©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Characterization of Two Drosophila Guanylate Cyclases Expressed in the Nervous System (*)

Wencheng Liu (1), Jaeseung Yoon (2), Martin Burg (1), Lin Chen (1), William L. Pak (1)(§)

From the (1) Department of Biological Science, Purdue University, West Lafayette, Indiana 47907 and the (2) Department of Genetic Engineering, Kyung Hee University, Yongin-Gun, Kyungki-Do, South Korea

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated, by interspecies hybridization, two classes of Drosophila cDNA each encoding a different guanylate cyclase (GC). One of them encodes an subunit homolog of soluble GC, designated DGC1, and the other encodes a receptor-type GC, designated DrGC. The dgc1 cDNA encodes a protein of 676 amino acids and maps to 99B. In situ hybridization to adult tissue sections showed that dgc1 mRNA is found mainly in the cell bodies of the optic lobe, central brain, and thoracic ganglia. The DGC1 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.


INTRODUCTION

Guanylate cyclase (GC)() 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)).

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, 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).

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 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).

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, dgc1, 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.


MATERIALS AND METHODS

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.

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.


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 dgc1 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 dgc1 cDNA is given at the top. The thickline in the middle and the shaded box represent, respectively, the dgc1 cDNA sequence and its coding sequence. Nucleotide sequence differences between dgc1 and dgc1 cDNAs are shown in open boxesbelow the thick line, and the corresponding amino acid sequence differences between the deduced DGC1 and DGC1 proteins are shown below the shaded box. Seven additional nucleotides, at six different positions, were found in the dgc1 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 DGC1 sequence corresponds to the stop codon TAG in the dgc1 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).

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 NaHPO, 3 mM KHPO, 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% HO) 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 DGC1 antiserum or DGC1 antiserum preincubated with excess DGC1-GST fusion protein for 1 h at room temperature.


RESULTS

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.

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 dgc1 (Drosophila soluble-type guanylate cyclase ) and the encoded protein as DGC1.

Comparison of the dgc1 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 dgc1 cDNA, and the shaded box below it represents the coding region. In comparison to dgc1 cDNA, dgc1 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 dgc1 terminates nine codons earlier than that of dgc1. Moreover, the deduced DGC1 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).

A more significant difference, however, is that in chromosomal in situ hybridization, dgc1 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 dgc1 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. dgc1 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 dgc1 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).

The same 0.3-kb EcoRI fragment was used to synthesize riboprobes to examine tissue distribution of dgc1 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 DGC1 protein, indicating that the antiserum specifically recognized the DGC1 protein.

Immunocytochemical localizations of the DGC1 protein in adult tissue sections were carried out using the DGC1-GST antiserum. The antiserum preincubated with DGC1-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 DGC1 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).

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.


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.




DISCUSSION

We have identified, by low-stringency interspecies hybridization, two guanylate cyclases of Drosophila: a soluble type, DGC1, and a receptor type, DrGC.

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, DGC1 (Fig. 1). To determine tissue distributions of dgc1 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 DGC1 protein is not likely to play a major role in photoreceptors.

Abundant expression of DGC1 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).

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, DGC1 is likely to be important in investigating the roles of sGC in the Drosophila nervous system.

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.


FOOTNOTES

*
This work was supported by National Eye Institute Grant EY00033. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank/EMBL Data Bank with accession number(s) U23485.

§
To whom correspondence should be addressed. Tel.: 317-494-8202; Fax: 317-494-0876.

The abbreviations used are: GC, guanylate cyclase; kb, kilobase pair(s); PBS, phosphate-buffered saline: MOPS, 4-morpholineethanesulfonic acid; GST, glutathione S-transferase.

S. Shah and D. R. Hyde (University of Notre Dame) have independently isolated and analyzed the same class of GC cDNA clones, manuscript submitted for publication.

McNeil et al. (1995) (Oregon Health Sciences University) have also independently isolated and analyzed the same class of GC cDNA clones.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Angerer, L. M., Stoler, M. H., and Angerer, R. C.(1987) in In situ Hybridization: Application to Neurobiology (Valentino, K. L., Eberwine, J. H., and Barchas, J. D., eds) pp. 42-70, Oxford University Press, New York
  2. Arnold, W. P., Mittal, C. K., Katsuki, S., and Murad, F.(1977) Proc. Natl. Acad. Sci. U. S. A. 74, 3203-3207 [Abstract]
  3. Bacigalupo, J., Johnson, E. C., Vergara, C., and Lisman, J. E.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7938-7942 [Abstract]
  4. Baumann, A., Frings, S., Godde, M., Seifert, R., and Kaupp, U. B. (1994) EMBO J. 13, 5040-5050 [Abstract]
  5. Bohme, G. A., Bon, C., Schutzman, J-M., Doble, A., and Blanchard, J-C. (1991) J. Pharmacol. 199, 379-381
  6. Brown, T. E., Rubin, L. J., Ghalayini, A. J., Tarrer, A. P., Irvine, R. F., Berridge, M. J., and Anderson, R. E.(1984) Nature 311, 160-163 [Medline] [Order article via Infotrieve]
  7. Bloomquist, B. T., Shortridge, R. D., Schneuwly, S., Perdew, M., Montell, C., Stellar, H., Rubin, G., and Pak, W. L.(1988) Cell 54, 723-733 [Medline] [Order article via Infotrieve]
  8. Bredt, D. S., and Snyder, S. H.(1992) Neuron 8, 3-11 [Medline] [Order article via Infotrieve]
  9. Buechler, W. A., Nakane, M., and Murad, F.(1991) Biochem. Biophys. Res. Commun. 174, 351-357 [Medline] [Order article via Infotrieve]
  10. Burg, M. G., Sarthy, P. V., Koliantz, G., and Pak, W. L.(1993) EMBO J. 12, 911-919 [Abstract]
  11. Chang, M. S., Lowe, D. G., Lewis, M., Hellmiss, R., Chen, E., and Goeddel, D. V.(1989) Nature 341, 68-72 [CrossRef][Medline] [Order article via Infotrieve]
  12. Chinkers, M., Garbers, D. L., Chang, M. S., Lowe, D. G., Chin, H., Goeddel, D. V., and Schulz, S.(1989) Nature 338, 78-83 [CrossRef][Medline] [Order article via Infotrieve]
  13. Cornwell, T. L., and Lincoln, T. M.(1989) J. Biol. Chem. 264, 1146-1155 [Abstract/Free Full Text]
  14. Craven, P. A., and DeRubertis, F. R.(1978) J. Biol. Chem. 253, 8433-8443 [Abstract]
  15. Currie, M. G., Fok, K. F., Kato, J., Moore, R. J., Hamra, F. K., Duffin, K. L., and Smith, C. E.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 947-951 [Abstract]
  16. de Sauvage, F. J., Keshav, S., Kuang, W.-J., Gillett, N., Henzel, W., and Goeddel, D. V.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9089-9093 [Abstract]
  17. Devereux, J.(1992) The GCG Sequence Analysis Software Package, Version 7.0, Genetic Computer Group, Inc., University Research Park, Madison, WI
  18. Drewett, J. G., and Garbers, D. L.(1994) Endocr. Rev. 15, 135-162 [Medline] [Order article via Infotrieve]
  19. Engels, W. R., Preston, C. R., Thompson, P., and Eggelston, W. B. (1986) Bethesda Research Laboratories Focus 8, 6-8
  20. Fein, A., Payne, R., Corson, D. W., Berridge, M. J., and Irvine, R. F. (1984) Nature 311, 157-160 [Medline] [Order article via Infotrieve]
  21. Feinberg, A. P., and Vogelstein, B.(1984) Anal. Biochem. 137, 266-267 [Medline] [Order article via Infotrieve]
  22. Fesenko, E. E., Kolesnikov, S. S., and Lyubarsky, A. L.(1985) Nature 313, 311-313
  23. Gall, J. G., and Pardue, M. L.(1971) Methods Enzymol. 21, 470-478
  24. Garthwaite, J.(1991) Trends Neurosci. 14, 60-67 [CrossRef][Medline] [Order article via Infotrieve]
  25. Garthwaite, J., Charles, S. L., and Chess-Willians, R.(1988) Nature 336, 385-388 [CrossRef][Medline] [Order article via Infotrieve]
  26. Gigliotti, S., Cavaliere, V., Manzi, A., Tino, A., Graziani, F., and Malva, C.(1993) Dev. Biol. 159, 450-461 [CrossRef][Medline] [Order article via Infotrieve]
  27. Goy, M. F.(1991) Trends Neurosci. 14, 293-299 [CrossRef][Medline] [Order article via Infotrieve]
  28. Guan, K. L., and Dixon, J. E.(1988) Anal. Biochem. 192, 262-267
  29. Hafen, E., and Levine, M.(1986) in Drosophila: A Practical Approach (Roberts, D. B., ed) pp. 139-158, IRL Press, Oxford
  30. Haley, J. E., Wilcox, G. C., and Chapman, P. F.(1992) Neuron 8, 211-216 [Medline] [Order article via Infotrieve]
  31. Hanks, S. K., Quinn, A. M., and Hunter, T.(1988) Science 241, 42-52 [Medline] [Order article via Infotrieve]
  32. Harteneck, C., Koesling, D., Soling, G., Schultz, G., and Bohme, E. (1990) FEBS Lett. 272, 221-223 [CrossRef][Medline] [Order article via Infotrieve]
  33. Humbert, P., Niroomand, F., Fischer, G., Mayer, B., Koesling, D., Hinsch, K-H., Gausepohl, H., Frank, R., Schultz, G., and Bohme, E. (1990) Eur. J. Biochem. 190, 273-278 [Abstract]
  34. Itoh, N., Salvaterra, P., and Itakura, K.(1985) Dros. Inf. Service 61, 89
  35. Kamisaki, Y., Saheki, S., Nakane, M., Palmieri, J. A., Kuno, T., Chang, B. Y., Waldman, S. A., and Murad, F.(1986) J. Biol. Chem. 261, 7236-7241 [Abstract/Free Full Text]
  36. Koesling, D., Herz, J., Gausepohl, H., Niroomand, F., Hinsch, K. D., Mulsch, A., Bohme, E., Schultz, G., and Frank, R.(1988) FEBS Lett. 239, 29-34 [CrossRef][Medline] [Order article via Infotrieve]
  37. Koesling, D., Harteneck, C., Humbert, P., Bosserhoff, A., Frank, R., Schulz, G., and Bohme, E.(1990) FEBS Lett. 266, 128-132 [CrossRef][Medline] [Order article via Infotrieve]
  38. Koesling, D., Bohme, E., and Schultz, G.(1991) FASEB 5, 2785-2791 [Abstract/Free Full Text]
  39. Lipman, D. J., and Pearson, W. R.(1985) Science 227, 1435-1441 [Medline] [Order article via Infotrieve]
  40. Lowe, D. G., Chang, M. S., Hellmiss, R., Chen, E., Singh, S., Garbers, D. L., and Goeddel, D. V.(1989) EMBO J. 8, 1377-1384 [Abstract]
  41. Mahmoudi, M., and Lin, V. K.(1989) Biotechnique 7, 331-333 [Medline] [Order article via Infotrieve]
  42. McNeil, L., Chinkers, M., and Forte, M.(1995) J. Biol. Chem. 270, 7189-7196 [Abstract/Free Full Text]
  43. Miller, K.(1987) Bethesda Research Laboratories Focus 9, 14-15
  44. Minke, B., and Selinger, Z.(1991) Prog. Retinal Res. 11, 99-124
  45. Montell, C., Jones, K., Hafen, E., and Rubin, G.(1985) Science 230, 1040-1043 [Medline] [Order article via Infotrieve]
  46. Nakane, M., Seheki, S., Kuno, T., Ishii, K., and Murad, F.(1988) Biochem. Biophys. Res. Commun. 156, 1000-1006 [Medline] [Order article via Infotrieve]
  47. Nakane, M., Arai, K., Saheki, S., Kuno, T., Buechler, W., and Murad, F. (1990) J. Biol. Chem. 265, 16841-16845 [Abstract/Free Full Text]
  48. O'Dell, T. J., Hawkins, R. D., Kandel, E. R., and Arancio, O.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11285-11289 [Abstract]
  49. Palmer, R. M. J., Ferrige, A. G., and Moncada, S.(1987) Nature 333, 664-666 [CrossRef]
  50. Paupardin-Tritsch, D., Hammond, C., Gerschenfeld, H. H., Nairn, A. C., and Greengard, P. C. (1986a) Nature 323, 812-814 [Medline] [Order article via Infotrieve]
  51. Paupardin-Tritsch, D., Mammond, C., and Gerschenfeld, H. M. (1986b) J. Neurosci. 6, 2715-2723 [Abstract]
  52. Payne, R.(1986) Photobiochem. Photobiophys. 13, 373-397
  53. Pearson, W. R., and Lipman, D. J.(1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2444-2448 [Abstract]
  54. Regulski, M., and Tully, T.(1993) Abstract in Neurobiology of Drosophila, pp. 126, Cold Spring Harbor Press, Cold Spring Harbor, NY
  55. Sambrook, J., Fritsch, E. F., and Maniatis, T.(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  56. Sanger, F., Nicklen, S., and Coulson, A. R.(1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  57. Schulz, S., Singh, S., Bellet, R. A., Singh, G., Tubb, D. J., Chin, H., and Garbers, D. L.(1989) Cell 58, 1155-1162 [Medline] [Order article via Infotrieve]
  58. Schulz, S., Green, C. K., Yuen, P. S. T., and Garbers, D. L.(1990) Cell 63, 941-948 [Medline] [Order article via Infotrieve]
  59. Schulz, S., Chrisman, T. D., and Garbers, D. L.(1992) J. Biol. Chem. 267,16019-16021 [Abstract/Free Full Text]
  60. Shibuki, K., and Okada, D.(1991) Nature 349, 326-328 [CrossRef][Medline] [Order article via Infotrieve]
  61. Shortridge, R. D., Yoon, J., Lending, C. R., Bloomquist, B. T., Perdew, M. H., and Pak, W. L.(1991) J. Biol. Chem. 266, 12474-12480 [Abstract/Free Full Text]
  62. Singh, S., Lowe, D. G., Thorpe, D. S., Rodriguez, H., Kuang, W. J., Dangott, L. J., Chinkers, M., Goeddel, D. V., and Garbers, D. L.(1988) Nature 334, 708-712 [CrossRef][Medline] [Order article via Infotrieve]
  63. Smith, D. B., and Johnson, K. S.(1988) Gene (Amst.) 67, 31-40 [CrossRef][Medline] [Order article via Infotrieve]
  64. Stryer, L.(1986) Annu. Rev. Neurosci. 9, 87-119 [CrossRef][Medline] [Order article via Infotrieve]
  65. Thorpe, D. S., and Garbers, D. L.(1989) J. Biol. Chem. 264, 6545-6549 [Abstract/Free Full Text]
  66. Towbin, H., Staehelin, T., and Gordon, J.(1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4355 [Abstract]
  67. Verma, A., Hirsch, D. J., Giatt, C. E., Ronnet, G. V., and Snyder, S. H.(1993) Science 259, 381-384 [Medline] [Order article via Infotrieve]
  68. Vincent, S. R., and Hope, B. T.(1992) Trends Neurosci. 15, 108-113 [CrossRef][Medline] [Order article via Infotrieve]
  69. Waldman, S. A., and Murad, F.(1987) Pharmacol. Rev. 39, 163-189 [Medline] [Order article via Infotrieve]
  70. Wilcox, J. N., Augustine, A., Goeddil, D. V., and Lowe, D. J.(1991) Mol. Cell. Biol. 11, 3454-3462 [Medline] [Order article via Infotrieve]
  71. Yoon, J., Shortridge, R. D., Bloomquist, B. T., Schneuwly, S., Perdew, M. H., and Pak, W. L.(1989) J. Biol. Chem. 264, 18536-18543 [Abstract/Free Full Text]
  72. Yoshikawa, S., Miyamoto, I., Aruga, J., Furuichi, T., Okano, H., and Mikoshiba, K.(1993) J. Neurochem. 60, 1570-1573 [Medline] [Order article via Infotrieve]
  73. Yuen, P. S., and Garbers, D. L.(1992) Annu. Rev. Neurosci. 15, 193-225 [CrossRef][Medline] [Order article via Infotrieve]
  74. Zhuo, M., Small, S. A., Kandel, E. R., and Hawkins, R. D.(1993) Science 260, 1946-1950 [Medline] [Order article via Infotrieve]
  75. Zhuo, M., Hu, Y., Schulz, C., Kandel, E. R., and Hawkins, R. D.(1994) Nature 368, 635-639 [CrossRef][Medline] [Order article via Infotrieve]
  76. Zinn, K., McAllister, L., and Goodman, C. S.(1988) Cell 53, 577-587 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.