(Received for publication, September 14, 1994; and in revised form, December 16, 1994)
From the
Membrane forms of guanylyl cyclase are single-transmembrane proteins that are activated by the binding of specific peptide ligands to their extracellular domains. In this report, we describe the identification and characterization of a Drosophila cDNA clone encoding a protein, DrGC-1, with high sequence identity to members of this family of receptor proteins. The protein contains a single, hydrophobic domain predicted to represent a transmembrane segment separating an extracellular domain with significant sequence identity (30%) to sea urchin egg peptide receptors from intracellular domains containing a protein kinase-like domain followed by a region with high sequence identity (65%) to cyclase catalytic domains found in receptor guanylyl cyclases from both vertebrates and invertebrates. In contrast to other members of this family, DrGC-1 is predicted to contain a carboxyl-terminal extension of 430 residues that has no homology to any described protein. Northern analysis indicates that DrGC-1 transcripts are present at variable levels in all stages of development. In situ hybridization demonstrates that high levels of uniformly distributed transcript are present in 0-2-h embryos. Later in embryogenesis (14-18 h), elevated levels of hybridization appear to be preferentially associated with muscle fibers.
Cyclic GMP (cGMP) is a ubiquitous intracellular second messenger responsible for mediating a wide variety of physiological responses. In vertebrates, these responses include smooth muscle relaxation, natriuresis, phototransduction, inhibition of hormone secretion, and stimulation of intestinal fluid secretion (reviewed in Drewett and Garbers, 1994). In invertebrates, enhanced sperm motility and respiration induced by egg peptides, molting induced by eclosion hormone, and muscle relaxation and glycogen breakdown induced by crustacean hyperglycemic hormone are all associated with increases in intracellular cGMP levels (Goy, 1991). In each case, elevation of intracellular cGMP is due to the activation of soluble or membrane forms of guanylyl cyclase by extracellular stimuli. Elevation of cGMP then results in alterations in the activity of target molecules such as phosphodiesterases, cGMP-dependent protein kinases, and ion channels, which ultimately generate the physiological responses of the cell (Lincoln and Cornwell, 1993).
Soluble forms of guanylyl cyclase are
heterodimeric proteins that are activated by binding of nitric oxide
(NO) ()(Drewett and Garbers, 1994). NO is generated by NO
synthase. Isoforms of this enzyme are strongly regulated by
Ca
/calmodulin so that hormones that increase
intracellular Ca
concentrations stimulate the enzyme
(Bredt and Snyder, 1994). Increased enzyme activity results in the
production of NO gas which then diffuses to neighboring cells. Binding
of NO to the heme group of soluble guanylyl cyclases in these cells
results in enzyme activation and an increase in intracellular cGMP.
While sequence analysis suggests that each subunit of the soluble
guanylyl cyclase contain a catalytic domain, co-expression of both
subunits is required for enzymatic activity.
Membrane forms of guanylyl cyclase are single-transmembrane proteins that are activated by the binding of specific peptide ligands to their extracellular domains (Drewett and Garbers, 1994). Members of this family include receptors for sea urchin egg peptides that regulate sperm motility, vertebrate natriuretic peptides that regulate cardiovascular homeostasis, guanylins that regulate intestinal electrolyte balance, and a retinal protein having no identified ligand. All of these proteins contain, immediately intracellular to the transmembrane domain, a protein kinase-like domain containing most of the invariant residues found in authentic protein kinases (Hanks et al., 1988). These domains lack key residues thought to be involved in phosphate transfer, however, and appear to lack protein kinase activity. Since deletion of this region results in constitutive activation, this region is thought to serve as an autoinhibitory domain. It also serves to anchor a protein serine phosphatase that may be involved in receptor desensitization (Chinkers, 1994). The guanylyl cyclase catalytic domain found near the carboxyl terminus of each of these receptors is similar in sequence to the catalytic domains of adenylyl cyclases and soluble forms of guanylyl cyclase.
Despite increasing information concerning the molecular characteristics of proteins that serve to catalyze the formation of cGMP in response to extracellular signals, significant questions dealing with the cellular role of this signaling pathway remain to be addressed. For example, the developmental role of receptor-activated cGMP pathways, if any, remains to be identified. In addition, pathways activated by cGMP might interact with other signaling pathways operating within a responding cell. In order to investigate these possibilities, we have begun to characterize cGMP pathways in Drosophila, an organism in which the combined approaches of both classical and molecular genetic analysis can be used to address such issues within the context of a well characterized developmental system. Here, we report the identification and characterization of a Drosophila cDNA encoding a protein that represents a member of the receptor guanylyl cyclase family. The restricted spatial and temporal expression of transcripts encoding this molecule during embryogenesis suggests a role for receptor-mediated increases in cGMP in specific developmental processes.
Figure 1: Schematic diagram of partial-length cDNA clones encoding DrGC-1. The predicted topography of the DrGC-1 cDNA is represented by a box. 5`-NR and 3`-NR represent 5`- and 3`-untranslated regions. ECD, KD, and CYC represent the extracellular, kinase-like, and cyclase domains, respectively. The hatched box indicates the predicted transmembrane region. Overlapping partial clones GC-1, GC-3, and GC-8 were used to construct the full-length cDNA. The stippled region in GC-1 represents an AT-rich region. The restriction map of the composite DrGC-1 clone is shown. The bold line indicates the coding region of this cDNA.
Phage DNA isolations were carried out by a
glycerol gradient method (Maniatis et al., 1982). Plasmid DNA
isolations were carried out by alkaline lysis (Maniatis et
al., 1982). DNA probes used for library screening were labeled
with [-
P]dCTP by random priming (Boehringer
Mannheim random priming kit) or by nick translation (Maniatis et
al., 1982).
Restriction fragments from positive clones were
subcloned into Bluescript (Stratagene). Longer fragments were subjected
to Exonuclease III treatment to generate shorter fragments of
200-400 bp for sequencing. Sequencing of both strands was
performed by the dideoxynucleotide method (Sanger et al.,
1977) using Sequenase (United States Biochemical Corp.) and S-labeled dATP. Reactions were run on 5% denaturing
polyacrylamide gels. T7 and T3 primers were used for sequencing unless
otherwise indicated.
The sequences of the forward primer (complementary to bases 1976-1995 of DrGC-1) and reverse primer (complementary to bases 3082-3102 of DrGC-1) were: GC-4, GCA C GA ATT CAT AAA GGG CAT GAT TCT A (EcoRI); GC-5, TGT G AA GCT TTA GCG CCT CTC CGT TGC T (HindIII).
PCR was conducted in a thermocycler by denaturing at 96 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min for 40 cycles. The 1.2-kb PCR products from 0-2 h, 10-14 h, and adult fly head samples were isolated and purified using the GeneClean kit (Bio 101). They were then digested with EcoRI and HindIII and subcloned into the corresponding sites of pBluescript (Stratagene) for sequencing.
Poly(A) RNA was prepared from total RNA using the Promega poly(A) tract
kit (Promega, Madison, WI). Poly(A)
RNA from different
developmental stages (10 µg) was electrophoresed on a 1.2% agarose,
6% formaldehyde gel in MOPS buffer, 1 mM EDTA, 1 mM sodium acetate, pH 7.0) for 5-6 h at 70 V and transferred to
a Nytran membrane (Schleicher and Schuell) in 10
SSC buffer.
The blot was subjected to UV cross-linking to fix the RNA and stained
with methylene blue (Herrin and Schmidt, 1988) to visualize the
samples. The blot was hybridized with probe at 42 °C in 50%
formamide, 5
SSC, 1% SDS, 5
Denhardt's solution,
0.5 M sodium phosphate, pH 7.2, and washed in 0.1
SSC,
0.2% SDS at 65 °C.
In situ hybridization to polytene chromosomes was carried out as described (Quan et al., 1989) except that cDNAs were labeled by sulfonation and detected using the Sulfoprobe DNA Detection System (Sigma).
Six additional clones encoding DrGC-1 were identified in a 12-24-h embryonic cDNA plasmid library, screened with 5` sequences encoding the kinase-like domain of DrGC-1 (see ``Experimental Procedures''). Restriction mapping and sequence analysis of the longest clone from this library, GC-8, indicated that it contained a 3-kb insert that, at its 3` end, contained approximately 300 bp of sequence found in GC-1 encoding the kinase-like domain of DrGC-1 (Fig. 1). In contrast to GC-1, 5` sequences in GC-8 did not contain an AT-rich region but instead contained an open reading frame which, when combined with GC-1 and GC-3, represented the complete coding sequence for DrGC-1. Thus, the three partial cDNA clones representing DrGC-1 (GC-1, GC-3, and GC-8) overlap to encompass a 6-kb sequence containing a single open reading frame of 1525 amino acids. The composite clone has 563 bp of 5`-untranslated sequence, a 4575-bp coding region, and 0.9 kb of 3`-untranslated sequence. Restriction fragments of these clones were ligated together to create a full-length clone.
Since the partial cDNAs used to construct the full-length
cDNA were isolated from two different sources (adult heads and
12-24-h embryos), non-qunatitative reverse transcribed PCR was
used to confirm that the composite cDNA represented an authentic
transcript. Two oligonucleotide primers were designed extending from
residue 658 to 1034 of the composite cDNA and spanning the junctions of
all three partial cDNAs. PCR was performed on first strand cDNA reverse
transcribed from poly(A) mRNAs isolated from several
developmental stages and adult fly heads. Agarose gel electrophoresis
of each reaction product showed a fragment of the expected size (Fig. 2). The amplification products from 0 to 2 and 10 to 14 h
and adult fly head transcripts were purified, subcloned, and sequenced.
In each case, the nucleotide sequence obtained corresponded to that of
the DrGC-1 cDNA composite clone. Thus, the composite cDNA represents an
authentic transcript present both during embryogenesis and in adult
flies.
Figure 2:
PCR
analysis of DrGC-1 transcripts PCR amplification of DrGC-1 was carried
out on reverse transcription products of poly(A) RNA
from different developmental stages. The PCR products were analyzed on
a 1% agarose gel. The position of the 1.2-kb PCR product is indicated
by an arrow. The + control lane contains PCR
amplification product of pSVL-DrGC-1 plasmid DNA and the - control lane amplification products from reactions with no added
template.
Figure 3: Nucleotide and deduced amino acid sequence of DrGC-1. Nucleotide and deduced amino acid sequences of DrGC-1 are shown. Nucleotide 1 is the first nucleotide of the ATG translation start codon. The translation termination codon is indicated by a dot. Nine potential N-linked glycosylation sites (Asn-X-Ser/Thr) in the putative extracellular region of DrGC-1 are underlined. The putative transmembrane region between residues 493 and 514 is also underlined.
The deduced amino acid sequence of the protein encoding DrGC-1 is similar to other vertebrates and invertebrate receptor/guanylyl cyclases, containing all the signature domains of these receptor proteins. These include an extracellular domain, a single putative transmembrane domain, a protein kinase-like domain, and a cyclase catalytic domain. DrGC-1 has an average overall sequence similarity of 30% with the other members of the receptor-guanylyl cyclase family and the highest degree of overall identity (38%) with the sea urchin Strongylocentrotus purpuratus guanylyl cyclase cDNA. Comparison of the amino acid sequence of DrGC-1 with the sequences of the sea urchin egg peptide receptor, with another guanylyl cyclase receptor recently isolated from Drosophila (termed DMGCR, Gigliotti et al., 1993), and with rat GC-A is shown in Fig. 4.
Figure 4: Comparison of the amino acid sequence of DrGC-1 with other receptor guanylyl cyclases. The deduced amino acid sequence of the Drosophila guanylyl cyclase (DGC-1) is aligned with the sea urchin S. purpuratus guanylyl cyclase (SPCYC), the rat guanylyl cyclase-ANP receptor (GCA), and a partial clone encoding a Drosophila membrane guanylyl cyclase (DMGCR). The identical residues between all four sequences are boxed.
Hydropathic analysis using the Kyte-Doolittle algorithm reveals a major hydrophobic stretch of 22 amino acids between residues 493 and 514 of DrGC-1 suggesting the presence of a single membrane spanning segment (not shown). The putative transmembrane region has the same relative position when compared with the transmembrane domains of other membrane guanylyl cyclases. This 22-amino-acid sequence is followed by Arg Lys residues similar to the stop transfer sequence found in other guanylyl cyclases and may represent the stop transfer signal anchoring the membrane protein during biosynthesis (Sabatini, et al., 1982). The protein appears to be divided by this hydrophobic segment into a 492-residue extracellular domain and a 1001-residue intracellular domain.
The putative extracellular region of DrGC-1 has 30% identity with the corresponding region of the sea urchin egg peptide receptor. It has much less similarity with mammalian guanylyl cyclase receptors; 21% with GC-A and 24% with GC-B and GC-C. The amino-terminal region also contains nine potential asparagine-linked glycosylation sites at positions 74, 184, 222, 338, 383, 394, 416, 428, and 458.
All the members of the membrane guanylyl cyclase family identified so far contain a protein kinase-like domain just intracellular to the predicted transmembrane domain. Protein kinases contain 33 highly conserved or invariant residues that have been divided into 11 subdomains (Hanks et al., 1988). DrGC-1 has 23 of these key 33 residues conserved in its kinase-like domain. The glycine-rich loop GXGXXG conserved in most of the protein kinases is present as GXXXG in DrGC-1. This is characteristic of receptor guanylyl cyclases, in which the glycine-rich motif is altered or absent. The invariant Asp in subdomain VI of protein kinases is present as an Asn residue in DrGC-1; lack of an Asp in this position is also a characteristic deviation of guanylyl cyclase receptors from authentic protein kinases. Mutagenesis of Asp to Asn in c-kit (murine white spotting locus) (Tan et al., 1990) and in fps (Moran et al., 1988) resulted in a loss of protein kinase activity. Similarly, erbB3, which has an Asn residue in this position, lacks protein kinase activity when expressed in insect cells (Carraway et al., 1994).
The predicted cyclase catalytic domain of DrGC-1 has 64% identity with rat GC-A, 61% with the sea urchin receptor, and 50% identity with DMGCR. Within this domain is a 165-amino-acid region that is highly conserved among guanylyl cyclases, mammalian adenylyl cyclases, and a bacterial adenylyl cyclase from Rhizobium meliloti (Parma et al., 1991). This 165-residue region has invariant amino acids in four blocks or subdomains (I-IV, Fig. 5). The two most conserved stretches of amino acid sequences in the catalytic domain of the receptor guanylyl cyclases (VYKVETIGDAYM in subdomain II and MPRYCLFG in subdomain III) are present in DrGC-1.
Figure 5: Alignment of the four highly conserved subdomains present in a number of cyclases with those present in DrGC-1. Alignment of the four highly conserved subdomains present within the 165 amino acid common cyclase domain of the guanylyl cyclase from Drosophila melanogaster (DGC-1), a partial clone encoding a Drosophila membrane guanylyl cyclase (DMGCR), rat brain ANP-A receptor (GCA), rat intestinal enterotoxin receptor (GCC), sea urchin (S. purpuratus) membrane guanylyl cyclase (SPCYC), a soluble guanylyl cyclase from Drosophila (DSGC), and the light (BLSGC) and heavy (BHSGC) subunits of the soluble guanylyl cyclase from bovine lung. Amino acids are denoted by their single-letter notation. Numbers in parentheses denote the number of amino acids in less conserved regions. Numbers on the left side of the sequence indicates the amino acid position of the first residue (with position 1 for the initiator methionine).
The sequence analysis described above indicates that DrGC-1 encodes a receptor guanylyl cyclase. In addition, DrGC-1 is predicted to contain an additional 430 amino acids at its carboxyl terminus. This extended carboxyl tail is rich in uncharged polar amino acids; about 14% of the terminal 430 residues are serine or glutamine. Although shorter carboxyl-terminal extensions in GC-C and in the retinal guanylyl cyclase have been suggested to be involved in anchoring to the cytoskeleton (Drewett and Garbers, 1994), this large, carboxyl-terminal region of DrGC-1 is not related to these guanylyl cyclase receptors or to any other proteins in sequence data bases.
The functional role of this COOH-terminal extension is not known. It may serve to target the protein to a particular subcellular location, or could be involved in the regulation of cyclase activity. This region contains several potential phosphorylation sites. Interestingly, the Drosophilarutabaga gene encodes an adenylyl cyclase that also contains an extended carboxyl region of >1000 residues beyond the translation termination site present in mammalian adenylyl cyclases (Levin et al., 1992). This region of the rutabaga gene contains many polyglutamine tracts that constitute an OPA repeat sequence found in a number of Drosophila genes (Wharton et al., 1985); its function remains to be determined.
Northern analysis of transcripts present at various stages of embryogenesis, larval, and pupal stages indicated the presence of a 7-kb transcript at all stages examined (Fig. 6A). The size of this transcript is approximately 1 kb more than the length of the cDNA isolated. This suggests that additional 5`-untranslated sequences are missing from the isolated cDNA. The abundance of this transcript, however, appears to be developmentally regulated. DrGC-1 is expressed at very high levels in early cleavage stage embryos (0-2 h) followed by relatively lower expression at later stages (2-6 and 6-10 h) of embryogenesis. During later stages of embryogenesis (10-14 and 14-18 h), DrGC-1 expression is again expressed at high levels. The message is present at very low levels in larval and pupal stages. Northern analysis of transcripts prepared from adult heads and bodies demonstrate equivalent levels of expression of the 7-kb transcript (Fig. 6B).
Figure 6:
Northern blot analysis of DrGC-1 and
DrGC-2 expression in adult flies. A, poly(A) RNA (5 µg) from different developmental stages including
embryonic, larval, and pupal stages was electrophoresed on a 1-2%
agarose gel and transferred to Nytran. This blot was then hybridized at
high stringency to a 5` 0.7-kb
P-labeled
Asp
/BamHI fragment of the DrGC-1 cDNA and
subsequently subjected to autoradiography. The lower panel,
indicated by the arrow, shows the autoradiograph of the
developmental Northern probed with Drosophila ribosomal
protein 49 (RP49) to demonstrate equal loading of lanes. B,
poly(A)
RNA (10 µg) from adult fly heads and
bodies was electrophoresed on a 1-2% agarose gel and transferred
to Nytran. This blot was then hybridized at high stringency to a 5`
0.7-kb
P-labeled Asp
/BamHI fragment
of the DrGC-1 cDNA and subsequently subjected to
autoradiography.
The spatial expression pattern of the DrGC-1 gene during embryogenesis was determined by whole mount in situ hybridization using digoxygenin-labeled probes (Fig. 7). High levels of uniformly distributed specific hybridization were observed in 0-2-h embryos (Fig. 7A). Levels of DrGC-1 transcript are almost undetectable by this method from gastrulation until late stages of embryogenesis (Fig. 7B). Elevated levels of hybridization were again observed at these later stages (14-18 h). Here, hybridization appears to be preferentially associated with somatic and visceral muscle fibers (Fig. 7, C and D). RNase-treated embryos hybridized to identical probes showed no significant hybridization (Fig. 7, E and F). In addition, embryos processed without added probe also showed no hybridization (not shown).
Figure 7: Whole-mount, in situ hybridization of DrGC-1 probes to Drosophila embryos. In situ hybridization to different stages of embryos was carried out using digoxigenin-labeled random primed DrGC-1 probes as described. Hybridization was detected by using anti-digoxigenin conjugated to alkaline phosphatase. The four panels below show stained embryos of different developmental stages as indicated. The arrows indicate staining in the body wall musculature. A, early 1-h embryo showing maternal expression of DrGC-1. B, 5-h embryo. C, late stage 16-h embryo. D, high magnification of 16-h embryo showing muscle staining. E, RNase-treated 1-h embryo. F, RNase-treated 16-h embryo.
Both Northern and in situ hybridization analysis indicate that DrGC-1 transcripts are present at high levels during the earliest stages of embryogenesis (0-2 h) when zygotic genes are not active. This indicates that DrGC-1 is placed into forming oocytes as a maternal message. Many maternally expressed genes encode components of a variety of signal transduction cascades that are important in early determination events such as the establishment of the embryonic axes (St. Johnston and Nusslein-Vollhard, 1991). This early expression of DrGC-1 may reflect a requirement for receptor-mediated increases in cGMP in the modulation of early developmental events. At later stages of embryogenesis, in situ hybridization analysis indicates that DrGC-1 transcripts are preferentially expressed in a restricted set of tissues. This is most prominent at late stages, specifically in muscle fibers. The temporal pattern of DrGC-1 expression at these later stages appears to correlate well with the period of myogenesis in developing embryos. Specific localization to developing musculature may reflect a role for DrGC-1-mediated increases in cGMP in the determination of muscle cells or in the normal physiology of embryonic muscle fibers similar to the vasorelaxation mediated by cGMP in both vertebrates and invertebrates.
The Drosophila genome has now been demonstrated to encode both membrane and soluble forms of guanylyl cyclase. Two receptor guanylyl cyclases have been identified in this study, and the structure and expression of one of these proteins has been described in detail. In addition, a membrane guanylyl cyclase gene mapping to region 32 of the Drosophila genome has recently been described (Gigliotti et al., 1993). A cDNA encoding a soluble, head-specific guanylyl cyclase has also been isolated from Drosophila head libraries (Yoshikawa et al., 1993). This soluble cyclase is preferentially distributed in the central nervous system and retina and is hypothesized to have a role in the fly visual system. The identification of several guanylyl cyclases in Drosophila suggests the importance of the cGMP signaling pathway in this organism, validating the use of Drosophila as an experimental system in which to study the role of signaling pathways that utilize cGMP as a second messenger. This system also provides a variety of molecular and genetic approaches that will allow further studies aimed at testing specific hypotheses on the role of cGMP in the development and physiology of this organism.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L35598[GenBank].