©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Two Drosophila Genes That Encode the and Subunits of the Brain Soluble Guanylyl Cyclase (*)

Seema Shah , David R. Hyde (§)

From the (1)Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We identified two Drosophila genes (dgc1 and dgc1) that encode the soluble guanylyl cyclase and subunits, respectively. The putative Dgc1 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 Dgc1 protein is 86 kDa and exhibits 59% amino acid identity with the rat 1 protein. However, the Dgc1 protein has an additional 118 amino acids inserted near the amino terminus, which makes it significantly larger than the rat 1. The Dgc1 protein was immunolocalized to the optic lobes and throughout the brain neuropil, with no detectable expression in the retina. The Dgc1 and Dgc1 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 Dgc1 and Dgc1 are soluble guanylyl cyclase and subunits that are capable of forming a functional guanylyl cyclase heterodimer.


INTRODUCTION

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 () 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).

While both the 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) .

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 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.


EXPERIMENTAL PROCEDURES

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 Dgc1, 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 Dgc1 and Dgc1 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 Dgc1, 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 GC1. The amino acid single letter codes for Dgc1, soluble rat lung GC1 (rGC1) (18), and the soluble Drosophila Dgc1 proteins (33) are shown. The Dgc1 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 Dgc1 and the rat GC1 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 Dgc1 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 Dgc1 sequence. The Dgc1 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 Dgc1 and the soluble rat lung GC-S subunit (rGC1) (18) are shown. The Dgc1 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 Dgc1 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 Dgc1 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 Dgc1 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 Dgc1 proteins in stably transfected human kidney 293 cells. Human kidney 293 cells that were not transfected (293 cells) or stably transfected with Dgc1 (293 cells w/Dgc1), Dgc1 (293 cells w/Dgc1), or both (293 cells w/Dgc1 & Dgc1) 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 Dgc1 antiserum or the Dgc1 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 Dgc1 protein was detected in both of the lines that were transfected with the Dgc1 cDNA. The 84-kDa Dgc1 protein was also detected in both of the lines that were transfected with the Dgc1 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 Dgc1 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 Dgc1 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 Dgc1 and Dgc1 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).

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 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 .


RESULTS

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.

The predicted amino acid sequence for the soluble guanylyl cyclase subunit, Dgc1, 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 Dgc1 and rat soluble guanylyl cyclase 1 subunits share 35% identity and an additional 8% conserved amino acid substitutions (Fig. 1). The Dgc1 sequence also exhibits 35% amino acid identity with the rat 2 sequence (data not shown). Dgc1 is 93% identical with the 46 invariant amino acids in the guanylyl cyclase catalytic domain(2) . The Dgc1 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 Dgc1 and absent in Dgc1. Thus, the Dgc1 and Dgc1 cDNA sequences are 99.4% identical at the nucleotide level within the open reading frame.

The predicted amino acid sequence for the Dgc1 soluble guanylyl cyclase subunit is shown in Fig. 2. The Dgc1 protein and the rat soluble guanylyl cyclase 1 subunits share 59% identity and another 8% conserved amino acid substitutions (Fig. 2). The Dgc1 sequence possesses less identity with the rat kidney soluble 2 sequence (27%; data not shown, Ref. 16). However, Dgc1 is 98% identical with the 46 invariant amino acids in the guanylyl cyclase catalytic domain(2) . The putative Dgc1 protein has a molecular mass of 86 kDa and lacks hydrophobic regions. While most guanylyl cyclase subunits are larger than the subunits, Dgc1 is larger than any of the known subunits. This is due to an additional 118 amino acids located near the amino terminus of Dgc1 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 dgc1 genes in situ to polytene chromosomes. Both genes are located on the third chromosome, with the dgc1 gene at 99B and the dgc1 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 Dgc1 and Dgc1 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 Dgc1 cDNAs were biotinylated, hybridized to third instar larval chromosomes, and detected as described under ``Experimental Procedures.'' The Dgc1 cDNA hybridized to the 99B region on the third chromosome (arrow in A), while the Dgc1 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 dgc1 mRNAs, we examined their mRNA expression using Northern blots. We synthesized in vitro antisense RNA probes to the Dgc1 and Dgc1 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 Dgc1 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 Dgc1 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 Dgc1 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 Dgc1 cDNAs (data not shown). However, the 3.8-kb cDNA had a different 5`-untranslated sequence than the other Dgc1 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 Dgc1 mRNAs (Fig. 4B). Therefore, the dgc1 gene is likely to be alternatively spliced, yielding three mRNAs that encode the same 76-kDa protein. The Dgc1 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 Dgc1 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 Dgc1 open reading frame (A), the 5`-untranslated region of the 3.8-kb Dgc1 cDNA (B), or the Dgc1 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 Dgc1 proteins in Drosophila heads. Frozen Drosophila head sections (8 µm) were stained with either the Dgc1 (A and B) or Dgc1 (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 Dgc1 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 Dgc1 antiserum stains throughout the neuropil (n) of the adult brain. The brain cortex (c) has very little if any detectable signal. C, the Dgc1 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 Dgc1 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 Dgc1 (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 Dgc1 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 dgc1 mRNAs (see ``Discussion''). The Dgc1 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 Dgc1 protein is expressed in wild-type and eya heads but not detected in wild-type bodies.


Figure 5: Immunoblot analysis of the Dgc1 and Dgc1 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 Dgc1 or a Dgc1 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 Dgc1 antiserum, while the Dgc1 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 Dgc1 polyclonal antisera to localize the soluble guanylyl cyclase subunits in the adult head. The Dgc1 antiserum stained the retina and underlying optic lobes (lamina, medulla, lobula, and lobula plate) of the adult visual system (Fig. 6A). In addition, the Dgc1 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 Dgc1 immunodetection is consistent with the predicted localization based on the Northern blots and immunoblots. While the Dgc1 antiserum failed to stain the adult retina, it did stain the optic lobes (Fig. 6C). This suggests that the Dgc1 protein is localized to either the photoreceptor axons or the secondary and tertiary neurons of the visual system. Similar to the Dgc1 pattern, the Dgc1 antiserum also stained much of the adult brain neuropil (Fig. 6D).

The Dgc1 and Dgc1 Proteins Function as a Soluble Heterodimeric Guanylyl Cyclase

We examined whether Dgc1 and Dgc1 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 Dgc1 or the Dgc1 cDNAs in the pCIS2M vector(30) . We also stably cotransfected both the Dgc1 and Dgc1 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 Dgc1 cDNA expressed only the 74-kDa Dgc1 protein (Fig. 7A), while the Dgc1 transfected lines correctly expressed the 84-kDa protein (Fig. 7B). It is interesting to note that the cotransfected lines expressed more Dgc1 and Dgc1 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).

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 Dgc1 and Dgc1 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 Dgc1 and Dgc1 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 Dgc1 and Dgc1 proteins possess guanylyl cyclase activity and are stimulated like the previously characterized soluble guanylyl cyclase subunits.


DISCUSSION

We identified a soluble guanylyl cyclase and subunit from Drosophila melanogaster. The Dgc1 protein closely resembles the previously identified subunits in its molecular mass and its similarity in amino acid sequence, particularly in the catalytic domain. While Dgc1 possesses a greater level of amino acid identity with the other subunits than Dgc1 has with the other subunits, the additional 118 amino acids in the amino-terminal half of Dgc1 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 Dgc1 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 Dgc1 protein. Therefore, the function of this region must await further biochemical or genetic analyses.

Immunolocalization revealed that both the and subunits are expressed in the Drosophila brain; however, their distribution is not completely overlapping. Most notably, the 74-kDa Dgc1 protein is expressed in the retina, while no significant level of Dgc1 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 Dgc1 protein is not expressed in the retina, an additional soluble guanylyl cyclase subunit must exist to generate a functional heterodimer with the 74-kDa Dgc1 in the retina. Even though Dgc1 and Dgc1 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 Dgc1 interacts with Dgc1 in some neurons and with an unidentified subunit in others.

The exact origin of the 155-kDa protein detected with the Dgc1 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 dgc1 gene based on two criteria. First, cDNAs representing the different Dgc1 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 Dgc1 transfected cell lines (Fig. 7). This suggests that the retina-specific 155-kDa protein shares a common epitope with Dgc1. This epitope is most likely not within the highly conserved catalytic domain for three reasons. First, the Dgc1 fusion protein used to generate the polyclonal antiserum contained very little of the catalytic domain. Second, the Dgc1 antiserum did not detect the Dgc1 protein on immunoblots, which also possesses the highly conserved catalytic domain. Third, an antiserum generated against the Dgc1 guanylyl cyclase catalytic domain failed to detect the 155-kDa protein on immunoblots and failed to stain the retina (data not shown).

The Dgc1 and Dgc1 subunits behaved biochemically like the previously characterized guanylyl cyclase subunits, with activity requiring coexpression of both Dgc1 and Dgc1 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 Dgc1 and Dgc1 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, Dgc1 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.

While we could not determine whether Dgc1 and Dgc1 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 Dgc1 and Dgc1 functional heterodimer in transfected cells suggests that their expression coincides in Drosophila and they form a functional heterodimer in vivo. If Dgc1 interacts with Dgc1 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) .

Two lines of evidence strongly suggest that the dgc1 and the previously isolated dgc1 genes are the same. First, the Dgc1 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 Dgc1 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 dgc1 to the 99B region. Liu et al.(38) independently isolated the dgc1 gene and their DNA sequence and chromosomal map position are consistent with our results. Taken together, the previously isolated dgc1 and our dgc1 genes are identical.

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 Dgc1 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 (Dgc1) 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 Dgc1 protein in both wild-type and eya heads. Second, immunostaining demonstrated that the Dgc1 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 Dgc1 protein expression in the optic lobes and central brain neuropil.

The identification of both the soluble guanylyl cyclase and genes in Drosophila will permit a molecular and genetic analysis of the role of these proteins. Because the Dgc1 and Dgc1 subunits are expressed throughout the adult brain, it is possible that the corresponding dgc1 and dgc1 mutations may be lethal. Lethal mutations were already identified in the 99B and 100B regions. Germ line transformation of wild-type dgc1 and dgc1 genes and the subsequent suppression of lethality would demonstrate that the lethal mutation is in the dgc gene. The dgc1 and dgc1 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 Dgc1.

  
Table: Guanylyl cyclase activity in stably transfected human embryonic kidney 293 cells

Cytosolic fractions were isolated from human kidney 293 cells that were not transfected or stably transfected with either the Dgc1 or Dgc1 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).



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant EY08058 (to D. R. H.). 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) U27117 and U27123.

§
To whom correspondence should be addressed: Dept. of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556. Tel.: 219-631-8054; Fax: 219-631-7413.

The abbreviations used are: kb, kilobase(s); IPTG, isopropyl-1-thio--D-galactopyranoside; TBS, tris-buffered saline.


ACKNOWLEDGEMENTS

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.


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