Our results demonstrate for the first time the presence of a GPCR in the nematode C. elegans
with homology to human GnRHR1 and AKHRs of insects (Figs. , , , , , ; Tables &). The nematode GPCR superfamily consists of 170 rhodopsin-like receptors, 650 seven-TM chemoreceptors, and other similar proteins, and represents the largest gene family accounting for more than 5% of the entire C. elegans
]. Evidence that this GPCR [WormBase: F54D7.3] is orthologous to the human GnRHR1 and Dm-AKHR is supported by the findings that, 1) the next closest potential orthologue to human GnRHR1 (UDP-glucuronosyltransferase; [GenBank: NM_073809
]) has considerably less identity (amino acid = 13.8%, E-score = 7.1) to human GnRHR1 than F54D7.3 (amino acid = 22.2%, E-score = 0.45). Likewise, the next closest potential orthologue to Dm-AKHR (hypothetical protein Y116A8B.5; [GenBank: CAA16290
]) has less identity (amino acid = 21.8%, E-score = 2e-18) to Dm-AKHR than F54D7.3 (amino acid = 28.7%, E-score = 2e-45); 2) the alignment of F54D7.3 with human GnRHR1 and two other Class A GPCRs, rhodopsin and vasopressin receptor type 1A, showed that the functionally important amino acids of F54D7.3 were significantly more similar to the same sites in human GnRHR (56%; Table ). Likewise, the alignment of F54D7.3 with Dm-GnRHR also indicated significant similarity between the functionally important amino acids (66%; Table ), and 3) this GPCR was actively transcribed in the adult worm (Fig. ) and its translated protein was localized to the germline, fertilized eggs, intestine, and pharynx (Figs. , , , ). Likewise, in Drosophila melanogaster
AKHR was most highly expressed in ovaries, digestive system, brain, tracheae and fat body cells, although immunoreactivity appeared to be more cytoplasmic in nature [20
Phylogenetic analysis supports the idea that the evolutionary relationships of Ce-GnRHR place it somewhere between the vertebrate GnRHRs and insect AKHRs, though it is more closely allied with AKHRs (Fig. ; Table ). The strict consensus tree of select Class A and B GPCRs (Fig. ) provides minimal bootstrap support for many of the deeper nodes, but three conservative conclusions relevant to our study of Ce-GnRHR may be drawn from the phylogenetic analysis: (1) Ce-GnRHR is more closely related to insect AKHRs than to chordate GnRHRs or corazonin receptors, (2) vertebrate GnRHRs comprise a distinct group of receptors, separate from all other GnRHR-like receptors, including Ce-GnRHR, and, (3) the classification of Ce-GnRHR [F54D7.3] as a GnRHR-like receptor (as opposed to another class of GPCR) is supported.
The Ce-GnRHR ligand has not been identified. Intriguingly, though, we have shown that human GnRH increases both egg laying (17%) and viable offspring (42%) in C. elegans
(Vadakkadath Meethal et al., 2004 [21
] and unpublished data), although it is unclear at this stage whether human GnRH1 binds Ce-GnRHR. GnRHR and GnRH/GnRH-like oligopeptides have been identified in all mammalian and non-mammalian vertebrate species [11
] studied to date, as well as other vertebrates and invertebrates (octopus, tunicates, lamprey, fish, frogs, etc) [23
]. While a variety of invertebrate species, including numerous insects and the oyster, Crassotrea gigas
, have been shown to possess GnRHR orthologues, insects bind a distinct, non-GnRH-like peptide (AKH) [13
]. In insects, AKHs are secreted from endocrine cells of the corpora cardiaca into the hemolymph [26
] and mobilize energy reserve from storages (from fat body) and regulate energy homeostasis [27
] by signaling through AKHRs.
Molecular phylogenetic analyses of the past decade have garnered increasingly strong support for the group Ecdysozoa [28
], the major phyla of which are Arthropoda and Nematoda. Under this phylogenetic hypothesis, it is not unreasonable to expect a nematode GnRHR will bind an AKH-like peptide. Yet, an invertebrate GnRH peptide has been biochemically characterized in the mollusk Octopus vulgaris
. Given the placement of Ce-GnRHR in the topology of our phylogenetic hypothesis – between the AKHRs and the molluscan GnRHR (oyster), whose ligand is unknown, but may very well be GnRH – another possibility is that Ce-GnRHR is a receptor capable of binding AKH- and
GnRH-like ligands. A final, equally plausible possibility is that Ce-GnRHR binds an altogether different hormone specific to nematodes, which is supported by the relatively low conservation of ligand binding amino acid residues for both human GnRHR and Dm-AKHR (Table ).
Ce-GnRHR localized to the nucleus of germline and intestinal cells, as well as to the myofilamant lattice of the pharyngeal musculature (Fig. ). Although GnRHR1 is traditionally thought of as a plasma membrane receptor [30
], it is becoming increasingly evident that GnRHR1 can be internalized from the plasma membrane to the nucleus [18
]. Indeed, the nuclear localization of GnRHR1 has been reported in rapidly proliferating cells such as pancreatic and breast cancer cells [17
]. Ligand binding to GnRHR may be the stimulus for the nuclear internalization of the receptor since GnRH has been shown to be rapidly internalized to the nuclear membrane prior to entry into the nucleus [18
]. Although we did not detect intense Ce-GnRHR staining in germline and intestinal plasma membranes (Fig. ), it remains to be determined whether Ce-GnRHR localized to the nucleus (Fig. ) is from de novo
receptor synthesized in the cytosol or from Ce-GnRHR translocalization from the plasma membrane. Nevertheless, the localization of Ce-GnRHR to the nucleus provides an exceptional molecular marker of nuclear growth as germ cells progress through the gonadal arm prior to fertilization (Fig. ).
In vertebrates, GnRH neurons originate from the olfactory placode during organogenesis [32
]. GnRH secretion from hypothalamic neurons is tightly influenced by environmental conditions [37
], and this environmental sensing mechanism regulates reproduction [41
]. Indeed, it has been demonstrated that GnRH1 increases the excitability of olfactory receptor neurons, that the terminal nerve functions to modulate the odorant sensitivity of olfactory receptor neurons and that this signaling is tightly linked to reproduction [43
]. The localization of Ce-GnRHR to the germline, pharynx, and intestine (Figs. , , ) is suggestive of a role in modulating reproductive function in accordance with environmental conditions. Like the human, the nematode also regulates reproduction dependent upon environmental signals [44
] and it is well demonstrated that reproduction in C. elegans
is strictly controlled by environmental cues such as food and temperature. Under adverse conditions (starvation, high population densities and high temperature), C. elegans
can enter a reproductively inactive alternative third larval stage called dauer [45
]. The decision to enter the developmentally arrested dauer larval stage is triggered by a combination of signals from sensory neurons in response to environmental cues [45
]. Although signaling between olfactory neurons and the reproductive system has been demonstrated in C. elegans
], it is unclear what signaling pathways are involved. It does however suggest the presence of an endocrine system that regulates reproduction in response to environmental conditions.
We propose a model whereby a putative signaling peptide (GnRH, GnRH-like peptide, and/or AKH) in C. elegans
may be released into the body of the worm from neurons in the head during favorable conditions, where it can then act to signal through Ce-GnRHR located on the pharyngeal musculature, intestine and germ cells (Figs. , , ). In this way, this peptide signaling can simultaneously initiate both pharyngeal pumping and reproduction when food is plentiful. Interestingly, the dauer-inducing pheromone detected by sensory neurons in C. elegans
signals by a complex pathway to the germline, pharynx, intestine, and the ectoderm [46
]. Indeed, it has recently been shown that octopus GnRH (oct-GnRH) has a contractile effect on the radula retractor muscle which expresses oct-GnRHR [23
], and that oct-GnRH mRNA-expressing cell bodies and immunoreactive fibers are present on the superior buccal lobe suggesting that oct-GnRH is involved in feeding behavior generated by contractions of the muscle of the buccal mass [49
]. The coupling of food intake to reproduction by such a mechanism would allow for the rapid development and subsequent reproduction of the worm. It is intriguing that Ce-GnRHR is closely related evolutionary with human GnRHR and insect AKHR, involved in regulating reproduction and metabolism, respectively. Ce-GnRHR may provide a molecular link between reproduction and metabolism.