The Cloning of a Caenorhabditis Elegans Guanylyl Cyclase and the Construction of a Ligand-sensitive Mammalian/Nematode Chimeric Receptor*

(Received for publication, March 13, 1997, and in revised form, April 11, 1997)

Eric J. Baude Dagger §, Vivek K. Arora Dagger , Sidney Yu Dagger , David L. Garbers Dagger §par and Barbara J. Wedel Dagger **

From the Dagger  Department of Pharmacology and the § Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, 75235-9050

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Substantial guanylyl cyclase activity was detected in membrane fractions prepared from Caenorhabditis elegans (100 pmol cGMP/min/mg at 20 °C or 500 pmol cGMP/min/mg at 37 °C), suggesting the potential existence of orphan cyclase receptors in the nematode. Using degenerate primers, a cDNA clone encoding a putative membrane form of the enzyme (GCY-X1) was obtained. The apparent cyclase was most closely related to the mammalian natriuretic peptide receptor family, and retained cysteine residues conserved within the extracellular domain of the mammalian receptors. Expression of the cDNA in COS-7 cells resulted in low, but detectable guanylyl cyclase activity (about 2-fold above vector alone). The extracellular and protein kinase homology domain of the mammalian receptor (GC-B) for C-type natriuretic peptide (CNP) was fused to the catalytic domain of GCY-X1 and expressed in COS-7 cells to determine whether ligand-dependent regulation would now be obtained. The resulting chimeric protein (GC-BX1) was active, and CNP elevated cGMP in a concentration-dependent manner. Subsequently, a search of the genome data base demonstrated the existence of at least 29 different genes from C. elegans that align closely with the catalytic domain of GCY-X1, and thus an equally large number of different regulatory ligands may exist.


INTRODUCTION

Guanylyl cyclases appear to fall into two general families. (i) The membrane forms contain an extracellular ligand binding domain, a single transmembrane segment, and an intracellular protein kinase-like and cyclase catalytic domain; and (ii) the soluble forms contain two heterologous subunits, both of which contain a consensus cyclase catalytic domain (1, 2). These general features of the soluble and membrane forms appear to be conserved across many species, although expression of various invertebrate cDNAs encoding putative membrane guanylyl cyclases in mammalian cultured cells has not resulted in detectable enzyme activity (3-5).

Caenorhabditis elegans has proven a highly effective animal model for unraveling the functions of specific signaling pathways, the power resting particularly in the well defined lineage of each cell and the use of genetics. While cGMP has been measured (6) and a cGMP-dependent protein kinase has been purified from nematodes (7), C. elegans has been largely ignored as a means to define the functions of the various guanylyl cyclase/cGMP signaling pathways even though recent work on disruption of the cyclic nucleotide-gated ion channel has resulted in defective sensory neuron function (8, 9).

In preliminary studies on crude membrane fractions from C. elegans, guanylyl cyclase activity was easily measured, and PCR1 subsequently resulted in the identification of a cDNA clone encoding a putative guanylyl cyclase that was most closely related to the mammalian natriuretic peptide receptor family. The conservation of cysteine residues within the extracellular domain when compared with the mammalian natriuretic peptide receptors also suggested that the putative cyclase represented an orphan receptor. When GCY-X1 was expressed in COS-7 cells, although activity was low, it was reproducibly detected. To determine whether the nematode cyclase was capable of ligand-dependent regulation, we produced a chimeric protein consisting of the extracellular domain, transmembrane segment, and protein kinase homology domain of the mammalian guanylyl cyclase-B receptor (GC-B) and the putative catalytic domain of the C. elegans cyclase. This chimeric protein was active when transiently expressed in mammalian cells. Furthermore, the ligand (CNP) that binds to the mammalian cyclase, stimulated the activity of the nematode cyclase in a concentration-dependent manner. The fact that the cyclase catalytic domain of C. elegans can be regulated by the mammalian peptide supports the theory that GCY-X1 is an orphan receptor and suggests that a large family of apparent C. elegans guanylyl cyclases are also orphan receptors.


MATERIALS AND METHODS

Fractionation of C. elegans

The Bristol N2 strain of C. elegans was grown in liquid culture and frozen at -80 °C (10). An aliquot of frozen worms (0.25 ml) was ground with mortar and pestle under liquid nitrogen and resuspended in 1 ml of ice-cold homogenization buffer (10% glycerol, 50 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA, 2 mM dithiothreitol, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, 10 µg/ml aprotinin). The worm suspension was centrifuged at 2,000 × g for 10 min at 4 °C. The pellet was discarded, and the supernatatant fluid was centrifuged for 20 min at 100,000 × g and 4 °C to generate a membrane fraction (pellet) and a cytosolic fraction (supernatant fluid). Membranes were washed twice with homogenization buffer and resuspended by sonication.

Guanylyl Cyclase Assays

The nematode membrane fractions were assayed for cyclase activity after resuspension. For expression in COS-7 cells, cells were transiently transfected with eukaryotic expression vectors using the DEAE-dextran method (11). Cells were harvested 48 h after transfection in the presence of homogenization buffer and resuspended by sonication. Particulate fractions were isolated by centrifugation for 20 min at 100,000 × g. Membranes were washed twice with homogenization buffer and resuspended by sonication. The assay for guanylyl cyclase activity was performed in the presence of 5 mM MnCl2 and 0.1% Triton X-100 (12). Reaction mixtures containing 25 mM HEPES, pH 7.4, 50 mM NaCl, 0.25 mM 3-isobutyl-1-methylxanthine (IBMX), 0.1% bovine serum albumin, 10 mM NaN3, 1 mM dithiothreitol, 100 µM GTP and 0.5 µCi [alpha -32P]GTP (3000 Ci/mmol, DuPont NEN) in a total volume of 100 µl were incubated for 10 min at 37 °C unless indicated otherwise. [alpha -32P]cGMP was determined as described previously (12).

Cloning of the 3'-End of the GCY-X1 cDNA

Degenerate primers previously used to amplify guanylyl cyclase sequences from rat (13) were used to obtain guanylyl cyclase sequences from a mixed stage C. elegans cDNA library (Stratagene). The sense primer (TGTGGACIGCICCIGA(A/G)C(A/T)(A/G/C/T)(C/T)) is based on a conserved region within the protein kinase-like domain of membrane guanylyl cyclases and the antisense primer (TAIGC(A/G)TCICCIA(C/T)IGT(C/T)TCITT(A/G)TA) is based on a conserved region within the catalytic domain. The PCR reaction was performed as described (13) and yielded a single 940-base pair band. The PCR product was digested with XbaI and EcoRI and ligated into EcoRI- and XbaI-digested pGEM3Z (Promega). Thirty subclones were sequenced using the Prism kit (Applied Biosystems) and were shown to be identical. The cDNA fragment was radiolabeled and used to screen a mixed stage C. elegans cDNA library (Stratagene). Six positive phage clones were isolated, and the Bluescript plasmid containing the insert was excised according to the manufacturer instructions. The clone with the longest insert (PBS.16A, 4.8 kilobases) was sequenced completely in both directions. The putative guanylyl cyclase cDNA was designated GCY-X1 (the gene has not yet received a nomenclature within the C. elegans genome project).

Isolation of the 5'-End of GCY-X1 by Reverse Transcription-PCR

For RNA isolation, 1 ml of packed frozen C. elegans were thawed on ice, homogenized in RNA STAT-60 (Tel-Test) with a Brinkman Polytron homogenizer and passed twice through a 22-gauge needle. After addition of 2 ml of chloroform, the mixture was vortexed and centrifuged at 8,000 × g for 15 min. The upper phase was precipitated with isopropyl alcohol, and the precipitate was collected by centrifugation, washed with 70% ethanol, and dissolved in diethylpyrocarbonate-treated sterile water. Polyadenylated RNA was purified using the Fast Track kit (Invitrogen). 500 ng of polyadenylated RNA were reverse transcribed using the Superscript system (Life Technologies). The first strand cDNA was subjected to PCR using a sense primer corresponding to the SL-1 (14) or SL-2 RNA sequences (15) and a GCY-X1 antisense primer (GGGGGTACCGATGCACATAGGATAATCACA). The resulting PCR product was subcloned into pGEM3Z to give pSL1.700. The PCR product was sequenced in both directions.

Construction of Eukaryotic Expression Vectors for GCY-X1

To introduce a mammalian translation initiation sequence (16) just 5' to the predicted initiator methionine, PCR was performed on pSL1.700 with the primers (GGGCTGCAGGCCACCATGCTCAGATGGCTAACTCTACTT) and (CCCTCTAGAGTCGACTGTAACGAAATTTCTCGCAGCCAC). The product was digested with XbaI and PstI and ligated to pGEM3Z. The resulting construct was digested with SalI and ligated with the SalI/XhoI fragment of pBS.16A to create pKZ.GC. The full-length GCY-X1 cDNA was isolated by XbaI/PstI digestion of pKZ.GC and was ligated to XbaI/PstI-restricted pCMV5 (17) to give pCMV5.GCY-X1.

Construction of a Chimeric GC-B/GCY-X1 Expression Vector

Primers (GGGGGTACCAGCATATTGGATAATTTACTCAAAAGA) and (CCCGTCGACCTATGCAAATTCTTCGCCAAA) were used in the PCR with pBS.16A as template to amplify a GCY-X1 fragment containing an Acc65I site. The PCR product was digested with Acc65I and SalI and ligated into pBluescript (KS). The resulting construct was sequenced and digested with Acc65I and SalI. The insert was cloned into Acc65I- and SalI-digested pCMV5.GC-B to create pCMV5.GC-BX1.

Intact Cell cGMP Measurements

Cells transiently transfected with guanylyl cyclase expression vectors were washed with Dulbecco's modified Eagle's medium (DMEM) and incubated with DMEM containing 0.25 mM IBMX for 10 min at 37 °C. The medium was then switched to DMEM/0.25 mM IBMX and various concentrations of CNP (Peninsula Laboratories), and cells were incubated for 10 min at 37 °C. Perchloric acid (0.5 N) was added to stop the reaction, and the amount of cGMP formed was measured by radioimmunoassay (12).

Production of Antisera

A 15-mer peptide (GYKDVEIPDFGEEFA) corresponding to the predicted carboxyl terminus of GCY-X1 was coupled to the purified protein derivative (Serumstaateninstitut, Copenhagen, Denmark), and antiserum was produced (18). The peptide conjugate was injected into 8-pound female New Zealand White rabbits.

Western Blot Analysis

Membranes prepared from transiently transfected cells were subjected to SDS-PAGE. The proteins in the gel were electrophoretically transferred to nitrocellulose, which was incubated with primary antisera (1:1,000) and subsequently with a goat anti-rabbit antibody conjugated to horseradish peroxidase (Biosource International). The immunoreactive bands were visualized with the ECL reagent (Amersham Life Science).

Gene Localization

A polytene blot of 958 yeast artificial chromosomes (YAC) (gift of Alan Coulson, MRC laboratory for Molecular Biology) was probed with radiolabeled fragments corresponding to GCY-X1 (Arg50-Ser195 or Ser195-Thr497). The blot was prehybridized at 65 °C in 30 mM sodium phosphate, pH 6.2, 100 mM NaCl, 0.075% (w/v) sarkosyl, 17% (w/v) dextran sulfate and washed at 60 °C in 15 mM sodium phosphate, pH 6.2, 100 mM NaCl, 0.5 mM EDTA. The filter was subjected to autoradiography to visualize the positively hybridizing YACs.


RESULTS AND DISCUSSION

Guanylyl Cyclase activity in C. elegans

Guanylyl cyclases in most cells exist in both a soluble and particulate fraction. To initially characterize the localization as well as the relative activities of guanylyl cyclases of C. elegans, worms were homogenized and fractionated by high speed centrifugation. Surprisingly, given the lack of any report on cyclase activity in the nematode, cGMP was formed at a rate of 50-600 pmol/min/mg membrane protein, dependent on the assay temperature (Fig. 1). The activity of rat lung, the most active mammalian tissue for soluble enzyme activity, is about 100 pmol/min/mg protein (19), whereas invertebrate spermatozoa possess the highest activities of guanylyl cyclase found in particulate fractions, about 10 nmol/min/mg protein (20). The guanylyl cyclase activity in C. elegans membranes is relatively higher at lower temperatures (20-30 °C) than at higher temperatures (35-40 °C) when compared with GC-B (Fig. 1). Since C. elegans prefers soil temperatures that are nearer 20 °C, these temperature-dependent differences could be physiologically relevant.


Fig. 1. Temperature dependence of guanylyl cyclase activity in membranes of C. elegans. Guanylyl cyclase activity was compared between membranes of the invertebrate C. elegans and membranes of COS-7 cells expressing the mammalian GC-B receptor. The assay was performed in the presence of Mn2+/Triton X-100 for 10 min.
[View Larger Version of this Image (15K GIF file)]

Isolation of a Guanylyl Cyclase cDNA

The guanylyl cyclase activity in membrane fractions of C. elegans suggested a potential role for cGMP signaling in the nematode. We therefore used PCR to amplify receptor guanylyl cyclase sequences from a C. elegans cDNA library. Degenerate oligonucleotide primers directed against conserved regions of the mammalian receptor guanylyl cyclases were used for the amplification. From various PCR clones, only a single sequence (GCY-X1) was obtained and used to screen a C. elegans cDNA library. Several cDNA clones were isolated, but even the longest cDNA lacked the 5'-end, as evidenced by the lack of a splice leader RNA sequence and a predicted signal peptide sequence. To isolate the 5'-end, PCR was performed on first-strand cDNA using a sense primer corresponding to the C. elegans trans-splice leader RNAs, SL-1 or SL-2, and an antisense GCY-X1 primer. Only the product obtained with the SL-1 primer was derived from GCY-X1 sequence. The first predicted methionine occurs 30 nucleotides after the SL-1 sequence and is in frame with the previously determined GCY-X1 sequence (Fig. 2). Stop codons appear in the other two reading frames preceding the first methionine. Following this Met is a hydrophobic stretch of amino acids that would comprise a signal peptide sequence, and the mature protein is predicted to begin at Gly24 (21). The predicted GCY-X1 protein has an Mr 145,000 and a single transmembrane segment (22). There are eight consensus N-glycosylation sites within the putative extracellular domain. GCY-X1 is predicted to contain about 560 amino acids extracellularly, 23 amino acids within the membrane-spanning region, and 600 amino acids intracellularly. Between the juxtamembrane region and the Gly loop of the kinase-like domain, a 150-amino acid stretch occurs that bears no identity to sequences in the GenBankTM/EBI Data Bank. This region is rich in basic amino acids (12 Lys), and contains eight consensus protein kinase A phosphorylation sites as well as one consensus protein-tyrosine kinase phosphorylation site (23). Both GC-A and GC-B are known to be regulated by phosphorylation/dephosphorylation reactions (24-26), and therefore, this region of GCY-X1 may indicate an important regulatory region. The intracellular domain is about 55% identical to GC-A, and phylogenetic analysis places the C. elegans cyclase closest to the natriuretic peptide receptors (GC-A and GC-B) (Fig. 3). Furthermore, cysteine residues conserved in the extracellular domain of the natriuretic peptide receptors are also conserved in GCY-X1 (data not shown).


Fig. 2. The predicted protein sequence of GCY-X1. The putative signal peptide is in lowercase letters, while the sequence of the mature protein is in uppercase letters. Cysteine residues are highlighted in bold, potential N-glycosylation sites are in italics, and the putative transmembrane segment is underlined and in bold.
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Fig. 3. Dendrogram showing that GCY-X1 is most closely related to the mammalian natriuretic peptide receptor family. Analysis was with DNAstar software.
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Gene Localization

A polytene blot with 958 YAC clones allowing 95% coverage of the C. elegans genome (gift of Alan Coulson, MRC laboratory for Molecular Biology) was hybridized with two different GCY-X1 cDNA probes. Both probes hybridized to the same YAC clones, Y67H12 and Y39E12 (data not shown). A YAC clone, Y62H3, that overlaps with the 5' portions of Y67H12 and Y39E12 resulted in no signal, and thus the gene for GCY-X1 lies between unc-73 and unc-38 on chromosome I (data not shown) (27).

Genomic Sequence Data Base

Subsequently, a large number (>29) of C. elegans genomic DNA sequences homologous to guanylyl cyclases were found within the C. elegans genome sequencing project. An alignment of a highly conserved region within the catalytic domains of 17 of these putative guanylyl cyclases is given in Fig. 4. Determination of whether or not these sequences actually encode guanylyl cyclase activity is critical, although there have been consistent failures to express invertebrate putative membrane forms of guanylyl cyclases in mammalian cultured cells (3-5).


Fig. 4. Partial alignment of 17 different sequences from the C. elegans genome data base (33) with the conserved, putative catalytic region of GCY-X1. The respective region of the catalytic domain of GC-A, shown at the top, is highly conserved in all bona fide guanylyl cyclases. The gene nomenclatures of the predicted guanylyl cyclases in C. elegans were assigned roughly in their order of discovery in the genome sequencing project. GCY-31, GCY-32, and GCY-33 are putative soluble guanylyl cyclases.
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GCY-X1 cDNA Encodes a Guanylyl Cyclase

To formally demonstrate that GCY-X1 is a guanylyl cyclase, it is necessary to show that the respective protein catalyzes the conversion of GTP to cGMP. Several groups attempting to express other invertebrate drosophila (4, 5) or sea urchin membrane cyclases (3) have failed. The cyclases most closely related to GCY-X1, GC-A (28) or GC-B (29), however, express high enzymatic activity in various mammalian cultured cells. When GCY-X1 was expressed in COS-7 cells and enzyme activity of membranes estimated in the presence of Mn2+/Triton X-100, cyclic GMP was formed at a rate of about two times the rate of vector-transfected cells (Fig. 5B). Although substantially lower than that seen with the mammalian cyclases, the elevation was consistently observed. The relatively low activity could be due to low expression levels (see Fig. 5B, inset). The addition of micromolar concentrations of ANP, BNP, or CNP to the isolated COS-7 cell membranes did not stimulate GCY-X1 when assays were conducted in the presence of Mg2+. Given that Koller et al. (30) have succeeded in producing mammalian GC-A/GC-B chimeric enzymes capable of responding to ligand, we designed experiments that would test the hypothesis that GCY-X1 was an orphan receptor. A GC-B/GCY-X1 chimeric expression vector was constructed. The rat GC-B cDNA clone contains an Acc65I site preceding the hinge region of the protein, an amphipathic helix proposed to mediate dimerization of GC-A (31). This fortuitous restriction site facilitated the swapping of a cDNA fragment encoding the presumed dimerization motif and catalytic domain of GC-B with the respective cDNA fragment of GCY-X1. The resulting chimera (GC-BX1), shown schematically in Fig. 5A, comprises amino acids 1-798 of GC-B and amino acids 1111-1233 of GCY-X1. GC-BX1 was expressed at levels much higher than GCY-X1 (Fig. 5B, inset). cGMP elevations in COS-7 cells transfected with GC-B or GC-BX1 were examined as a function of CNP concentration. The mammalian/nematode chimeric enzyme responded to the added CNP at concentrations equivalent to that of GC-B (Fig. 6) (32). EC50 values for GC-B and GC-BX1 were identical (3 × 10-8 M).


Fig. 5. A, schematic presentation of GC-B, GC-BX1, or GCY-X1. The chimera GC-BX1 consists of the extracellular, transmembrane, and kinase homology domain of GC-B, as well as the hinge region and putative catalytic domain of GCY-X1. ECD, extracellular domain; TM, transmembrane domain; KHD, kinase homology domain; H, hinge region; and CAT, catalytic domain. B, guanylyl cyclase activity in membranes of COS-7 cells transfected with pCMV5, pCMV.GC-B, pCMV.GC-BX1 or pCMV.GCY-X1. Enzymatic activity was determined in the presence of Mn2+/Triton X-100. Inset, Western blot showing the expression levels of GC-BX1 or GCY-X1 in COS-7 cell membranes. The predicted Mr for GCY-X1 is 145,000 and for GC-BX1 is 117,916. The antiserum was directed against a peptide corresponding to the predicted carboxyl terminus of GCY-X1.
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Fig. 6. Elevations of cGMP levels in COS-7 cells transfected with GC-B or GC-BX1 as a function of CNP concentration. Bars represent the standard error of means of quadruplicate determinations of a single representative experiment.
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This is the first time an invertebrate membrane guanylyl cyclase activity has been successfully expressed in a mammalian cultured cell, confirming the hypothesis that GCY-X1 is a guanylyl cyclase. Although the ligand for GCY-X1 is not yet known, the extracellular domain most closely resembles the mammalian natriuretic peptide receptors, suggesting that if the C. elegans cyclase is a receptor, the ligand may fall into the natriuretic peptide family. Koller et al. (30) had previously demonstrated that the extracellular domains of GC-A and GC-B could be interchanged with retention of ligand sensitivity but that such chimeric constructs with GC-C (receptor for heat-stable enterotoxin and only about 10% identical at the amino acid level within the extracellular domain) were unsuccessful. Thus, it was postulated that if GCY-X1 is an orphan receptor, given it appears most closely related to the mammalian natriuretic peptide receptors, the cyclase catalytic domain of the nematode enzyme could be sensitive to regulation by a mammalian extracellular, ligand-binding domain. This was the case, and therefore by extension, it is suggested that many or all of the cyclases that are part of the large family (>29) of apparent cyclases in the nematode represent orphan receptors.

The large number of cyclases in the nematode also raises the question of whether a much larger family of cyclases exists in the mammal. Only six membrane forms of cyclase have been reported in the mammal, three of which remain orphans (2, 13), despite a mammalian genome about 30 times larger than that of C. elegans. PCR and low stringency hybridization have been used to search for other mammalian guanylyl cyclases, but the closest apparent relatives to GCY-X1 continue to be the natriuretic peptide receptors. The positive results with the chimeric receptor constructed here support the hypothesis that GCY-X1, in fact, is the C. elegans natriuretic peptide receptor homologue.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) L80003[GenBank].


   Supported by a Medical Scientist Training Program Fellowship.
par    To whom correspondence should be addressed.
**   Supported by a Deutsche Forschungsgesellschaft Fellowship.
1   The abbreviations used are: PCR, polymerase chain reaction; GC-A-F, mammalian guanylyl cyclase-A-F; GCY-X1, nematode guanylyl cyclase-X1; GC-BX1, chimeric protein between GC-B and GCY-X1; ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide; CNP, C-type natriuretic peptide; IBMX, 3-isobutyl-1-methylxanthine; DMEM, Dulbecco's modified Eagle's medium; YAC, yeast artificial chromosomes.

ACKNOWLEDGEMENTS

We are grateful to Lynda Doolittle for sequencing the cDNA and Debbie Miller for synthesizing the oligonucleotide primers. We also thank David Foster for carefully reading the manuscript.


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