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Tunicate Gonadotropin-Releasing Hormone (GnRH) Peptides Selectively Activate Ciona intestinalis GnRH Receptors and the Green Monkey Type II
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     Department of Biology (J.A.T., N.M.S.), University of Victoria, Victoria, British Columbia, Canada V8W 3N5

    Clayton Foundation Laboratories for Peptide Biology (J.E.R.), The Salk Institute, La Jolla, California 92037

    Abstract

    In vertebrates, GnRH binds to its receptor and stimulates predominantly Gq/11-mediated signal transduction in gonadotropes. However, little is known about the GnRH receptor and its signaling pathway in tunicates, a group that arose before the vertebrates. Although tunicates have had duplications of a few genes in the last 600 million years, the early vertebrates had duplications of the full genome. Also unknown is the nature of GnRH signaling in the tunicate, which lacks both a pituitary gland and sex steroids. However, we know that tunicates have GnRH peptides because we previously reported six GnRH peptides encoded within the tunicate genome of Ciona intestinalis. Here we clone and sequence cDNAs for four putative GnRH receptors from C. intestinalis. These are the only invertebrate GnRH receptors found to date. Each Ciona GnRH receptor was expressed in COS-7 cells, incubated with each of the six C. intestinalis GnRHs and assayed for a signaling response. GnRH receptors 1, 2, and 3 responded to Ciona GnRH peptides to stimulate intracellular cAMP accumulation. In contrast, only GnRH receptor 1 activated inositol phosphate turnover in response to one of the Ciona GnRHs. The green monkey type II GnRH receptor cDNA was tested as a comparison and a positive control. In conclusion, the four GnRH receptors encoded within the C. intestinalis genome were all transcribed into messenger RNA, but only three of the Ciona GnRH receptors were biologically active in our assays. The Ciona GnRH receptors almost exclusively activated the cAMP pathway.

    Introduction

    GnRH IS AN ANCIENT PEPTIDE that mediates the release of LH and FSH from the pituitary gland, which in turn induce gametogenesis and steroidogenesis in the vertebrate gonads. A highly conserved molecule, GnRH is 10 amino acids in length, with 24 family members identified to date (1). The GnRH receptor (GnRHR) was first cloned from a murine gonadotrope cell line, T3–1 (2, 3) and was shown to be a member of the seven-transmembrane G protein-coupled receptors (GPCRs). It is now clear that most vertebrates each have more than one GnRHR subtype that coincides with expression of up to three GnRH ligands in each species. One human GnRH ligand is GnRH1 (also known as mammalian GnRH); it is identified in early bony fish, lungfish, amphibians, and mammals (4, 5, 6). The other human GnRH form is GnRH2 (also known as chicken GnRH-II), which was first isolated from chicken (7) but is found in almost all jawed vertebrates from sharks to humans (4). Identification and characterization of each GnRHR is crucial for determining the GnRH target sites and signaling pathways. The multiple GnRH ligands and receptors found within Ciona intestinalis (a sea squirt) may be involved in a number of distinct functions.

    Genes in protochordates are of interest because they represent the foundation from which vertebrates evolved. The origin of vertebrates is thought to be an ancestral protochordate such as tunicates. One hypothesis is that the vertebrates evolved as a result of more than one duplication of the genome, which is supported by the presence of one cluster of Hox genes in tunicates but four clusters in mouse and human (8). The recent sequencing of two tunicate genomes, C. intestinalis and C. savignyi (whole-genome shotgun databases located at http://www.jgi.doe.gov/ciona and http://www.broad.mit.edu/annotation/ciona, respectively) (8), has added to the debate regarding the evolutionary origin of higher chordates in general and of GnRH and GnRHR molecules in particular. One can now examine the possible origin of a number of molecules including GnRH and its cognate receptor that may be ancestral to those in vertebrates.

    Recently we characterized two genes from a tunicate, C. intestinalis, in which each gene encoded three distinct GnRH peptides for a total of six deduced peptides (1). Also, we characterized three other distinct tunicate GnRH peptides: one was deduced from the C. savignyi genomic sequence (1) and two were isolated as peptides from Chelyosoma productum (9). In addition, a GnRH-like peptide of 16 amino acids, which we refer to as tunicate-X, was annotated in the C. intestinalis genome as the homolog of a gonadoliberin-II precursor. All the tunicate GnRH peptides are closely related to vertebrate GnRH peptides with high conservation of key amino acids. The apparent lack of steroid receptors and key enzymes essential for sex steroid synthesis in the C. intestinalis genome (8) precludes GnRH from an involvement in stimulation and release of reproductive steroids. We previously detected estradiol in tunicate gonads by RIA (10), but tunicate extracts may have interfered with the assay designed for human steroids. In tunicates, GnRH is evidently involved in reproductive functions and may act directly on the gonads and/or gonoducts instead of stimulating the hypophyseal release of LH and FSH as in higher chordates. Previous studies by our laboratory (1), are consistent with findings by Terakado (11) that have shown injection with physiological concentrations of tunicate GnRHs into gravid C. intestinalis induces the release of gametes from the atrial siphon. These injections may mimic the release of tunicate GnRH located in nerves that terminate in a blood sinus near both the testis and ovary (12).

    Given that native GnRH peptides are bioactive in C. intestinalis, we were interested in establishing whether these peptides transmit their signal through a GnRHR similar to those possessed by vertebrates. We found four putative GnRHR homologs encoded in the C. intestinalis genome. Two of the GnRHRs were previously identified by cDNA structure for C. intestinalis (13), but the receptors were tested with only two GnRHs from Chelyosoma productum. It is important to determine whether the receptors are activated by Ciona GnRHs because a recent report showed that a receptor isolated from Drosophila appeared to be structurally related to a GnRHR, but was activated by adipokinetic hormone (AKH) rather than GnRH (14).

    In this study we examine whether tunicates have functional GnRHRs. We provide the cDNA sequences for four putative GnRHRs from C. intestinalis and compare these sequences to the genomic structure to establish the gene organization and exon boundaries. Two of the receptor sequences are novel and the other two sequences confirm the structures of a previous report. To determine receptor function, we expressed each receptor in COS-7 cells and incubated them with each of nine native tunicate GnRHs, one novel GnRH (tunicate-X), and one related peptide AKH. We tested receptor activation by assaying the levels of GnRH-induced second messengers: inositol phosphates (IPs) and cAMP. We also examined whether critical motifs are conserved for ligand binding, binding pocket formation, G protein coupling and receptor activation. Hereafter we use the abbreviated name Ciona for the species C. intestinalis; if other species of Ciona are mentioned, the full genus and species are given.

    Materials and Methods

    Gene organization

    Gene arrangements were discovered initially using the database from the Department of Energy Joint Genome Institute’s C. intestinalis genome project (http://www.jgi.doe.gov/programs/ciona.htm) and N. Satoh and Y. Satou’s Ghost C. intestinalis cDNA database (http://ghost.zool.kyoto-u.ac.jp/indexr1.html). Human, rat, and horse GnRHR amino acid sequences were used to search the available TBLASTN input form. Using the default parameters (BLOSUM 62 matrix), each search generated closely matched fragments. The DNA regions for the matching fragments of four putative receptors were compiled, examined for exon/intron boundaries, and analyzed for a complete open reading frame. Primers were designed and the complete receptor cDNAs were amplified using PCR and sequenced as stated below. To complete any missing regions in the receptors, 5'- or 3'-rapid amplification of cDNA ends (RACE) was used.

    Animals

    Adult C. intestinalis (subphylum Tunicata, class Ascidiacea) were obtained from Woods Hole Biological Station (Woods Hole, MA) and treated under the guidelines of the Animal Care Committee at the University of Victoria. The tissues were dissected and frozen in liquid nitrogen.

    Isolation of mRNA and synthesis of cDNA

    The mRNA was isolated from tissues and embryos using a micropoly(A) pure mRNA isolation kit (Ambion, Inc., Austin, TX). Then mRNA was reverse transcribed in a 50-μl reaction that contained mRNA, 2 mM oligo dT, 2 mM deoxynucleoside triphosphates, 1x first-strand reaction buffer, 0.01 M dithiothreitol, 5 U RNase inhibitor, and 100 U Superscript II reverse transcriptase (Invitrogen, San Diego, CA). The reaction was incubated at 42 C for 90 min, and the enzyme was heat inactivated at 90 C for 10 min. A negative reverse transcription reaction was run for each tissue sample without reverse transcriptase to check for genomic DNA contamination.

    For RACE-PCR, approximately 200–300 ng of mRNA was used to prepare RACE-ready cDNA using the RLM-RACE kit (Ambion) according to the manufacturer’s instructions, except that the DNA was redissolved in distilled water.

    PCR and sequencing of cDNA

    Oligonucleotides were designed to regions encoding candidate GnRHRs based on the compiled sequences for Ciona GnRHR genes 1–4. Each 50-μl reaction contained 2.5 U Platinum Taq polymerase high-fidelity (Invitrogen), 1x high-fidelity PCR buffer, 2.5 mM MgSO4, 0.2 mM deoxynucleoside triphosphates (Invitrogen), and 0.4 μM of each Kozak forward (f) and stop reverse (r) primer (Table 1). PCRs were performed under the following conditions: initial denaturation at 94 C for 2 min, 33 cycles of denaturation at 94 C for 30 sec, annealing at 57 C for 30 sec, extension at 72 C for 2 min, and a 5-min final extension. The PCR amplicons were separated by electrophoresis on a 1.3% agarose gel and visualized with ethidium bromide staining using an Eagle Eye II still video system (Stratagene, La Jolla, CA). Bands were selected, isolated (QIAGEN, Valencia, CA), and cloned or cloned directly as amplicons into pGEM Vector-T (Promega Corp., Madison, WI) and sequenced. The SequiTherm EXCEL II DNA sequencing kit using the Sanger sequencing method was performed by the University of Victoria Sequencing Centre. Each gene was amplified using M13 forward and reverse priming sites present on pGEM-T and sequenced on a LI-COR 4200-Global IR2.

    PCR for tissue distribution

    The tissue expression (TE) primer pairs (Table 1) were used in each tissue expression PCR. Each 50-μl reaction contained 1 U Platinum Pfx DNA polymerase (Invitrogen), 1x Pfx amplification buffer, 1 mM MgSO4, 0.3 mM deoxynucleoside triphosphates (Invitrogen), and 0.4 μM of each f and r primer (Table 1). PCRs were performed under the following conditions: initial denaturation at 94 C for 2 min, 35 cycles of denaturation at 94 C for 30 sec, annealing at 58 C for 30 sec, extension at 68 C for 2 min 30 sec, and a 3-min final extension. The PCR amplicons were separated by electrophoresis on a 1.3% agarose gel as stated above.

    Receptor and green fluorescent protein vector design

    A clone of green monkey GnRHR (gm-gnrhr) was a gift from Dr. Jimmy Neill (University of Alabama, Birmingham, AL). The cDNAs containing full-length open reading frames of each Ciona intestinalis-gnrhr (Ci-gnrhr) and gm-gnrhr were altered to include a strong context Kozak sequence (A/G)NNATGG) (where N is any amino acid) (15) using the same PCR conditions as previously mentioned and then cloned into pcDNA3.1(–) (Invitrogen) in front of an internal ribosome entry site and an enhanced green fluorescent protein (eGFP) coding region. This system generates coexpression of the receptor protein and eGFP allowing for live selection of COS-7 cells with high transfection efficiency.

    In addition, we fused the eGFP coding sequence directly to the 3' end of the cDNAs for Ci-gnrhr1 and Ci-gnrhr4 to verify that the receptors were expressed as a protein. After we determined that Ci-GnRHR4 was not bioactive, we used an alternate Kozak sequence (GCCACCatgG) for the translation start site for Ci-gnrhr4 and fused the cDNA directly to eGFP as above to verify that the receptor was translated into protein.

    IP accumulation assay

    No Ciona cell lines were available to facilitate our receptor signaling assays, so we chose COS-7 cells, which have been well used in previous GnRHR assays (16, 17, 18). COS-7 cells (Invitrogen) were seeded and grown into monolayer cultures in T-75 cm2 flasks in growth medium containing DMEM (Invitrogen) supplemented with 0.1 mM nonessential amino acids (Invitrogen) and 10% fetal bovine serum (Invitrogen) at 37 C in 5% CO2. After 3 d the monolayers were trypsinized and seeded in 24-well tissue culture treated plates (Corning-Costar Corp., Cambridge, MA) at a density of 65,000 cells/well and grown overnight in growth medium. At 85–95% confluence, usually 24 h after seeding, the cells were washed and incubated with serum-free medium (VP-SFM, Invitrogen) and then transfected with 0.8 μg/well of receptor-encoded plasmid DNA using lipofectamine as per the manufacturer’s protocol (Invitrogen). After another 24 h, the cells were washed with labeling medium (Medium 199, Invitrogen) containing 0.3% bovine albumin (Sigma-Aldrich, St. Louis, MO) and subsequently labeled with 0.9 μCi/well of myo-[2-3H]Inositol (Amersham, Piscataway, NJ) in labeling medium for about 24 h. The wells were screened for high transfection efficiency comparing cellular levels of endogenously marked eGFP using an inverted microscope with a fluorescein isothiocyanate filter set. The cells were washed and preincubated for 30 min at 37 C in labeling medium containing 20 mM LiCl and then stimulated with various concentrations of ligands for 1 h at 37 C with gentle agitation. After stimulation, the medium in each well was removed, and 200 μl of 0.1 M formic acid were added to each well to lyse the cells. Quantitation of IPs was performed in cell extracts by the multiwell filtration method described by Chengalvala et al. (19).

    cAMP accumulation assay

    COS-7 cells were grown, seeded, and transfected in 24-well plates as described above. The cells were allowed to grow in VP-SFM for 48 h after transfection, after which wells with high transfection efficiency were selected. These cells were washed with Hank’s buffered salt solution supplemented with 20 mM HEPES and 300 μM 3-isobutyl-1-methylxanthine and preincubated for 15 min at 37 C and then stimulated with various concentrations of ligands for 1 h at 37 C with gentle agitation. Intracellular cAMP concentrations were measured using a cAMP direct enzyme immunoassay as per the manufacturer’s protocol (Amersham).

    Data analysis

    All IP samples were measured in triplicate and cAMP samples in duplicate within each assay. All assays were repeated in at least three independent experiments. Data analysis was performed using nonlinear regression. The GnRH concentrations inducing EC50 were calculated using PRISM4 software (GraphPad Software, Inc., San Diego, CA). The GnRH concentrations inducing EC50 presented in Tables 4 and 5 were calculated from the mean ± SEM of at least three independent experiments. The data were analyzed by one-way ANOVA followed by Newman-Keuls test. P < 0.05 was considered statistically significant.

    Phylogenetic analysis

    The deduced amino acid sequences of the four C. intestinalis GnRHRs were aligned with GnRHR and GnRHR-like proteins from other species using the ClustalW program. The sequences were: two human (Homo sapiens) GnRHRs type I (GenBank accession no. NP000397) and type II (Q96P88); rubber eel (Typhlonectes natans) GnRHR (NF174481); African clawed frog (Xenopus laevis) type II GnRHR (AF257320); bullfrog (Rana catesbeiana) GnRHR-3 (AF144062); house mouse (Mus musculus) type I GnRHR (NP034453); African green monkey (Cercopithecus aethiops) type II GnRHR (Q95MH6); striped sea bass (Marone saxitilis) GnRHR (AAF28464; amberjack (Seriola dumerili) GnRHR (CAB65407; fruitfly (Drosophila melanogaster) adipokinetic hormone receptor (AF077299); and the human (Homo sapiens) 1-adrenergic receptor (AAQ91331 as an outgroup. The phylogenetic tree was generated based on the ClustalW alignment using a topological algorithm with PHYLIP software (available at the EMBnet Node: http://www.genebee.msu.su/emb.html). Branch lengths are to scale.

    GnRH peptide synthesis

    Ten tunicate GnRH peptides (Table 2) were synthesized automatically on a CS-biopeptide synthesizer (model CS536, CS Bio Co., Inc. San Carlos, CA) on a methyl benzhydrylamine resin using the Boc-strategy at the Salk Institute. The peptides were cleaved with hydrofluoric acid, concomitantly deprotected, and then purified as described by Adams et al. (1). The purity of the peptides was also characterized by capillary zone electrophoresis performed on a Beckman P/ACE System 2050 controlled by an IBM personal system/2 (model 50Z; IBM, White Plains, NY) connected to a ChromJet integrator (Spectra Physics, San Jose, CA). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the peptides was measured on an ABI-Perseptive DE-STR instrument (PE Applied Biosystems, Foster City, CA).

    AKH from Bombyx mori was purchased from Bachem (King of Prussia, PA).

    Results

    Two novel C. intestinalis cDNAs encode seven-transmembrane putative GnRHRs

    In silico analysis revealed four candidate GnRHR genes in C. intestinalis. Each of four cDNAs, encoding distinct GnRHR-like receptors, was amplified from poly(A)+ mRNA extracted from the neural complex by RT-PCR. Two GnRHRs were unique sequences that did not match any known GnRHR proteins to date. The other two GnRHR cDNAs were similar to previously identified Ciona GnRHRs (13) in which one had 98% amino acid identity with Ci-GnRHR1 (AB103333) and the other had 96% amino acid identity with Ci-GnRHR2 (AB103334) (Fig. 1). Our two novel GnRHRs were designated Ci-GnRHR3 and Ci-GnRHR4. The isolated Ci-gnrhr1 confirmed a 2043-bp cDNA encoding a protein of 450 amino acids, whereas the cDNA for Ci-gnrhr2 was 1675 bp encoding a protein of 401 amino acids. The novel full-length Ci-gnrhr3 cDNA consisted of 1752 bp encoding a protein of 454 amino acids, and the novel Ci-gnrhr4 cDNA consisted of 1666 bp encoding a protein of 366 amino acids. The four Ciona GnRHR cDNAs encoded proteins with the hydrophobicity profile characteristic of GPCRs: seven-transmembrane domains connected by alternating intracellular and extracellular loops, and extracellular N-terminal and intracellular C-terminal domains (Fig. 1).

    To establish the identity of the receptors, the four Ciona GnRHR amino acid sequences were entered into the TBLASTN input form and searched against the NCBI databases. The sequence identity of Ciona GnRHRs with each other was analyzed (Table 3). The highest matches of the novel receptors to other GnRHRs were to the rubber eel GnRHR (AAD49750 in which Ci-GnRHR3 and Ci-GnRHR4 exhibited 36 and 52% amino acid identity, respectively.

    The nucleotide sequences for the two novel Ciona GnRHR cDNAs, Ci-gnrhr3 and Ci-gnrhr4, are in the GenBank nucleotide databases under the GenBank accession no. AY742890 and AY742891. The open reading frames for Ci-gnrhr1 and Ci-gnrhr2 that we identified are submitted under the GenBank accession no. AY742888 and AY742889, respectively.

    Ciona GnRHR genes maintain consistent exon arrangement but not intron size

    The receptor cDNAs were aligned with the C. intestinalis genome to determine the intron/exon boundaries. This technique revealed gene sizes of 8378, 7419, 15795, and 5960 bp for Ci-gnrhr1, Ci-gnrhr2, Ci-gnrhr3, and Ci-gnrhr4, respectively. Each gene for Ci-gnrhr1, Ci-gnrhr2, and Ci-gnrhr3 consists of eight exons separated by seven introns, whereas the Ci-gnrhr4 gene contains seven exons separated by six introns (Fig. 2). The corresponding sizes of exons three, four, and five are similar for all C. intestinalis GnRHRs. The 3'-exon deviates most in size among the four Ci-gnrh receptor transcripts.

    Ciona GnRHR proteins have conserved domains like vertebrate GnRHRs

    The four amino acid sequences of Ciona GnRHRs were aligned with type I human, type II human, and type II monkey GnRHRs (Fig. 1). There is high conservation in all seven putative transmembrane domain (TMD) regions with some conservation in intracellular loop (ICL) 2 and extracellular loops (ECL) 1 and 2. Three of the Ciona GnRHRs possess potential glycosylation sites in the N-terminal domain (at positions 16, 27, and 31 for Ci-GnRHR1; 31, 38, and 44 for Ci-GnRHR3; 10 and 15 for Ci-GnRHR4). The N-terminal extracellular region and C-terminal tail show no conservation with the vertebrate receptors. Ci-GnRHR1, Ci-GnRHR2, and Ci-GnRHR3 each possess a C-terminal tail longer than the monkey type II receptor. Ci-GnRHR4 has a short 20 amino acid C-terminal tail more analogous in length to type I mammalian GnRHRs.

    Ci-GnRHRs share conserved residues and motifs with vertebrate GnRHRs; the conserved residues for Ci-GnRHR1 are highlighted in Fig. 3. Several residues previously characterized to be involved in ligand binding or formation of the binding pocket are conserved in all tunicate GnRHRs: Asp2.61(98) (D), Trp2.64(101) (W), and Asn2.65(102) (N) in TMD2; Lys3.32(121) (K) in TMD3; Trp6.48(280) (W), Tyr6.51(283) (Y), and Tyr6.52(284) (Y) in TMD6; and Trp6.59(291) (W) in ECL3. Also, Ci-GnRHRs have highly conserved Cys (C) residues in ECL1 and ECL2. Highly conserved microdomains that may be involved with receptor activation are present, with Asn1.50 (N) in TMD1, Asp2.50 (D) or Asn2.50 (N) in TMD2, and the DRxxxI/V motif within and adjacent to TMD3. This motif is modified to DRxxxL in Ci-GnRHR4.

    The four GnRHR transcripts are widely distributed in adult Ciona

    Transcripts for each Ciona GnRHR were found in the neural complex, gonad, heart, intestine, endostyle, and branchial sac (Fig. 4). The control reaction that lacked template was negative for all four receptor transcripts.

    Cloning the Ci-GnRHR3 revealed a splice variant present in the neural complex of mature specimens. The splice variant molecule named Ci-gnrhr3 contains a 271-bp deletion in exons 1 and 2 (from nucleotide positions 213–484). Comparison of Ci-gnrhr3 coding regions with the full-length Ciona GnRHRs reveals that this deletion is predicted to cause a shift in the open reading frame, leading to protein truncation eight amino acids downstream of the splice site, resulting in an incomplete receptor protein.

    Only one Ciona GnRH receptor activates the IP signaling pathway

    To determine whether each Ci-gnrhr cDNA encoded a functional GnRHR, we expressed each receptor cDNA in COS-7 cells. The control and transfected COS-7 cells were exposed to 10–5 to 10–11 M concentrations for each of 10 native Ciona GnRH peptides and the related peptides (Table. 2). Untransfected COS-7 cells or those transfected with eGFP only were unable to elicit a dose response with either IP or cAMP with any of the peptides tested.

    Tunicate (t) GnRH-6 was able to stimulate IP accumulation in Ci-GnRHR1 (Fig. 5A). None of the ligands tested were able to stimulate 3H-IP accumulation with the other Ci-GnRHRs (Table 4).

    Two tGnRH ligands stimulate the type II gm-GnRHR in IP pathway

    To compare IP stimulation profiles with a prototypical mammalian type II GnRHR, the type II gm-GnRHR was assayed and shown to be activated with GnRH2 > tGnRH-5 > tGnRH-3; statistical analysis revealed that there was no significant difference among the three peptides (Fig. 5B).

    Three Ciona GnRHRs signal through the cAMP pathway

    The limited IP activation response led us to examine whether the Ciona receptors would couple to Gs and elicit a cAMP response after stimulation. COS-7 cells were transfected with each Ciona GnRHR cDNA, and cAMP production was measured in response to native Ciona GnRHs and vertebrate GnRH2. For Ci-GnRHR1 the potency was tGnRH-6 > GnRH2 > tGnRH-7 > tGnRH-8 > tGnRH-3 > tGnRH-5 > tGnRH-4; statistical analysis showed that tGnRH-6 was the most potent (see Fig. 6A and Table 5). With Ci-GnRHR2, the order of effectiveness was tGnRH-8 > tGnRH-7 > tGnRH-6 > tGnRH-4 > GnRH2 > tGnRH-5 > tGnRH-3; analysis showed that there was no significant difference with the GnRH peptides (see Fig. 6B and Table 5). Ci-GnRHR3 was active with: tGnRH-5 > tGnRH-3 > GnRH2 > tGnRH-6 > tGnRH-7 > tGnRH-4 > tGnRH-8; statistical analysis showed that tGnRH-5 and tGnRH-3 were the most potent of the tunicate GnRHs (see Fig. 6C and Table 5). Ci-GnRHR4 did not demonstrate cAMP accumulation with any of the tGnRHs nor did tGnRH-X or AKH activate any of the Ci-GnRHRs.

    Ciona GnRHRs form an independent branch in phylogenetic tree

    The primary amino acid sequences of the four Ciona GnRHRs were aligned with selected types I, II, and III vertebrate GnRHRs, as previously classified (20), to construct a phylogenetic tree (Fig. 7). The four Ciona receptors form their own independent branch, compared with the types I, II, or III vertebrate GnRHRs. Ci-GnRHR1 and Ci-GnRHR2 cluster to the same branch of the tree, whereas Ci-GnRHR3 and Ci-GnRHR4 form independent branches within the tree.

    Discussion

    C. intestinalis encodes four GnRHRs

    In the present study, we found four GnRHR transcripts were encoded within the tunicate C. intestinalis genome. Each Ciona GnRHR transcript encodes a protein with an extracellular N terminus, seven hydrophobic stretches corresponding to putative -helical TMDs linked by alternating ICLs and ECLs, and an intracellular C-terminal tail. Ciona GnRHR1, GnRHR2, and GnRHR3 each have a long tail (77–115 amino acids) characteristic of mammalian type II and other nonmammalian GnRHRs, whereas Ci-GnRHR4 has a short C-terminal tail (20 amino acids), which is more analogous to the mammalian type I receptor structure.

    The Ciona receptors have a number of features common to many GnRHRs (Fig. 1). Three Ciona GnRHRs but not Ci-GnRHR2 have consensus glycosylation sites (N-X-S/T) on their N-terminal extracellular extension. Alteration of rodent GnRHR glycosylation did not affect the binding affinity but did decrease the number of receptors expressed on the cell surface (21, 22). Like the rhodopsin family of GPCRs, all Ciona GnRHRs possess conserved cysteine residues, two of which may form a disulfide bond between the first two ECLs (23). All Ciona GnRHRs possess additional cysteine residues in the N-terminal extension and ECLs, which may form a second disulfide bond, as in the human type I GnRHR (24).

    All Ciona GnRHRs possess either Asn2.50 or Asp2.50 in TMD2. In the mouse GnRHR, Asn2.50 is thought to interact with Asp7.49 in TMD7 (25). The interactions between these two TMDs stabilize the receptor structure possibly through a water molecule as demonstrated with other GPCRs (26). Ciona GnRHRs possess His7.49 as a replacement for Asp7.49 in TMD7, a feature unique to the Ciona GnRHRs.

    A motif important in GnRHR activation, located at the junction between TMD3 and ICL2 (20, 27, 28), is well conserved in the Ciona GnRHRs. Compared with DRxxxI/V in vertebrates, three Ciona GnRHRs have a DRxxxI motif but Ci-GnRHR4 has a slight modification with DRxxxL.

    Ciona receptors have key residues for GnRH binding and formation of the binding pocket

    All four Ciona receptors appear to be true GnRH receptors based on conservation of critical motifs involved with ligand binding or formation of the binding pocket, although proof of GnRH binding or activation is essential in the definition of a GnRHR. Residues Asp2.61(98), Trp2.64(101), Asn2.65(102), Lys3.32(121), and Trp6.48(280) in human GnRHRs are all present in homologous positions in all Ciona GnRHRs (Fig. 1). Asp2.61(98) is proposed to form a salt bridge with Lys3.32(121) and have multiple interactions with GnRH possibly through His2, Trp3, and Ser4 (20, 29). Site-directed mutagenesis of Trp2.64(101) to Ala demonstrated a major reduction in ligand-induced signal transduction; Trp2.64(101) was proposed to be involved in the formation of the ligand binding pocket (30). Davidson et al. (31) suggested that Asn2.65(102) forms a hydrogen bond with Gly10-NH2 of GnRH and/or is involved in the ligand binding pocket. The exact role of Lys3.32(121) in GnRH binding remains uncertain. Lys3.32(121) was initially proposed to interact with either His2 or Trp3 of GnRH through hydrogen bonding (32). Subsequent studies demonstrated that Asp2.61(98) interacts with His2 and may form an interhelical interaction with Lys3.32(121) that acts to position the side chain of Lys3.32(121) to interact also with the ligand and/or stabilize the binding pocket (29). Chauvin et al. (33) showed that mutation of Trp6.48 to Ser or Arg reduced ligand binding and abolished GnRH-induced IP production. They suggested that Trp6.48 had a hydrophobic interaction with Trp3 in GnRH within the binding pocket.

    The human CWTPYYLLGL/IWYWF motif in the extracellular end of TMD6 (Cys6.47(279)-Phe6.60(292)] is partially conserved in all Ciona GnRHRs. This motif is thought to form an aromatic cage, which may be important for ligand affinity and G protein-mediated signal transduction (34). Ala mutants of aromatic residues Tyr6.51(283), Tyr6.52(284), and Trp6.59(291) in the human GnRHR abolished both agonist and antagonist binding and signal transduction (35). Hovelmann et al. (35) proposed that perturbation of these residues may destroy the ligand binding pocket and/or overall receptor structure, which may target the misfolded protein for destruction by the proteosome. Trp6.57(289) is conservatively replaced by Tyr in Ci-GnRHR1, Ci-GnRHR2, and Ci-GnRHR3 but is altered to Met in Ci-GnRHR4. Tyr6.58(290) is changed to Asp in Ci-GnRHR1 and Ci-GnRHR2 and to His in Ci-GnRHR3 and Ci-GnRHR4. Tyr6.58(290) is thought to interact with Tyr5 in the GnRH agonist ([D-Trp6]-GnRH) (35).

    The human type I GnRHR favors binding GnRH1 due to electrostatic interactions between Asp7.32(302) and the Arg8 that induces a high affinity -II-bend in the ligand that facilitates the positioning of GnRH1 within the ligand binding pocket (36). It seems unnecessary for Ciona GnRHRs to contain Asp7.32 because all Ciona GnRH ligands have another residue at position eight. Marmoset, green monkey, rhesus monkey, and human type II GnRHRs all lack Asp7.32, which indicates a plasticity for another residue at this position for type II GnRHRs.

    Four GnRHR mRNAs are found ubiquitously in Ciona

    Transcripts of each Ciona GnRHR were found in all tissues sampled including the neural complex, gonad, heart, intestine, endostyle, and branchial sac. We previously found that each GnRH gene is coexpressed in the above tissues (data not shown). These results suggest that GnRHRs may have local paracrine or autocrine functions in the tunicate. A screen of cDNA produced from four-cell and gastrulation stages of the developing Ciona embryo showed early expression of both Ci-gnrhr3 and Ci-gnrhr4 (results not shown), implying that these receptors may have an early role during development of the embryo.

    One Ciona GnRH activates Ci-GnRHR1 in IP path

    Results of the receptor activation assay showed that only tGnRH-6 was able to induce IP accumulation in one of the Ciona GnRHRs. Ci-GnRHR1 was shown to be the only Ciona GnRHR that was able to couple to the IP signaling pathway and elicit a dose response within the transfected COS-7 cells. Hence, tGnRH-6 was able to bind Ci-GnRHR1 and evoke an IP signaling response with a log EC50 value of –6.90.

    cAMP signaling response is dominant in the Ciona GnRHRs

    Ci-GnRHR1, Ci-GnRHR2, and Ci-GnRHR3 were able to activate a cAMP dose response with all of the tGnRHs tested. For Ci-GnRHR1, tGnRH-6 was 1000-fold more potent than tGnRH-3, -4, and -5. However, Ci-GnRHR2 did not show significant difference in potency of the GnRH peptides. In contrast, Ci-GnRHR3 had two highly potent GnRH peptides (tGnRH-3 and tGnRH-5) that were 1000-fold more effective than tGnRH-4, -6, -7, and -8.

    A Gs protein recognition sequence in the ICL1 of the mouse GnRHR was identified as specific residues at the proximal end with a BBxxB sequence motif (where B was a basic residue and x was any amino acid) or a combination of positive charges at the distal end (37). Arora et al. (37) showed that positive charges were important for receptor-Gs interaction and any unbalanced charge substitutions to the distal region drastically impaired Gs coupling and resulted in a sharp decrease in cAMP accumulation. Ci-GnRHR1 and Ci-GnRHR2 each have a BxxBxB sequence, and Ci-GnRHR3 has a BBBxB sequence in ICL1. These slightly modified but heavily basic sequences may be responsible for Gs coupling and the GnRH-induced cAMP responses observed. Ci-GnRHR4 contained only two adjacent basic residues within ICL1, which may contribute to the negative cAMP response observed.

    To date, Ci-GnRHR1 is the only known invertebrate GnRHR that activates both the IP3 and cAMP pathway. Previously, a GnRHR-like protein was identified in Drosophila melanogaster and was initially thought to be activated by GnRH (38). Later studies revealed that AKH, a peptide structurally similar to GnRH, activated this receptor (14). As a precaution we tested silkworm AKH against all the Ciona GnRHRs and found it unable to induce either IP production or cAMP accumulation, even at 10 μM concentrations.

    The different stimulation profiles displayed by the functional Ciona GnRHRs suggest distinct roles for each receptor. tGnRH-3 and tGnRH-5 were shown to have the highest potency at inducing gamete release from the live animals and were also the most effective at activating Ci-GnRHR3. It is plausible that each receptor has a specific function with Ci-GnRHR3 being responsible for the induction of gamete release within the gravid animal, but the ubiquitous expression suggests that further investigation is needed.

    Kusakabe et al. (13) showed similar expression results with Ci-gnrhr1 and Ci-gnrhr2 detected in the neural complex, gonad, stomach, intestine, endostyle, and branchial sac. In our studies, we were able to find Ci-gnrhr2 transcripts in the heart and ovary as well (data not shown). Kusakabe et al. (13) identified a partial gene encoding Ci-gnrhr3 but were unable to predict the full-length coding sequence or isolate a cDNA clone representing this third GnRHR transcript. We show that Ci-gnrhr3 is a processed transcript in all the tissues we examined. They were able to detect inward currents using Xenopus oocytes injected with Ci-gnrhr1 mRNA after stimulation with tGnRH-1 isolated from Chelyosoma productum (13). Conversely, we were unable to elicit a response to tGnRH-1 from Ci-GnRHR1 expressed in COS-7 cells through the IP pathway.

    Our previous study showed that three of nine tGnRHs (tGnRH-3, -5, and -7) were able to weakly stimulate the human type I GnRHR at 10–6 M (1). However, we found that the Ciona GnRHRs share features common to type II receptors as evidenced by the high matches to African clawed frog and the rubber eel GnRHRs. Therefore, we tested the tGnRH and related peptides on the African green monkey type II receptor. IP accumulation assays showed that tGnRH-3 and tGnRH-5 were able to stimulate the monkey GnRHR at high concentrations (see Table 4). Also, the structurally related peptide, 1-mating factor, was able to activate the gm-GnRHR at very high concentrations (10–4.5 M) (data not shown).

    Phylogeny of the Ciona GnRHRs and polymorphism

    The sequences for Ci-GnRHR1 and Ci-GnRHR2 submitted by Kusakabe et al. (13) showed higher sequence similarity to the published genome than our two sequences. However, a high level of allelic polymorphism was found in C. intestinalis individuals in the sequencing of their genome (8). Our animals were obtained from a different population compared with Kusakabe, and this probably accounts for the observed differences.

    Our Ciona receptor protein sequences were compared with other type I, type II, and type III GnRHRs and were found to cluster to their own branch of the cladogram. This indicates that the Ciona GnRHRs are more similar among themselves than to any previously identified GnRHR types. When each receptor was searched against the NCBI database, the closest external species match was either the rubber eel or the African clawed frog type II GnRHR, both proposed by Millar et al. (20) to cluster with type II GnRHRs. Like many nonmammalian GnRHRs, the Ciona receptors possess a C-terminal tail that has been implicated in rapid agonist-induced receptor internalization, coupling to G protein signaling paths and increased membrane expression (39). The high sequence identity between Ci-GnRHR1 and Ci-GnRHR2 suggests that these two receptors are the result of a more recent duplication event. Ci-GnRH3 and Ci-GnRH4 appear to have been generated from earlier gene duplications, perhaps in an ancestral tunicate due to their low sequence conservation with the other Ciona receptors.

    The functional data suggest that Ci-GnRHR2, Ci-GnRHR3, and Ci-GnRHR4 either lack the critical Gq/11 coupling motifs and do not couple well with monkey Gq/11 or the ability to activate Gq/11 is a recently acquired characteristic for GnRHRs in evolution. It appears that cAMP is the dominant pathway used by three Ciona GnRHRs. Like Ci-GnRHR1, the bullfrog, mouse, and type II gm-GnRHRs were also shown to couple to both the IP and the adenylyl cylase signaling pathways, suggesting that Ci-GnRHR1 may be an ancient ortholog (37, 40, 41).

    Ci-GnRHR4 does not have the ability to activate either IP or cAMP accumulation, at least in response to the tested peptides, and is distinctly different from all the other Ciona receptors. Ci-gnrhr4 possesses only seven exons, whereas the others each have eight. It has a much shorter C-terminal tail with only 20 amino acid residues, whereas the rest have longer tails (more than 75 residues). Ci-GnRHR4 is also the only receptor with DRxxxL in TMD3 and Met6.57 instead of Trp6.57. To be certain that GnRHR-4 is expressed as a protein, we fused the eGFP coding sequence to the 3'-end of the receptor cDNA and found the protein product was expressed. It is possible that Ci-GnRHR4 is able to be activated by another ligand, has improper posttranslational processing, or has the ability to activate an alternate signaling pathway that was not tested.

    Tunicates are classified as both invertebrates and protochordates, which make them an important group for understanding the transition from invertebrates to vertebrates (8). This study reveals that functional GnRHRs exist in animals without a pituitary gland or sex steroids. Because the tunicate larvae share many chordate features with higher chordates including humans, delineation of the function of each Ciona GnRHR reveals clues to the function and origin of all GnRHR subtypes and may uncover functions that are masked in the vertebrates.

    Acknowledgments

    We are indebted to Amanda Wilson for help with the receptor assays in N. Sherwoods’s laboratory and L. Cervini, Dr. J. Erchegyi, G-C Jiang, R. Kaiser, S. Lahrichi, and C. Miller in J. Rivier’s laboratory for the synthesis and chemical characterization of the peptides used in this study.

    Footnotes

    Nucleotide sequences for Ci-gnrhr1, Ci-gnrhr2, Ci-gnrhr3, and Ci-gnrhr4 were submitted to GenBank and appear with GenBank accession no. AY742888, AY742889, AY742890, and AY742891, respectively.

    This work was supported by grants from Natural Sciences and Engineering Research Council of Canada and Canadian Institutes of Health Research Canada (to N.M.S.) and National Institutes of Health R01-HD039899 (to J.E.R.).

    Abbreviations: AKH, Adipokinetic hormone; ECL, extracellular loop; eGFP, enhanced green fluorescent protein; f, forward; gm-GnRHR, green monkey GnRHR; GnRHR, GnRH receptor; GPCR, G protein-coupled receptor; ICL, intracellular loop; IP, inositol phosphate; r, reverse; RACE, rapid amplification of cDNA ends; t, tunicate; TE, tissue expression; TMD, transmembrane domain.

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