches isolated from
the ventral photoreceptor of Limulus. cGMP has also been shown
to mediate ion channel activity in neurons on the ventral face of the
parietal ganglion of the snail Helix aspersa (Paupardin-Tritsch et al., 1986b). Yoshikawa et
al.(1993) have reported on cDNA cloning of a head-enriched
In this work we attempted to isolate additional
Drosophila GC cDNAs expressed in the adult nervous system. We
describe two classes of cDNA resulting from this effort. One class,
dgc
Initial
screening yielded over 200 clones that could be grouped into 13
non-overlapping classes on the basis of mutual cross-hybridization and
chromosomal locations. Clones of two of these classes, 99B and 76C,
were investigated further because preliminary RNA blot studies
indicated that they are expressed in the head. Only one cDNA clone
(GC13 in Fig. 5) of the 76C class was isolated in the original
screen. Accordingly, 0.8-kb EcoRI fragments of this clone were
used to screen the previously screened head cDNA library as well as an
embryonic library (Zinn et al., 1988) to isolate eight
additional clones of this class.
For
immunocytochemistry, flies were embedded in OCT compound (Tissue Tek),
and 10-µm tissue sections were cut and transferred onto subbed
slides. The sections were fixed in 4% paraformaldehyde (Polyscience) in
PBS (10 mM Na
Of the 13 non-overlapping classes of cDNA and genomic clones
isolated by screening Drosophila libraries with rat GC probes
(see ``Materials and Methods''), we focused on two because of
preliminary indications that clones of these classes are expressed in
the head. These two will be referred to as 99B and 76C classes on the
basis of their chromosomal localizations.
Comparison of the dgc
A more significant difference,
however, is that in chromosomal in situ hybridization,
dgc
The same 0.3-kb EcoRI fragment
was used to synthesize riboprobes to examine tissue distribution of
dgc
Immunocytochemical localizations of the DGC
The spatial tissue distribution
of the drgc transcript in the adult fly was investigated using
riboprobes constructed from a 400-base pair BamHI fragment of
drgc cDNA (Fig. 5). Shown in Fig. 9A are
a head and a thoracic section hybridized with the antisense probe. It
may be seen that drgc mRNA is widely distributed in the head
and thorax with slightly higher labeling in the optic lobe and central
brain. Some labeling is also seen in the retina. In nervous tissues,
the neuropil as well as the cell bodies are labeled. Although not seen
in this particular section, the cardia and ventriculus are also
labeled. Fig. 9C is a female abdominal section
hybridized with the antisense probe showing strong labeling of the
oocytes and weaker labeling of the digestive tract. Negative controls
using sense probes showed no specific labeling (Fig. 9, B and D). Attempts have been made to express these drgc cDNA clones to generate antibodies and determine intrinsic
enzymatic activity without success.
We have identified, by low-stringency interspecies
hybridization, two guanylate cyclases of Drosophila: a soluble
type, DGC
A large body of evidence
now exists to indicate that the phototransduction pathway in
invertebrate photoreceptors involves a phosphoinositide-mediated
signaling cascade (Fein et al., 1984; Brown et al.,
1984; Payne, 1986; Bloomquist et al., 1988; Minke and
Selinger, 1991). However, the second messenger that opens the
light-activated channel(s) has not yet been identified. A few years
ago, Bacigalupo et al.(1991) reported that light-dependent
channels in excised patches of Limulus ventral photoreceptors
are opened by cGMP, implicating cGMP as a possible second messenger in
Limulus phototransduction. More recently, Baumann et
al.(1994) have reported cloning genomic DNA and cDNA encoding a
cGMP-gated channel expressed in the visual system of
Drosophila. If cGMP is utilized in phototransduction, one
would expect a guanylate cyclase(s) to be present in photoreceptors.
Consistent with this expectation, Yoshikawa et al.(1993)
suggested that dgc1 RNA is expressed in the Drosophila retina from comparison of Northern blots of wild type and eyes
absent mutants. The deduced protein product of dgc1 is
nearly identical in sequence to our soluble-type GC, DGC
Abundant expression of DGC
Very little is known about possible roles of
sGCs and NO synthases in the Drosophila nervous system.
However, it seems likely that they have roles in the Drosophila nervous system similar to those found in the mammalian brain. In
support of such a suggestion, a Drosophila NO synthase
expressed predominantly in the head has been identified from homology
to a rat NO synthase (Regulski and Tully, 1993). In view of its
abundant expression in the nervous system, DGC
The protein deduced from the other class of
cDNA isolated, drgc, has all the structural characteristics of
known receptor-type GCs: an extracellular ligand-binding region, a
transmembrane region, and an intracellular region divided into
kinase-like and cyclase domains (Fig. 5). Only one other
receptor-type GC of Drosophila has been reported previously
(Gigliotti et al., 1993). This GC, however, apparently is
expressed primarily during oogenesis and early embryonic development.
The presumptive ligand-binding domains of most receptor-type GCs are
characterized by the presence of several cysteines and potential
glycosylation sites (Chinkers et al., 1989; Schulz et
al., 1989, 1990). It has been proposed that extracellular
cysteines may be important in the ligand binding process (Yuen and
Garbers, 1992). Consistent with this picture, DrGC contains 12
cysteines and nine potential Asn-link glycosylation sites in the
extracellular region (Fig. 6). However, the extracellular domain
of DrGC displays no significant overall homology to corresponding
regions of any other rGCs, presumably reflecting the specificity of
this region for the particular ligand for this receptor.
The
kinase-like domain of receptor-type GCs was first identified from
conservation of amino acid residues between this domain of a receptor
type GC and several known protein kinases (Singh et al.,
1988). Subsequently, Yuen and Garbers(1992) showed that 22 of 33
residues that are invariant or conserved in a large number of known
protein kinases (Hanks et al., 1988) are also conserved in the
kinase-like domain of several receptor-type GCs tested. Twenty of these
22 residues are also identical or conserved in DrGC (asterisks in Fig. 7A).
In addition to the domains that are
present in other known receptor-type GCs, DrGC is characterized by the
presence of a 430-amino acid C-terminal region with no significant
homology to any known protein. The significance of this region is
unknown; it presumably represents a domain of specific importance to
DrGC.
Developmental RNA blot analysis (Fig. 8B) and
in situ hybridization of drgc riboprobes to adult
tissue sections (Fig. 9) showed that drgc RNA is present
throughout development and widely distributed in adult tissues,
including the optic lobe, central brain, thoracic ganglia, digestive
tract, and oocytes in the female abdomen (Fig. 9). The results
thus suggest that the DrGC product is widely utilized both in
development and in adults, including in adult nervous functions. Some
expression of drgc RNA is found also in the retina
(Fig. 9). Thus, a potential role of the DrGC product in
photoreceptors cannot be excluded.
For many genes that are expressed
in the central nervous system, tissue in situ hybridization
localizes RNA in the cortical regions of the nervous system, where the
neuronal cell bodies are located (e.g. see Fig. 3, Yoon
et al.(1989) and Fig. 5, Shortridge et
al.(1991)). Labeling of the central nervous tissues in drgc in
situ hybridization is distinct in that labeling is found
throughout the nervous system, including the neuropil (Fig. 9).
Thus, the drgc transcript is present not only in neuronal cell
bodies but also in neuronal processes. Oocytes are especially heavily
labeled in in situ hybridization, suggesting that the drgc product may act as a maternal transcript and plays a role in early
development.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Dr. D. L. Garbers for providing the rat
receptor-type GC cDNAs, Dr. P. Salvaterra for providing a
Drosophila head cDNA library, Dr. A. Bieber for providing a
Drosophila embryonic cDNA library, Dr. G. Koliantz and Dr. E.
Semionov for confirming the results of chromosomal localization,
Bharath Srinivasan for helpful discussions, and Ann Pellegrino for help
in preparation of the manuscript. We also thank Dr. D. Hyde and Dr. M.
Forte for communicating their results prior to publication.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit homolog of soluble GC,
designated DGC
1, and the other encodes a receptor-type GC,
designated DrGC. The dgc
1 cDNA encodes a protein of 676
amino acids and maps to 99B. In situ hybridization to adult
tissue sections showed that dgc
1 mRNA is found mainly in
the cell bodies of the optic lobe, central brain, and thoracic ganglia.
The DGC
1 protein was also localized primarily to the nervous
system by immunocytochemical staining, consistent with results of
in situ hybridization. However, no detectable expression of
this protein was found in the retina. The other class of cDNA,
drgc, maps to 76C and encodes a 1525-amino acid protein
displaying structural features similar to other known receptor-type
guanylate cyclases. However, it has a C-terminal 430 amino acid region
that has no homology to any known proteins. drgc RNA is
expressed at low levels throughout development and in adult heads and
bodies. In situ hybridizations to adult tissue sections showed
that drgc mRNA is expressed in a wide range of tissues,
including the optic lobe, central brain, thoracic ganglia, digestive
tract, and the oocyte.
(
)
catalyzes the
synthesis of cGMP, an important intracellular second messenger. It
responds, directly or indirectly, to a diverse spectrum of stimuli,
including hormones (Koesling et al., 1991; Goy, 1991) and
neurotransmitters (Bredt and Snyder, 1992; Garthwaite, 1991), and its
product, cGMP, has been shown to regulate a wide variety of effectors
from ion channels (Fesenko et al., 1985) to protein kinases
(Cornwell and Lincoln, 1989; Paupardin-Tritsch et al., 1986a;
Zhuo et al., 1994) in a host of tissues, ranging from smooth
muscle to the retina. Although the physiological role of cGMP in most
of these tissues remains poorly understood, its role in vertebrate
phototransduction has been well characterized. In this process, the
light-activated channels are gated by cGMP (Fesenko et al.,
1985). Light activation of cGMP-dependent phosphodiesterase results in
the hydrolysis of cGMP and closure of cGMP-gated channels to initiate
the light response of the photoreceptor. Resynthesis of cGMP by
guanylate cyclase reopens the channels, leading to the recovery of the
photoreceptor to the dark state (for review, see Stryer(1986)).
and
(Kamisaki et al., 1986; Humbert et al., 1990). cDNAs
corresponding to both subunits of bovine and rat sGCs have been
isolated and analyzed (Koesling et al., 1988, 1990; Nakane
et al., 1988, 1990). These analyses have revealed that each
subunit contains a putative cyclase domain, which is also conserved in
the receptor-type GC. Coexpression of both subunits has been shown to
be required to yield a catalytically active enzyme in COS-7 cells in
culture (Harteneck et al., 1990; Buechler et al.,
1991).
subunit homolog of Drosophila soluble GC and suggested that it
is expressed in photoreceptors. In addition, cDNA corresponding to a
Drosophila nitric oxide synthase homolog, expressed abundantly
in the head, has been isolated by interspecies hybridization (Regulski
and Tully, 1993).
1, encodes an
subunit homolog of Drosophila soluble GC expressed predominantly in the nervous system. The
other class, drgc, encodes a receptor-type GC expressed
throughout development and widely in adult tissues including the
nervous system.
Standard Procedures
Plasmid and phage DNA
purification, DNA and RNA blotting, and Southern hybridization were
performed as described by Sambrook et al.(1989). DNA probes
were labeled with [P]dCTP using the random
primer method (Feinberg and Vogelstein, 1984).
Library Screening
A mixture of rat cDNA fragments
corresponding to the kinase-like and cyclase domains of GC-A (Chinkers
et al., 1989), GC-B (Schulz et al., 1989), and GC-C
(Schulz et al., 1990) was used as probes to screen a
Drosophila head cDNA (Itoh et al., 1985) and a
genomic library (Yoon et al., 1989) under low stringency
hybridization conditions. Filters were prehybridized in a solution of
30% formamide, 0.1% SDS, 5 Denhart's solution (1
Denhart's: 0.2 mg/ml each Ficoll, bovine serum albumin, and
polyvinylpyrrolidone), 5
SSC (1
SSC: 0.15 M
NaCl and 0.015 M sodium citrate, pH 7.0), and 50 µg/ml
denatured herring sperm DNA at 42 °C. Hybridization was carried out
overnight in the same solution with
P-labeled probes
added. After hybridization, filters were washed three times in 2
SSC, 0.1% SDS for 20 min each at 50 °C.
Figure 5:
Physical maps of drgc cDNA and
representative cDNA clones and a schematic diagram of the coding
region. The physical map of drgc cDNA is shown at the
top. Lines immediately below it correspond to cDNA
fragments used for RNA blot analysis (3.0 kb) and tissue in situ hybridization (0.4 kb). Physical maps of six representative cDNA
clones are shown in the middle, and a schematic diagram of the
coding region with deduced protein domains is shown at the bottom as a series of boxes. LBD, ligand-binding domain;
Kinase, kinase-like domain; Cyclase, cyclase domain;
shaded box, transmembrane domain. The unlabeled box at the C
terminus is a 430-amino acid domain without any significant homology to
any known protein. B, BamHI; E,
EcoRI; S, SalI.
RNA Isolation and RNA Blot Analysis
Total RNA was
isolated from 3 g of tissue from each developmental stage and each
adult body part examined, as described by Montell et
al.(1985). Poly(A) RNA was purified from total
RNA using oligo(dT) columns, separated electrophoretically in
formaldehyde agarose gel (Miller, 1987), and blotted to Nylon membrane
(Amersham). Prehybridization and hybridization were carried out
according to Mahmoudi and Lin(1989), and membranes were washed as
described previously (Shortridge et al., 1991).
Chromosomal in Situ Hybridization
Salivary
chromosome squashes were prepared from third instar larvae as described
by Gall and Pardue(1971). Probes were labeled with Bio-16-dUTP (ENZO
Biomedicals) using the random primer method of Feinberg and Vogelstein
(1984). Hybridization was performed as described by Engels et
al.(1986) except that Detek-I-HRP (ENZO) was used for signal
detection with 3`,3`-diaminobenzidine (0.6 mg/ml plus 0.03%
HO
) as substrate.
DNA Sequencing
DNA fragments to be sequenced were
subcloned into M13 mp18 using JM101 as host. A nested set of deletions
was generated using the Cyclone system (IBI). Sequencing of both
strands was carried out by the dideoxy chain termination method (Sanger
et al., 1977) using a T7 Sequenase kit (U. S. Biomedical
Corp.). The GCG program (Devereux, 1992) was used to analyze and
manipulate the DNA sequence, and FASTA (Lipman and Pearson, 1986) was
used to compare the deduced protein with those in the protein data base
and to analyze the deduced protein sequences.
Tissue in Situ
Hybridization
S-Labeled antisense and sense RNA
probes were prepared, using T7 or SP6 polymerase (Strategene), from
dgc
1 or drgc cDNA fragments (see
``Results'') that had been cloned into the pGEM-3Z vector
(Promega). Flies were embedded in frozen OCT (Tissue-Tek), and
10-µm tissue sections were cut on a cryostat. The sections were
processed as described by Hafen and Levine(1986) except for the
modifications in hybridization and wash conditions previously described
by Burg et al.(1993). After washing, the sections were treated
with RNase A (40 µg/ml in 0.5 M NaCl, 5 mM EDTA,
10 mM dithiothreitol, 50 mM Tris-Cl, pH 8.0) for 30
min at 37 °C to minimize nonspecific hybridization. Autoradiography
was performed essentially as in Angerer et al.(1987) using
Kodak NBT-2 autoradiographic emulsion.
Generation of Fusion Protein and Antisera
The
0.6-kb XhoI-HindIII fragment of the dgc1 cDNA (Fig. 1) was subcloned into the PGEX-KG vector (Smith
and Johnson, 1988; Guan and Dixon, 1988) using XL1-blue as the host
strain. The fusion protein was expressed and isolated according to the
procedure of Guan and Dixon(1988) except that 1 M
isopropyl-1-thio-
-D-galactopyranoside was used to induce
expression. 400 µl of the fusion protein (2 µg/µl) was
emulsified with an equal volume of Freund's adjuvant, and the
resulting mixture was injected into a rabbit. Two additional booster
injections were administered at 10-day intervals, and blood was
collected approximately 1 month after the first injection. Collected
blood was allowed to clot at 4 °C overnight, and the serum was
obtained by centrifuging the sample at 10,000
g for 10
min.
Figure 1:
Sequence differences between dgc1 and dgc1 cDNAs and deduced protein products. The
restriction map of dgc
1 cDNA is given at the top. The
thickline in the middle and the shaded
box represent, respectively, the dgc
1 cDNA sequence
and its coding sequence. Nucleotide sequence differences between
dgc1 and dgc
1 cDNAs are shown in open boxesbelow the thick line, and the corresponding
amino acid sequence differences between the deduced DGC1 and DGC
1
proteins are shown below the shaded box. Seven
additional nucleotides, at six different positions, were found in the
dgc
1 coding region in comparison to the dgc1 coding region. The six codons altered as a result of the above
differences and the corresponding amino acids are underlined in the nucleotide and deduced protein sequences, respectively. The
asterisk at the end of the DGC
1 sequence corresponds to
the stop codon TAG in the dgc
1 sequence. B,
BamHI; E, EcoRI; H,
HindIII; X, XhoI.
Western Blot Analysis and Immunocytochemistry
Head
and body samples of adult flies were obtained by freezing and vortexing
the flies and sieving the separated body parts, all at -20
°C. The samples were homogenized in 10 mM MOPS buffer, pH
6.5, 120 mM NaCl, and 1 mg/ml Peflobloc, using the Konte
homogenizing system. The homogenates were fractionated by 7.5%
polyacrylamide gel electrophoresis and transferred to nitrocellulose
filters (Schleicher & Schuell, 0.45 µm) in 20 mM Tris,
150 mM glycine, and 20% methanol (Towbin et al.,
1979). The filters were blocked in a TBST solution (10 mM
Tris, 140 mM NaCl, and 0.1% Tween 20) with 3% bovine serum
albumin and 2% goat serum and incubated for 1 h at room temperature in
the primary antiserum diluted 1:1,000 with HST solution (10 mM
Tris, pH 7.4, 1 M NaCl, 0.5% Tween 20, and 1% goat serum).
After washing in several changes of TBST solution for 1 h, the filters
were incubated in the secondary, alkaline phosphatase-conjugated goat
anti-rabbit antibody (Sigma), diluted with the HST solution, by
agitating for 1 h at room temperature. Immunolabeling was visualized by
reaction in 100 mM Tris, pH 9.5, 100 mM NaCl, and 5
mM MgCl containing 5-bromo-4-chloro-3-indolyl
phosphate and nitro blue tetrazolium chloride (Promega).
HPO
, 3 mM
KH
PO
, and 120 mM NaCl, pH 7.5) for 20
min, washed twice with PBS for 10 min each, and blocked with 5% goat
serum and 0.3% Triton X-100 in PBS, pH 7.5, for 30 min. They were
incubated overnight with primary antibodies (1:1,000 in PBS containing
1% goat serum and 0.3% Triton X-100) at 4 °C in a humidified
chamber. Then the sections were washed twice with PBST (PBS plus 0.3%
Triton X-100) containing 1% goat serum for 20 min and incubated with
secondary antibodies (1:1,000 horseradish peroxidase-conjugated goat
anti-rabbit serum in PBST) in a humidified chamber at room temperature
for 30 min, followed by a wash in PBST for 15 min. The immunolabels
were detected using 3`,3`-diaminobenzidine (0.6 mg/ml plus 0.03%
H
O
) as substrate, and the reaction was stopped
by washing in several changes of PBS. Negative controls consisted of
using either rabbit preimmune serum instead of DGC
1 antiserum or
DGC
1 antiserum preincubated with excess DGC
1-GST fusion
protein for 1 h at room temperature.
Characterization of the 99B Class Clones
There are three
cDNA clones (3.4, 2.9, and 2.5 kb in size) and a genomic clone in this
class. Sequencing these clones identified a single long open reading
frame which would encode a 676-amino acid protein.(
)
A search through the protein data base revealed that the
deduced protein sequence shares significant sequence homology with the
subunit of the mammalian soluble-type GC. We therefore designated
the gene and cDNA as dgc
1 (Drosophila soluble-type guanylate cyclase
) and the encoded protein as
DGC
1.
1 nucleotide and
protein sequences with those of the only Drosophila
subunit sGC cDNA reported to date, dgc1 (Yoshikawa et
al., 1993), revealed that they are nearly identical in both
nucleotide and amino acid sequences. However, there are some
differences, as summarized in Fig. 1. The thick line in
the middle of the figure represents the nucleotide sequence of
dgc
1 cDNA, and the shaded box below it
represents the coding region. In comparison to dgc1 cDNA,
dgc
1 cDNA contains additional bases GG, C, A, T, C, and G
at nucleotide positions 779, 853, 1740, 1744, 1755, and 2163,
respectively (Fig. 1). Because of these additional bases,
frameshifts occur, and the coding region of dgc
1 terminates nine codons earlier than that of dgc1.
Moreover, the deduced DGC
1 protein has seven less residues than
the DGC1 protein and contains three short stretches (25, 5, and 3
residues) of amino acids that differ from the corresponding regions of
the DGC1 protein (Fig. 1).
1 maps to 99B on the right arm of the third chromosome
(Fig. 2A), while dgc1 has been reported to
hybridize to 63A on the left arm of the third chromosome (Yoshikawa
et al., 1993). Hybridization of dgc
1 to the 99B
region has been verified using several different cDNA and genomic DNA
probes.
Figure 2:
Chromosomal localization and RNA blot
analysis of dgc1. A, chromosomal in situ hybridization. dgc
1 cDNA and genomic DNA probes were
biotin labeled and hybridized to the salivary gland polytene
chromosomes. The label (arrow) is found at 99B on the right
arm of the third chromosome. B, RNA blot analysis. 10 µg
of mRNA of each sample was used for this analysis. A 0.3-kb
EcoRI fragment from the 3`-end of dgc
1 cDNA
(Fig. 1) was used as probe to hybridize to poly(A)
RNA
blots of 0-12 h embryos (E), third instar larvae
(3), 7-day pupae (7), 9-day pupae (9), adult
heads (H), and adult bodies (B). A 2.5-kb transcript
is abundantly expressed in the head and also in the embryos, 7 day
pupae, 9 day pupae, and adult bodies at low
levels.
The developmental RNA expression pattern was examined using
the 3`-end 0.3-kb EcoRI fragment of dgc1 cDNA
(Fig. 1) as probe. This probe, chosen to avoid the conserved
cyclase-encoding domain, recognizes a 2.5-kb transcript expressed
predominantly in adult heads (Fig. 2B). Low levels of
expression are also found in adult bodies and at some earlier
developmental stages, e.g. in embryos and 9-day pupae
(Fig. 2B).
1 RNA by in situ hybridization to adult
cryosections. The antisense RNA probe showed specific hybridizations to
the cortical regions of the optic lobe, central brain, and thoracic
ganglia (Fig. 3, A and C). The photoreceptor
layers showed hybridization signals that are slightly, but not
significantly, above background. These labels were not found in control
sections probed with sense RNA (Fig. 3, B and
D).
Figure 3:
Tissue distribution of dgc1 RNA. The same 0.3-kb EcoRI used in Fig. 2B was
used to synthesize riboprobes to hybridize to adult cryosections. A and C are, respectively, dark-field micrographs of
horizontal sections of adult head and body hybridized with
S-labeled antisense probe. B and D are
corresponding controls hybridized with the sense probe. Note the strong
label in the cortical regions of the nervous system. ph,
photoreceptor; o, optic lobe; br, brain; tg,
thoracic ganglion. Scale bar: 200 µm in A and
B; 150 µm in C and
D.
An antiserum was generated against the peptide encoded
by the 0.6-kb XhoI-HindIII fragment of dgc1 cDNA (Fig. 1), expressed as a glutathione
S-transferase (GST) fusion protein. In Western blot analysis,
it recognized a single 76-kDa band which is present in the head but
only at very low levels in the body sample (data not shown), consistent
with RNA blot results (Fig. 2B). The size of the band
was also consistent with the estimated size of the DGC
1 protein,
indicating that the antiserum specifically recognized the DGC
1
protein.
1 protein
in adult tissue sections were carried out using the DGC
1-GST
antiserum. The antiserum preincubated with DGC
1-GST fusion protein
was used as a negative control (data not shown). The antiserum stained
the central brain, optic lobe, and thoracic ganglia (Fig. 4,
A and B), consistent with the results of tissue
in situ hybridization (Fig. 3, A and
C). No detectable staining of the retina was found.
Figure 4:
Immunocytochemical localization of
DGC1 protein. A and B are, respectively, adult
head and thoracic sections stained with a DGC
1 antiserum.
Scale bar: 200 µm. ph, photoreceptor; o,
optic lobe; br, brain; tg, thoracic
ganglion.
Characterization of 76C Class Clones
A total of
nine cDNA clones of this class were isolated from screening both a head
(Itoh et al., 1985) and an embryonic (Zinn et al.,
1988) cDNA library. Restriction maps of some of these cDNA clones are
shown in Fig. 5.(
)
The sequence information
for the coding region was obtained from the two largest overlapping
clones, GC13-25 and GC708. The composite cDNA is more than 6 kb
in length and contains a single long open reading frame which would
encode a 1525-amino acid protein (Fig. 6). The first methionine
codon, proposed as the translation initiation site, is found at the
564th nucleotide, and there is a stop codon three codons upstream of
this Met codon (Fig. 6).
Figure 6:
drgc cDNA and deduced protein
sequences. For both cDNA and protein sequences, the numbering begins
with the respective first residue. The first Met codon, taken as the
translation start site, is found at the 564th nucleotide position. A
stop codon is found nine bases upstream of the Met codon. The sequence
corresponding to the presumptive transmembrane domain is double
underlined. In the extracellular domain, upstream of the
presumptive transmembrane domain, potential Asn-linked glycosylation
sites are underlined, and cysteine residues are
boxed.
A search through protein data bases
revealed that the protein deduced from this cDNA shares structural
similarity (see Fig. 5) with previously characterized
receptor-type GCs, as documented below. For this reason, the cDNA and
the corresponding gene were designated as drgc (Drosophila receptor-type guanylate cyclase), and the deduced protein, as
DrGC. Hydropathy analysis (data not shown) revealed that the deduced
protein contains a single hydrophobic region of about 20 amino acids
(residues 492-512) that could serve as a transmembrane domain, as
has been found for all known rGCs. The first 490-amino acid region,
N-terminal to the transmembrane domain, presumably represents the
putative ligand-binding domain. Although this region shares 25%
sequence identity with the corresponding region of sea urchin rGC
(Thorpe and Garbers, 1989), it has no significant homology to the
corresponding regions of any other known GCs. The putative
transmembrane domain is followed by the kinase-like and cyclase domains
in the next 520 amino acids, as are characteristics of other known
rGCs. The amino acid sequences of the kinase-like and cyclase domains
of DrGC are compared with the corresponding regions of five other
receptor-type GCs in Fig. 7. The putative kinase domain shares,
respectively, 42, 39, 43, 33, and 45% sequence identity with the
corresponding regions of sea urchin GC (Thorpe and Garbers, 1989),
mammalian GC-A, -B, -C (Chinkers et al., 1989; Schulz et
al., 1989, 1990), and the previously identified Drosophila rGC, DMRGC (Gigliotti et al., 1993)
(Fig. 7A). The cyclase domain of DrGC shares 64, 64, 62,
42, and 59% sequence identity with the corresponding regions of sea
urchin GC, mammalian GC-A, -B, -C, and DMRGC, respectively
(Fig. 7B). Unlike any other known rGCs, the deduced
protein also contains a C-terminal 430-amino acid region with no
significant homology to any known protein.
Figure 7:
Amino acid sequence alignment between the
deduced DrGC protein and five other receptor-type GCs in the
(A) kinase-like and (B) cyclase domains. Gaps are
introduced in each sequence for optimal alignment. Amino acids that are
identical in at least two of the sequences are shaded. Percent
identities between DrGC sequence and each of the other sequences are
shown at the end of each sequence. In the case of the kinase-like
domain (A), the residues that are also invariant or conserved
in other known protein kinases are identified with an asterisk (*). SU-GC, sea urchin receptor-type GC (Thorpe and
Garbers, 1989); GC-A, rat brain receptor-type GC type A
(Chinkers et al., 1989); GC-B, rat brain
receptor-type GC type B (Schulz et al., 1989); GC-C,
enterotoxin receptor-type GC (Schulz et al., 1990);
DMRGC, Drosophila receptor-type GC (Gigliotti,
1993).
Chromosomal location of
drgc was determined by in situ hybridization of a
10-kb genomic fragment corresponding to the GC13 cDNA clone
(Fig. 5) to larval polytene chromosomes. The result localized
drgc to the 76C region on the left arm of the third chromosome
(Fig. 8A).
Figure 8:
Chromosomal localization and RNA blot
analysis. A, a 10-kb genomic DNA fragment corresponding to
GC13 cDNA clone (Fig. 5) was biotin-labeled and hybridized to polytene
chromosomes. The label is found at the 76C region of the left arm of
the third chromosome. B, the 3.0-kb EcoRI fragment of
GC708 clone (Fig. 5) was used to probe poly(A) RNA
blots of 0-12-h embryos (E), first and second instar
(1), third instar larvae (3), 7 day pupae
(7), 9 day pupae (9), adult heads (H), and
adult bodies (B). The probe recognizes a 7-kb transcript
expressed at low levels in all developmental stages and in adult heads
and bodies. 25 µg of mRNA of each sample was used for this
analysis.
The developmental drgc RNA
expression pattern was examined by probing RNA blots of the embryos,
first and second instar larvae, third instar larvae, 7-day pupae, 9-day
pupae, and adult heads and bodies with a 3.0-kb EcoRI fragment
of drgc cDNA (Fig. 5). The results showed that a 7-kb
transcript is present throughout development and in both adult heads
and bodies (Fig. 8B).
Figure 9:
Tissue distribution of drgc RNA.
Panels A and B are, respectively, dark-field
micrographs of horizontal sections of the head and thorax hybridized
with S-labeled antisense and sense RNA probes synthesized
from a 0.4-kb BamHI fragment of drgc cDNA (Fig. 5).
Similarly, panels C and D are horizontal sections of
female abdomen hybridized with the same antisense and sense probes,
respectively. The hybridization signals are found throughout the head
and thorax with slightly higher levels of labeling in the optic lobe
and central brain (A). In the female abdomen, strong labels
are found in the oocytes (C). These signals are not found in
sections probed with sense RNA probes (B and D).
br, brain; o, optic lobe; tg, thoracic
ganglion; oc, oocyte; d, digestive tract; f,
fatty bodies. Scale bar, 300
µm.
1, and a receptor type, DrGC.
1
(Fig. 1). To determine tissue distributions of dgc
1 RNA and protein products, we have carried out both tissue in
situ hybridization and immunocytochemical localization of the
protein product. Results of these studies are mutually consistent. Both
the RNA and protein products are found predominantly in nervous
tissues: the optic lobe, central brain, and thoracic ganglia (
Fig. 3
and Fig. 4). Their expression in the photoreceptors,
however, is not significantly above background. We conclude that the
DGC
1 protein is not likely to play a major role in photoreceptors.
1 in all other parts of the nervous
system, on the other hand, suggests that it plays an important role in
neural functions. In mammalian systems, soluble GCs have been shown to
be activated by nitric oxide, NO (Arnold et al., 1977; Craven
and DeRubertis., 1978), and sGCs and NO synthase have been co-localized
in certain areas of the central nervous system (Vincent and Hope, 1992;
Garthwaite, 1991). A growing body of evidence suggests that NO
generated in the postsynaptic neuron serves as a retrograde messenger
to activate a presynaptic soluble-type GC (O'Dell et
al., 1991; Garthwaite et al., 1988; Garthwaite, 1991),
and the resulting elevation in cGMP in the presynaptic neuron activates
cGMP-dependent protein kinases to trigger its physiological responses
(Zhuo et al., 1994). This sequence of events is thought to be
responsible, for example, for long-term potentiation in the hippocampus
(Bohme et al., 1991; O'Dell et al., 1991; Haley
et al., 1992; Verma et al., 1993; Zhuo et
al., 1993, 1994).
1 is likely to be
important in investigating the roles of sGC in the Drosophila nervous system.
/EMBL Data Bank with accession number(s) U23485.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.